Mat J.A.P. Daemen

Atherosclerotic lesions are predominantly observed in curved arteries and near side branches, where low or oscillatory shear stress patterns occur, suggesting a causal connection. However, the effect of shear stress on plaque vulnerability is unknown because the lack of an appropriate in vivo model precludes cause-effect studies.We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stresses in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or vortices, whereas the regions of increased shear stress were protected. Lowered shear stress lesions were larger (intima/media, 1.38+/-0.68 versus 0.22+/-0.04); contained fewer smooth muscle cells (1.9+/-1.6% versus 26.3+/-9.7%), less collagen (15.3+/-1.0% versus 22.2+/-1.0%), and more lipids (15.8+/-0.9% versus 10.2+/-0.5%); and showed more outward vascular remodeling (214+/-19% versus 117+/-9%) than did oscillatory shear stress lesions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity was higher in the lowered shear stress regions. Spontaneous and angiotensin II-induced intraplaque hemorrhages occurred in the lowered shear stress regions only.Lowered shear stress and oscillatory shear stress are both essential conditions in plaque formation. Lowered shear stress induces larger lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce stable lesions.

Abstract

One of the features of high-risk atherosclerotic plaques is a preponderance of macrophages. Experimental studies with hyperlipidemic rabbits have shown that ultrasmall superparamagnetic particles of iron oxide (USPIOs) accumulate in plaques with a high macrophage content and that this induces magnetic resonance (MR) signal changes. The purpose of our study was to investigate whether USPIO-enhanced MRI can also be used for in vivo detection of macrophages in human plaques.MRI was performed on 11 symptomatic patients scheduled for carotid endarterectomy before and 24 (n=11) and 72 (n=5) hours after administration of USPIOs (Sinerem) at a dose of 2.6 mg Fe/kg. Histological and electron microscopical analyses of the plaques showed USPIOs primarily in macrophages within the plaques in 10 of 11 patients. Histological analysis showed USPIOs in 27 of 36 (75%) of the ruptured and rupture-prone lesions and 1 of 14 (7%) of the stable lesions. Of the patients with USPIO uptake, signal changes in the post-USPIO MRI were observed by 2 observers in the vessel wall in 67 of 123 (54%) and 19 of 55 (35%) quadrants of the T2*-weighted MR images acquired after 24 and 72 hours, respectively. For those quadrants with changes, there was a significant signal decrease of 24% (95% CI, 33% to 15%) in regions of interest in the images acquired after 24 hours, whereas no significant signal change was found after 72 hours.Accumulation of USPIOs in macrophages in predominantly ruptured and rupture-prone human atherosclerotic lesions caused signal decreases in the in vivo MR images.

ObjectiveMacrophages are decisive in the chronic inflammatory processes that drive atherogenesis. The purpose of this study was to explore the presence and spatial distribution of polarized macrophage populations in human atherosclerosis.Methods & resultsWe used transcriptomics and immunohistochemistry to analyze macrophage subset dynamics in successive stages of atherogenesis. Developing lesions progressively accumulated both M1 and M2 cells, as was signified by the enhanced expression of associated markers at the transcriptional and protein level. Histologically, these markers were confined to overlapping, but spatially distinct CD68+ areas of the intima. We subsequently quantified the presence of these markers in relation to morphological determinants of plaque stability. In line with their pro-inflammatory characteristics, M1 macrophages dominated the rupture-prone shoulder regions of the plaque over M2 polarized cells, while the fibrous caps of lesions showed no significant differences between subsets. In contrast, vascular adventitial tissue displayed a pronounced M2 activation profile. As expected, areas of intraplaque hemorrhage clearly associated with CD163 staining. Rather than being limited to complicated lesions, this M2 marker was also readily detectable in stable plaques. Finally, foamy macrophages displayed an ambiguous repertoire that incorporates individual M1 and M2 markers.ConclusionM1 and M2 macrophage populations are present throughout atherogenesis. These subsets display disparity when it comes to their prevalence in morphological compartments of the vessel wall. Our current findings warrant continued investigation into the functional implications of polarized macrophage populations in human atherosclerosis.

<h3>SUMMARY:</h3> Intracranial vessel wall MR imaging is an adjunct to conventional angiographic imaging with CTA, MRA, or DSA. The technique has multiple potential uses in the context of ischemic stroke and intracranial hemorrhage. There remain gaps in our understanding of intracranial vessel wall MR imaging findings and research is ongoing, but the technique is already used on a clinical basis at many centers. This article, on behalf of the Vessel Wall Imaging Study Group of the American Society of Neuroradiology, provides expert consensus recommendations for current clinical practice.

We sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis. Atherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of HIF. To examine the presence of hypoxia in atherosclerotic plaques, the hypoxia marker pimonidazole was infused before carotid endarterectomy in 7 symptomatic patients. Also, the messenger ribonucleic acid (mRNA) and protein expression of HIF1α, HIF2α, HIF-responsive genes (vascular endothelial growth factor [VEGF], glucose transporter [GLUT]1, GLUT3, hexokinase [HK]1, and HK2), and microvessel density were determined in a larger series of nondiseased and atherosclerotic carotid arteries with microarray, quantitative reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. Pimonidazole immunohistochemistry demonstrated the presence of hypoxia, especially within the macrophage-rich center of the lesions. Hypoxia correlated with the presence of a thrombus, angiogenesis, and expression of CD68, HIF, and VEGF. The mRNA and protein expression of HIF, its target genes, and microvessel density increased from early to stable lesions, but no changes were observed between stable and ruptured lesions. This is the first study directly demonstrating hypoxia in advanced human atherosclerosis and its correlation with the presence of macrophages and the expression of HIF and VEGF. Also, the HIF pathway was associated with lesion progression and angiogenesis, suggesting its involvement in the response to hypoxia and the regulation of human intraplaque angiogenesis.

Blood vessels are exposed to multiple mechanical forces that are exerted on the vessel wall (radial, circumferential and longitudinal forces) or on the endothelial surface (shear stress). The stresses and strains experienced by arteries influence the initiation of atherosclerotic lesions, which develop at regions of arteries that are exposed to complex blood flow. In addition, plaque progression and eventually plaque rupture is influenced by a complex interaction between biological and mechanical factors—mechanical forces regulate the cellular and molecular composition of plaques and, conversely, the composition of plaques determines their ability to withstand mechanical load. A deeper understanding of these interactions is essential for designing new therapeutic strategies to prevent lesion development and promote plaque stabilization. Moreover, integrating clinical imaging techniques with finite element modelling techniques allows for detailed examination of local morphological and biomechanical characteristics of atherosclerotic lesions that may be of help in prediction of future events. In this ESC Position Paper on biomechanical factors in atherosclerosis, we summarize the current ‘state of the art’ on the interface between mechanical forces and atherosclerotic plaque biology and identify potential clinical applications and key questions for future research.

The clinical complications of atherosclerosis are caused by thrombus formation, which in turn results from rupture of an unstable atherosclerotic plaque. The formation of microvessels (angiogenesis) in an atherosclerotic plaque contributes to the development of plaques, increasing the risk of rupture. Microvessel content increases with human plaque progression and is likely stimulated by plaque hypoxia, reactive oxygen species and hypoxia-inducible factor (HIF) signalling. The presence of plaque hypoxia is primarily determined by plaque inflammation (increasing oxygen demand), while the contribution of plaque thickness (reducing oxygen supply) seems to be minor. Inflammation and hypoxia are almost interchangeable and both stimuli may initiate HIF-driven angiogenesis in atherosclerosis. Despite the scarcity of microvessels in animal models, atherogenesis is not limited in these models. This suggests that abundant plaque angiogenesis is not a requirement for atherogenesis and may be a physiological response to the pathophysiological state of the arterial wall. However, the destruction of the integrity of microvessel endothelium likely leads to intraplaque haemorrhage and plaques at increased risk for rupture. Although a causal relation between the compromised microvessel structure and atherogenesis or between angiogenic stimuli and plaque angiogenesis remains tentative, both plaque angiogenesis and plaque hypoxia represent novel targets for non-invasive imaging of plaques at risk for rupture, potentially permitting early diagnosis and/or risk prediction of patients with atherosclerosis in the near future.

Background— Inflammation has been closely linked to auto-immunogenic processes in atherosclerosis. Plasmacytoid dendritic cells (pDCs) are specialized to produce type-I interferons in response to pathogenic single-stranded nucleic acids, but can also sense self-DNA released from dying cells or in neutrophil extracellular traps complexed to the antimicrobial peptide Cramp/LL37 in autoimmune disease. However, the exact role of pDCs in atherosclerosis remains elusive. Methods and Results— Here we demonstrate that pDCs can be detected in murine and human atherosclerotic lesions. Exposure to oxidatively modified low-density lipoprotein enhanced the capacity of pDCs to phagocytose and prime antigen-specific T cell responses. Plasmacytoid DCs can be stimulated to produce interferon-α by Cramp/DNA complexes, and we further identified increased expression of Cramp and formation of neutrophil extracellular traps in atherosclerotic arteries. Whereas Cramp/DNA complexes aggravated atherosclerotic lesion formation in apolipoprotein E–deficient mice, pDC depletion and Cramp-deficiency in bone marrow reduced atherosclerosis and anti–double-stranded DNA antibody titers. Moreover, the specific activation of pDCs and interferon-α treatment promoted plaque growth, associated with enhanced anti–double-stranded–DNA antibody titers. Accordingly, anti–double-stranded DNA antibodies were elevated in patients with symptomatic versus asymptomatic carotid artery stenosis. Conclusions— Self-DNA (eg, released from dying cells or in neutrophil extracellular traps) and an increased expression of the antimicrobial peptide Cramp/LL37 in atherosclerotic lesions may thus stimulate a pDC-driven pathway of autoimmune activation and the generation of anti–double-stranded-DNA antibodies, critically aggravating atherosclerosis lesion formation. These key factors may thus represent novel therapeutic targets.

This study sought to examine the ultrastructure of microvessels in normal and atherosclerotic coronary arteries and its association with plaque phenotype. Microvessels in atherosclerotic plaques are an entry point for inflammatory and red blood cells; yet, there are limited data on the ultrastructural integrity of microvessels in human atherosclerosis. Microvessel density (MVD) and ultrastructural morphology were determined in the adventitia, intima-media border, and atherosclerotic plaque of 28 coronary arteries using immunohistochemistry for endothelial cells (Ulex europeaus, CD31/CD34), basement membrane (laminin, collagen IV), and mural cells (desmin, alpha-smooth muscle [SM] actin, smoothelin, SM1, SM2, SMemb). Ultrastructural characterization of microvessel morphology was performed by electron microscopy. The MVD was increased in advanced plaques compared with early plaques, which correlated with lesion morphology. Adventitial MVD was higher than intraplaque MVD in normal arteries and early plaques, but adventitial and intraplaque MVD were similar in advanced plaques. Although microvessel basement membranes were intact, the percentage of thin-walled microvessels was similarly low in normal and atherosclerotic adventitia, in the adventitia and the plaque, and in all plaque types. Intraplaque microvascular endothelial cells (ECs) were abnormal, with membrane blebs, intracytoplasmic vacuoles, open EC-EC junctions, and basement membrane detachment. Leukocyte infiltration was frequently observed by electron microscopy, and confirmed by CD45RO and CD68 immunohistochemistry. The MVD was associated with coronary plaque progression and morphology. Microvessels were thin-walled in normal and atherosclerotic arteries, and the compromised structural integrity of microvascular endothelium may explain the microvascular leakage responsible for intraplaque hemorrhage in advanced human coronary atherosclerosis.

Extracellular matrix (ECM) remodeling is one of the underlying mechanisms in cardiovascular diseases. Cathepsin cysteine proteases have a central role in ECM remodeling and have been implicated in the development and progression of cardiovascular diseases. Cathepsins also show differential expression in various stages of atherosclerosis, and in vivo knockout studies revealed that deficiency of cathepsin K or S reduces atherosclerosis. Furthermore, cathepsins are involved in lipid metabolism. Cathepsins have the capability to degrade low-density lipoprotein and reduce cholesterol efflux from macrophages, aggravating foam cell formation. Although expression studies also demonstrated differential expression of cathepsins in cardiovascular diseases like aneurysm formation, neointima formation, and neovascularization, in vivo studies to define the exact role of cathepsins in these processes are lacking. Evaluation of the feasibility of cathepsins as a diagnostic tool revealed that serum levels of cathepsins L and S seem to be promising as biomarkers in the diagnosis of atherosclerosis, whereas cathepsin B shows potential as an imaging tool. Furthermore, cathepsin K and S inhibitors showed effectiveness in (pre) clinical evaluation for the treatment of osteoporosis and osteoarthritis, suggesting that cathepsin inhibitors may also have therapeutic effects for the treatment of atherosclerosis.

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

Understanding the pathophysiology of atherogenesis and the progression of atherosclerosis have been major goals of cardiovascular research during the previous decades. However, the complex molecular and cellular mechanisms underlying plaque destabilization remain largely obscure. Here, we review how lesional cells undergo cell death and how failed clearance exacerbates necrotic core formation. Advanced atherosclerotic lesions are further weakened by the pronounced local activity of matrix-degrading proteases as well as immature neovessels sprouting into the lesion. To stimulate translation of the current knowledge of molecular mechanisms of plaque destabilization into clinical studies, we further summarize available animal models of plaque destabilization. Based on the molecular mechanisms leading to plaque instability, we outline the current status of clinical and preclinical trials to induce plaque stability with a focus on induction of dead cell clearance, inhibition of protease activity, and dampening of inflammatory cell recruitment.

Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions.

CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l(-/-)) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l(-/-) platelets into Apoe(-/-) mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l(-/-) platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l(-/-) platelets in contrast to Cd40l(+/+) platelets did not promote lesion formation. Remarkably, injection of Cd40l(+/+), but not Cd40l(-/-), platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l(-/-) platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions.

In the evaluation of cerebrovascular CO 2 reactivity measurements, it is often assumed that the diameter of the large intracranial arteries insonated by transcranial Doppler remains unaffected by changes in arterial CO 2 partial pressure. However, the strong cerebral vasodilatory capacity of CO 2 challenges this assumption, suggesting that there should be some changes in diameter, even if very small. Data from previous studies on effects of CO 2 on cerebral artery diameter [middle cerebral artery (MCA)] have been inconsistent. In this study, we examined 10 healthy subjects (5 women, 5 men, age 21–30 yr). High-resolution (0.2 mm in-plane) MRI scans at 7 Tesla were used for direct observation of the MCA diameter during hypocapnia, −1 kPa (−7.5 mmHg), normocapnia, 0 kPa (0 mmHg), and two levels of hypercapnia, +1 and +2 kPa (7.5 and 15 mmHg), with respect to baseline. The vessel lumen was manually delineated by two independent observers. The results showed that the MCA diameter increased by 6.8 ± 2.9% in response to 2 kPa end-tidal Pco 2 (Pet CO 2 ) above baseline. However, no significant changes in diameter were observed at the −1 kPa (−1.2 ± 2.4%), and +1 kPa (+1.4 ± 3.2%) levels relative to normocapnia. The nonlinear response of the MCA diameter to CO 2 was fitted as a continuous calibration curve. Cerebral blood flow changes measured by transcranial Doppler could be corrected by this calibration curve using concomitant Pet CO 2 measurements. In conclusion, the MCA diameter remains constant during small deviations of the Pet CO 2 from normocapnia, but increases at higher Pet CO 2 values.

Summary Neutrophils are important cellular sources of interleukin (IL) 17A and –F. Moreover, upon activation neutrophils are able to excrete chromatin embedded with components from their cytoplasmic granules to form ‘neutrophil extracellular traps’ (NETs). Recent studies suggested that NETs contribute to thrombosis by promoting fibrin deposition and platelet aggregation. IL17A may also promote thrombosis by enhancing platelet aggregation. In the present study we investigated the presence of neutrophils, NETs and IL17A and –F in coronary thrombosuction specimens obtained from patients after acute myocardial infarction. Neutrophils and NETs were identified using histochemical (H&amp;E, Feulgen procedure) and immunohistochemical stainings (Histone H1, myeloperoxidase, neutrophil elastase) in 15 fresh, 15 lytic and 15 organised thrombi. The presence and distribution of IL17A and –F was studied using (immuno)histochemical double staining and spectral image analysis, rtPCR and Western blot. High numbers of neutrophils are present (10–30% of the thrombus mass) in fresh and lytic, but not in organized thrombus. NETs were frequently observed in fresh (4/15) and lytic (12/15), but never in organised thrombus specimens. Double staining combining the Feulgen reaction with Histone- H1, MPO or neutrophil elastase confirmed colocalisation with DNA. Cytoplasmatic IL17A/F staining was found in the majority of the neutrophils, extracellularly and in NETs. Western blotting confirmed the presence of IL17A and IL17F in thrombus specimens. In conclusion, a large burden of neutrophils, neutrophil extracellular traps and IL17A and –F are important constituents of fresh and lytic thrombus after acute myocardial infarction. The specific colocalisation of these indicates a role during thrombus stabilisation and growth.

HomeCirculationVol. 130, No. 16Cause and Mechanisms of Intracranial Atherosclerosis Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessResearch ArticlePDF/EPUBCause and Mechanisms of Intracranial Atherosclerosis Katja Ritz, PhD, Nerissa P. Denswil, MSc, Olga C.G. Stam, MD, Johannes J. van Lieshout, MD, PhD and Mat J.A.P. Daemen, MD, PhD Katja RitzKatja Ritz From the Departments of Pathology (K.R., N.P.D., O.C.G.S., M.J.A.P.D.) and Internal Medicine (J.J.v.L.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Laboratory for Clinical Cardiovascular Physiology, AMC Center for Heart Failure Research, Academic Medical Centre, Amsterdam, The Netherlands (J.J.v.L.); and MRC/Arthritis Research UK Centre for Musculoskeletal Ageing Research, School of Life Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK (J.J.v.L.). Search for more papers by this author , Nerissa P. DenswilNerissa P. Denswil From the Departments of Pathology (K.R., N.P.D., O.C.G.S., M.J.A.P.D.) and Internal Medicine (J.J.v.L.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Laboratory for Clinical Cardiovascular Physiology, AMC Center for Heart Failure Research, Academic Medical Centre, Amsterdam, The Netherlands (J.J.v.L.); and MRC/Arthritis Research UK Centre for Musculoskeletal Ageing Research, School of Life Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK (J.J.v.L.). Search for more papers by this author , Olga C.G. StamOlga C.G. Stam From the Departments of Pathology (K.R., N.P.D., O.C.G.S., M.J.A.P.D.) and Internal Medicine (J.J.v.L.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Laboratory for Clinical Cardiovascular Physiology, AMC Center for Heart Failure Research, Academic Medical Centre, Amsterdam, The Netherlands (J.J.v.L.); and MRC/Arthritis Research UK Centre for Musculoskeletal Ageing Research, School of Life Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK (J.J.v.L.). Search for more papers by this author , Johannes J. van LieshoutJohannes J. van Lieshout From the Departments of Pathology (K.R., N.P.D., O.C.G.S., M.J.A.P.D.) and Internal Medicine (J.J.v.L.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Laboratory for Clinical Cardiovascular Physiology, AMC Center for Heart Failure Research, Academic Medical Centre, Amsterdam, The Netherlands (J.J.v.L.); and MRC/Arthritis Research UK Centre for Musculoskeletal Ageing Research, School of Life Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK (J.J.v.L.). Search for more papers by this author and Mat J.A.P. DaemenMat J.A.P. Daemen From the Departments of Pathology (K.R., N.P.D., O.C.G.S., M.J.A.P.D.) and Internal Medicine (J.J.v.L.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Laboratory for Clinical Cardiovascular Physiology, AMC Center for Heart Failure Research, Academic Medical Centre, Amsterdam, The Netherlands (J.J.v.L.); and MRC/Arthritis Research UK Centre for Musculoskeletal Ageing Research, School of Life Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK (J.J.v.L.). Search for more papers by this author Originally published14 Oct 2014https://doi.org/10.1161/CIRCULATIONAHA.114.011147Circulation. 2014;130:1407–1414Intracranial atherosclerosis, one of the leading causes of ischemic stroke, is associated with an increased risk for recurrent stroke and dementia.1,2 Individuals of Asian, Hispanic, and African American ancestry are especially affected. Recent European studies revealed a much higher prevalence of intracranial lesions than commonly presumed, suggesting that intracranial atherosclerotic disease is potentially the most common cause of ischemic stroke worldwide.1,3 Ischemic strokes are clinically categorized into 5 subtypes based on their underlying cause: large-artery atherosclerotic stenosis, small-artery disease (lacunes), cryptogenic, major-risk-source cardiogenic embolism, and unusual (eg, dissections, arteritis). Most nonlacunar ischemic strokes are thought to be thromboembolic, which presumably also accounts for most cryptogenic strokes. Embolic sources include minor-risk or covert cardiac sources, veins via paradoxical embolism, and nonocclusive atherosclerotic plaques in the aortic arch or cervical or cerebral arteries.4 Besides embolic strokes, 2 other mechanisms have been associated with intracranial atherosclerosis-related strokes, namely hypoperfusion through a stenotic artery causing watershed or border-zone stroke and plaque overgrowth of perforator artery ostia, which is associated with penetrating artery disease and lacunar infarcts and has been related to cryptogenic strokes.5–7 Even mild stenosis of intracranial atherosclerotic arteries (<50%) may therefore be clinically relevant, and high-resolution magnetic resonance imaging studies are needed to identify and determine the degree and location of stenosis in this patient group.5,8 The possibly causal role of nonstenotic plaques in ischemic stroke highlights the need for more insight into the mechanisms and occurrence of intracranial atherosclerosis.In the 1960s and 1970s, large, descriptive autopsy studies were conducted, providing classic morphological features of intracranial arteries. Despite the importance of intracranial atherosclerosis to stroke and dementia, there is a lack of more recent mechanistic studies. Therefore, we intend to draw attention to this neglected research field by providing an overview of available literature and a working model for intracranial atherosclerosis.Search StrategyA MEDLINE search was performed of literature published in English using the MeSH term intracranial atherosclerosis. A total of 8146 search hits were obtained for articles published before October 2013 and screened for relevance to the cause and mechanisms of intracranial atherosclerosis. Other references were extracted by cross-reference. Only studies including ≥10 patients were considered relevant. Because of space restrictions, we were able to include only a subset of references in this study; the remaining references can be found in Table I in the online-only Data Supplement.EpidemiologyIncidence and PrevalenceIn 45% to 62% of patients with ischemic stroke, intracranial plaques or stenoses were identified, which were causal in ≈10% to 20% of cases (reviewed elsewhere9). The estimated prevalence of symptomatic intracranial stenosis in literature ranges from 20% to 53%, depending on the study population, race, and method of choice (Table II in the online-only Data Supplement), and from 3% to 82% in asymptomatic patients. Most studies revealed a higher incidence of intracranial atherosclerosis in Asians and African Americans compared with white Americans. The few studies on white Europeans that are available suggest a high prevalence of intracranial plaques or stenosis. One French autopsy study detected intracranial plaques and stenosis in 62% and 43% of stroke cases, respectively, and in a Dutch study, 82% of asymptomatic patients showed calcification of the intracranial internal carotid artery (ICA) by computed tomography.3,8 Notably, magnetic resonance angiography, computed tomography angiography, and transcranial Doppler, which measure luminal changes, may underestimate the number of intracranial plaques, partly explaining the large differences in the literature.8Onset, Course, and DistributionIn the 1960s and 1970s, several large-scale autopsy studies performed in asymptomatic cohorts from fetuses to patients in their 10th decade of life revealed intracranial atherosclerotic changes from the first to second decade, progressing with age.10,11 Advanced atherosclerotic lesions are almost nonexistent up to the fourth decade.12 Overall, intracranial atherosclerosis develops ≈20 years later in life compared with atherosclerosis in extracranial arterial beds.13 Progression of atherosclerosis was not parallel in different vascular beds. Although aortic atherogenesis progressed linearly, intracranial atherosclerosis increased more slowly initially and paralleled aortic lesions thereafter.12 The steepest gradient for the incidence of intracranial atherosclerosis was reported in the sixth and seventh decades,10 with a steady increase beyond the eighth and ninth decades,11 whereas coronary atherosclerosis progressed more rapidly initially and attenuated between the fifth and eighth decades.12 Three percent to 4% of individuals >80 years of age exhibited only mild intracranial atherosclerotic changes.10,12 Intracranial atherosclerotic stenoses were described as dynamic lesions showing progression and regression, but they were less dynamic than coronary stenoses. Repeated magnetic resonance angiography over a 7-year period in patients with ischemic heart disease showed a 1.1% annual progression of the average intracranial stenosis. Differences among the intracranial vessels were evident with stable atherosclerosis in the intracranial ICA and dynamic lesions in the anterior (ACA), middle (MCA), and posterior (PCA) cerebral arteries, with 2.6% annual progression of average stenosis. Regression was noted in 14% of intracranial ICAs and 28% of ACAs, MCAs, and PCAs.14 A 5-year longitudinal study of 41 Japanese patients with ischemic heart disease reported the progression of cervical carotid artery stenosis in 5 patients (12%) and in only 1 patient with intracranial stenosis.15 During a 2- to 3-year follow-up of 40 stroke patients, 33% of MCA stenoses progressed and 8% regressed.16 Intracranial lesions were identified predominantly in the anterior circulation.17 Overall, American and European studies showed a similar pattern: The ICA was most commonly affected, followed by the MCA, basilar artery (BA), intracranial vertebral artery (VA), PCA, and ACA (Figure 1).10,18 The MCA appeared to be most commonly involved in Asians, followed by the ICA, BA, VA, PCA, and ACA.17 In all cohorts, cerebellar and communicating arteries were barely affected. Atherosclerosis in the intracranial ICA was observed mainly in the cavernous but also in the supraclinoid segment.18,19 The BA was commonly affected in the upper and lower parts and less affected in the middle part.20 MCA lesions were mainly found in the M2 segment.18Download figureDownload PowerPointFigure 1. Circle of Willis of a 90-year-old subject. Macroscopically, atherosclerotic lesions can be identified by the white vessels, whereas nondiseased arteries appear largely transparent. This case shows prominent atherosclerosis mainly in the internal carotid artery, vertebral artery, basilar artery, left middle cerebral artery, and posterior cerebral artery.MorphologyBasic StructureOne major characteristic that distinguishes healthy intracranial from extracranial arteries is that extracranial arteries such as the aorta and carotid arteries are elastic arteries rich in elastin filaments in the tunica media. In contrast, intracranial arteries are muscular arteries with few elastic fibers.21 The transition from elastic to muscular artery is at the level of the carotid bifurcation22 and embryological junctions between segments of the VA and ICA, which has been attributed to different embryological sites of origin of their primordial mesodermal cells.23 Compared with extracranial arteries of a similar size, a thinner media, less abundant adventitia, and only a few elastic fibers have been reported for intracranial vessels13 with a denser internal elastic lamina and without an external elastic lamina.13,21 The external elastic lamina is still present in the petrous portion of the ICA but disappears within the cavernous portion, which forms a hotspot of stenosis.24 A distinct vessel wall metabolism was suggested for intracranial arteries. Intima-media preparations of unaffected intracranial arteries showed lower contents of hexosamine, uronic acid, and sulfur; a lower proportion of hyaluronic acid and chondroitin sulfates in the total glycosaminoglycans; and a lower ratio of ester to total cholesterol, whereas the percentage of heparin sulfate was higher compared with normal aorta and coronary arteries.25 In addition, unaffected intracranial arteries of all ages revealed elevated antioxidant enzyme activity (manganese superoxide dismutase, copper-zinc superoxide dismutase, catalase) compared with extracranial arteries. Animal studies in rats and rabbits reported that the BA had less vesicles and caveolae and exhibited tight junctions between endothelial cells, leading to a reduced intimal permeability compared with the aorta and suggesting the presence of a barrier function.26,27 In monkeys and rabbits, a distinct composition of the glycocalyx on luminal endothelial cells was suggested in cerebral arteries; the carbohydrate-binding protein concanavalin A reacted with aorta, coronary, and carotid arteries but not with cerebral arteries. It has been speculated that a specific glycocalyx composition inhibits trapping of chylomicrons and very-low-density lipoprotein, resulting in a reduced deposition of apolipoproteins in the intima of intracranial vessels.28 In cats, histamine stimulation showed a 3-fold-stronger contraction of extracranial compared with intracranial arteries.29 Smooth muscle cells of rabbit intracranial arteries were relatively insensitive to sympathomimetic stimulation compared with systemic vessels.Vasa VasorumVasa vasorum are an essential component of extracranial vessels that facilitate nourishing and elimination of wastes; their existence in intracranial vessels is controversial. Most animal studies claim that vasa vasorum are absent beyond the first 1 to 2 cm of dural penetration in studies on Wistar rats, cats, and dogs. In cats, the presence of rete vasorum in the adventitia was suggested, being in continuity with the subarachnoid space, permeable to large proteins, and allowing diffusion between the cerebrospinal fluid and medial layer.30 Rete vasorum were not observed in humans. Vasa vasorum are found mainly in proximal intracranial segments; are associated with aging and vascular pathological processes such as vasculitis, atherosclerosis, aneurysm, and thrombosis; and may represent a response to vascular damage (reviewed elsewhere31). Vasa vasorum were reported in the MCA (M1 and A1 segments) and proximal parts of the ICA, VA, and BA.32,33 They were not seen beyond the first 1.5 cm proximal segments of the intracranial arteries after dural penetration, which corresponds to arteries with a thickness of <250 μm and to arteries surrounded by nutrient-rich cerebrospinal fluid.31,32 It is our experience that adventitial stripping, especially of smaller arteries, which is barely avoidable in autopsy studies, may lead to an underestimation of the presence of vasa vasorum. It has been suggested that vasa vasorum are required only beyond a critical vessel thickness to supplement luminal diffusion.32 The thin media and adventitia, which may facilitate luminal diffusion, and the absence of the external elastic lamina, which may allow greater diffusion from the cerebrospinal fluid, support this theory.31 Recent evidence suggests a critical role for the adventitia, including vasa vasorum in atherogenesis by initiating an inflammatory cascade. Along this line, later onset of more stable intracranial lesions may be explained partly by the initial absence of adventitial vasa vasorum, at least in normal intracranial arteries.31Plaque Characteristics and Age-Related ChangesIn intracranial arteries, aging was associated with a gradual loss of elastic fibers and muscular elements in the media and an increase in collagen tissue replacing medial muscle fibers.34 From the second to third decade, reduplication and splitting of the thick internal elastic lamina were observed frequently in combination with intimal thickening, which was most prominent from the fifth to sixth decade. In the same age group, fibrosis and hyalinization of media and adventitia prevailed.20 In the aorta and coronary arteries, fragmentation and reduplication of the internal elastic lamina were common in fetuses, infants, and young juveniles. In contrast to the aorta, lipids were rarely observed in intracranial arteries in patients <15 years of age.34Intracranial lesions not only developed later in life compared with extracranial vessels but developed mainly as fibrous plaques with fewer fatty streaks and complicated lesions. Complicated lesions, which contain calcifications or a plaque rupture, appeared after the fifth decade with a degree of involvement and lipid content similar to that observed in coronary arteries and were limited mainly to proximal segments of the ICA, VA, and BA.12,13 Hoff35,36 reported no major differences in chemical and enzymatic plaque characteristics in intracranial compared with extracranial arteries and no qualitative differences in apolipoproteins (apolipoprotein A1 from high-density lipoprotein, apolipoprotein B from low-density lipoprotein [LDL], apolipoprotein CIII from very-low-density lipoprotein).Intracranial arteries of human fetuses from hypercholesterolemic mothers showed fewer intimal macrophages and less intimal LDL, and oxidized LDL compared with extracranial arteries, which is suggestive of divergent atherogenic responses.37,38 Evidence for intracranial protective mechanisms comes from animal studies in rabbits. Hypercholesterolemia alone evoked a reduced intimal permeability and foam cell accumulation in the aorta but not in the BA, whereas the combination of hypercholesterolemia and hypertension also affected the permeability of intracranial arteries.27Our Histological DataOur review of the literature made clear that histological data in a recent cohort are lacking. Therefore, we screened 283 circle of Willis segments from 18 asymptomatic patients (mean age, 70.2±10.9 years; range, 51–90 years; male, 9; causes of death: cardiovascular disease, 8; malignancy, 3; subarachnoid hemorrhage, 2; Alzheimer disease, 2; sickle cell disease, 1; acute stroke, 1; HIV/hepatitis, 1) for basic structural features (Methods in the online-only Data Supplement). In accordance with previous literature, we identified mainly early lesions (63%) and a few advanced atherosclerotic lesions (15%). Calcifications were rare (6%). Two patients presented with complicated lesions (chronic total occlusion and intraplaque hemorrhage). Intracranial arteries, especially the smaller arteries (ACA, PCA, and cerebellar and communicating arteries), show a distinct structure such as the lack of vasa vasorum and an external elastic lamina and only a few medial elastic fibers compared with extracranial arteries. The larger intracranial arteries such as the ICA, MCA, VA, and BA, however, show an intermediate phenotype sharing structural features of both the larger extracranial and the smaller intracranial arteries (Table 1), which may explain the conflicting results in literature. Macrophage load was low in our series, which is in line which previous observations38 (0.9±0.7% CD68 positivity per plaque area compared with 1.8±2.4% in coronaries).39 In general, intracranial arteries exhibited fewer and more stable lesions, and the few advanced atherosclerotic lesions were identified predominantly in the large intracranial arteries such as the ICA, MCA, and VA.Table 1. Data From This Review on the Basic Characteristics of the Large Intracranial Arteries of 18 Asymptomatic PatientsICAVAMCABAType of lesion, % (n/N) Early57 (17/30)54 (15/28)68 (19/28)75 (24/32) Advanced33 (10/30)25 (7/28)25 (7/28)16 (5/32)High content of elastin fibers, % (n/N)37 (11/30)43 (12/28)29 (8/28)13 (4/32)Continuous EEL, % (n/N)17 (5/30)79 (22/28)21 (6/28)9 (3/32)Calcification, % (n/N)20 (6/30)18 (5/28)14 (4/28)9 (3/32)Vasa vasorum, % (n/N)53 (16/30)43 (12/28)11 (3/28)16 (5/32)Macrophages(mean±SD), %0.9±0.70.4±0.50.8±0.70.9±0.9BA indicates basilar artery; EEL, external elastic lamina; ICA, intracranial internal carotid artery; MCA, middle cerebral artery; and VA, intracranial vertebral artery. See Methods in the online-only Data Supplement for more information.Risk FactorsNonmodifiable Risk FactorsAgeAge is one of the most important independent risk factors for both intracranial and extracranial atherosclerosis. Several autopsy and imaging studies showed that aging is associated with increasing prevalence and severity of intracranial atherosclerosis among all investigated races with, as mentioned above, a distinct disease progression compared with extracranial arteries.3,10,19,40RaceAfrican Americans show a comparable or more severe degree of atherosclerosis compared with white Americans.41 In Asians, intracranial atherosclerosis developed earlier and more extensively compared with white Americans and Europeans.42 In symptomatic patients with transient ischemic attack or ischemic stroke, studies consistently reported the highest incidence and severity of intracranial atherosclerosis in Asians and Hispanics, followed by African Americans and whites. The reverse order was found for extracranial lesions.43,44 Differences in incidence and location of atherosclerosis were also observed within countries. Intracranial lesions in symptomatic patients were more common in North than in South China, which has been attributed to a more Westernized lifestyle in North China.45 Within African populations, Nigerians had lower atherosclerotic scores compared with Senegalese, Ugandans, and African Americans, with highest scores in the last group.46,47 Therefore, observed differences among races cannot be attributed to genetic factors only but are highly influenced by lifestyle and other risk factors. In addition, most studies do not take into account differences in the prevalence of vascular risk factors, being higher in Hispanic and African American populations and maybe resulting in an overrepresentation of specific races in intracranial atherosclerosis.48SexThe incidence of extracranial atherosclerosis is higher in men; this correlation is less evident in patients with intracranial lesions.19,40,49 This discrepancy may be attributed to the different disease course in men and women. Men showed a high increase in intracranial lesions in the fourth and fifth decades, which steadily progressed with age, whereas women exhibited relatively mild atherosclerotic lesions until the sixth decade, with rapidly increasing lesion formation thereafter. In the eighth and ninth decades, the degree of intracranial atherosclerosis was comparable between sexes, whereas women showed higher atherosclerotic scores in the ninth and tenth decades.10,45 It has been speculated that the observed sex differences can be explained by a distinct risk factor profile resulting from the influence of sex hormones such as the known hypocholesterolemic effect of estrogens.50OthersIt has been suggested recently that the circle of Willis and its communicating arteries protect the cerebral artery and blood-brain barrier from hemodynamic stress.51 In line with this hypothesis, variations in the circle of Willis were shown to influence the volume flow rates of the bilateral ICA and BA in healthy individuals and the development of atherosclerosis.52,53 Racial differences in atherogenesis that have been discussed before could not be linked to anatomic variations of the circle of Willis.54 No clear genetic risk factors such as 9p21 for coronary heart disease have been identified for intracranial atherosclerosis. Notably, most studies have been conducted in Asian cohorts; data for other countries, especially European countries, are lacking.Modifiable Risk FactorsHypertensionOne of the most important risk factors for atherosclerosis, especially intracranial lesions, is hypertension. Hypertension has been correlated to the degree of atherosclerosis in intracranial arteries in different ethnic cohorts.17,19,55 Some studies reported a higher incidence of hypertension in populations of African and Asian ancestry, which may explain their higher prevalence of intracranial atherosclerosis.43,46,56Diabetes MellitusDiabetes mellitus is a specific risk factor for intracranial lesions regardless of race in symptomatic and asymptomatic cohorts.3,8,19,40,44 In Koreans, diabetes mellitus was an independent risk factor for intracranial lesions only after 50 years of age50 and only in posterior, not anterior, circulation diseases in a prospective study.17 As for hypertension, the aforementioned higher incidence of intracranial lesions in patients of African and Asian ancestry may be partly attributed to an increased prevalence of diabetes mellitus.56Metabolic SyndromeRecent magnetic resonance angiography studies showed an independent association of the metabolic syndrome with intracranial atherosclerosis.17,44 In a prospective Korean study, the metabolic syndrome was related more to intracranial than to extracranial lesions and to posterior and not anterior circulation strokes.17DyslipidemiaDyslipidemia is a known risk factor for coronary atherosclerosis and myocardial infarction, but its role in intracranial atherosclerosis is less clear.48,57 High LDL cholesterol was associated mainly with extracranial lesions, whereas a high ratio of apolipoprotein B to apolipoprotein I and low levels of apolipoprotein AI, the major protein component of high-density lipoprotein, correlated with intracranial lesions.49,58 In China, low high-density lipoprotein cholesterol is one of the most common types of dyslipidemia and was associated with the development of intracranial artery stenosis in a cohort of acute ischemic stroke.59 Sex-specific differences were reported in 2 Asian studies. Hypercholesterolemia was an independent risk factor for intracranial atherosclerosis only in asymptomatic men, whereas elderly symptomatic women >63 years of age had significantly more intracranial atherosclerotic lesions and hyperlipidemia than men.45,50 Race and environmental factors may influence the effect of dyslipidemia on atherogenesis. Generally, individuals of Asian and African ancestry exhibited lower serum lipid levels than whites, which may be one factor explaining the lower incidence of extracranial and coronary atherosclerosis in both populations.11,43OthersExtracranial atherosclerosis was suggested as a risk factor for intracranial lesions. Extensive coronary atherosclerotic disease correlated with intracranial lesions,8,11,60 and patients with concurrent lesions had a higher risk of suffering further (fatal) vascular events.61 In a cohort of symptomatic intracranial atherosclerosis, 52% of cases were diagnosed with silent myocardial ischemia caused by coronary artery disease.62 The American Heart Association Stroke Council recommends testing for asymptomatic coronary artery disease in patients who have had ischemic events associated with intracranial atherosclerosis.9,63 In contrast, other studies reported correlations between coronary and carotid but not intracranial lesions64 or failed to show a correlation.15,18 A few studies suggested that smoking, especially duration of smoking, is a risk factor for intracranial lesions.3,49 However, large-scale studies of the effects of smoking on intracranial atherosclerosis are scarce. As for extracranial lesions, moderate hyperhomocysteinemia was a predictor for severity of intracranial atherosclerosis in Asian patients with cerebral infarction.65 A few reports associated Alzheimer disease, sickle cell disease, systemic lupus erythematosus, radiotherapy, bacterial meningitis, and Herpes zoster infection with intracranial atherosclerosis, but their contribution needs further research.66–71In conclusion, age, hypertension, diabetes mellitus, and probably the metabolic syndrome are the most consistent risk factors for intracranial atherosclerosis. Race may represent a predisposing factor, which is unfavorable in combination with other risk factors and especially lifestyle. Sex appears to influence intracranial atherosclerosis, and its effects are age dependent. Genetics may predispose to intracranial atherosclerosis, but large-scale association studies in different ethnic groups are lacking.MechanismsTwo major characteristics that distinguish intracranial and extracranial atherosclerosis are the later onset and the more stable plaque phenotype in intracranial arteries, which may be explained by the distinct characteristics of the intracranial arteries (Table 2 and Figure 2). These characteristics may also be linked to the role of intracranial arteries in regulating the cerebrovascular resistance. The control mechanisms of cerebral blood flow (CBF) encompass cerebrovascular responsiveness to O2 and CO2, cerebral autoregulation and neurogenic control of the cerebral vasculature, endothelium-mediated signaling, and neurovascular coupling meeting local cerebral metabolic demand.75 Cerebral artery endothelial cells and pericytes produce nitric oxide in direct proportion to the arterial CO2 partial pressure76 and contribute to the resting tone of cerebral arteries and arterioles.77 Impairment of cerebral autoregulation results in pressure-passive CBF; that is, CBF increases and decreases together with cerebral perfusion pressure, whereas reduced CO2 responsiveness affects the vasodilatory reserve of the brain (Figure I in the online-only Data Supplement). The traditional concept in peripheral circulations is that arterioles rather than large arteries are the main site of vascular resistance. However, for the brain, large extracranial vessels and surface vessels contribute ≈50% of cerebral vascular resistance,78 and both CBF

Atherothrombotic vascular disease is often triggered by a distinct type of atherosclerotic lesion that displays features of impaired inflammation resolution, notably a necrotic core and thinning of a protective fibrous cap that overlies the core. A key cause of plaque necrosis is defective clearance of apoptotic cells, or efferocytosis, by lesional macrophages, but the mechanisms underlying defective efferocytosis and its possible links to impaired resolution in atherosclerosis are incompletely understood. Here, we provide evidence that proteolytic cleavage of the macrophage efferocytosis receptor c-Mer tyrosine kinase (MerTK) reduces efferocytosis and promotes plaque necrosis and defective resolution. In human carotid plaques, MerTK cleavage correlated with plaque necrosis and the presence of ischemic symptoms. Moreover, in fat-fed LDL receptor-deficient (Ldlr-/-) mice whose myeloid cells expressed a cleavage-resistant variant of MerTK, atherosclerotic lesions exhibited higher macrophage MerTK, lower levels of the cleavage product soluble Mer, improved efferocytosis, smaller necrotic cores, thicker fibrous caps, and increased ratio of proresolving versus proinflammatory lipid mediators. These findings provide a plausible molecular-cellular mechanism that contributes to defective efferocytosis, plaque necrosis, and impaired resolution during the progression of atherosclerosis.

Vascular stiffness is a mechanical property of the vessel wall that affects blood pressure, permeability, and inflammation. As a result, vascular stiffness is a key driver of (chronic) human disorders, including pulmonary arterial hypertension, kidney disease, and atherosclerosis. Responses of the endothelium to stiffening involve integration of mechanical cues from various sources, including the extracellular matrix, smooth muscle cells, and the forces that derive from shear stress of blood. This response in turn affects endothelial cell contractility, which is an important property that regulates endothelial stiffness, permeability, and leukocyte–vessel wall interactions. Moreover, endothelial stiffening reduces nitric oxide production, which promotes smooth muscle cell contraction and vasoconstriction. In fact, vessel wall stiffening, and microcirculatory endothelial dysfunction, precedes hypertension and thus underlies the development of vascular disease. Here, we review the cross talk among vessel wall stiffening, endothelial contractility, and vascular disease, which is controlled by Rho-driven actomyosin contractility and cellular mechanotransduction. In addition to discussing the various inputs and relevant molecular events in the endothelium, we address which actomyosin-regulated changes at cell adhesion complexes are genetically associated with human cardiovascular disease. Finally, we discuss recent findings that broaden therapeutic options for targeting this important mechanical signaling pathway in vascular pathogenesis.

Vascular lipid accumulation and inflammation are hallmarks of atherosclerosis and perpetuate atherosclerotic plaque development. Mediators of inflammation, ie, interleukin (IL)-6, are elevated in patients with acute coronary syndromes and may contribute to the exacerbation of atherosclerosis.To assess the role of IL-6 in atherosclerosis, ApoE-/--IL-6-/- double-knockout mice were generated, fed a normal chow diet, and housed for 53+/-4 weeks. Mortality and blood pressure were unaltered. However, serum cholesterol levels and subsequent atherosclerotic lesion formation (oil red O stain) were significantly increased in ApoE-/--IL-6-/- mice compared with ApoE-/-, wild-type (WT), and IL-6-/- mice. Plaques of ApoE-/--IL-6-/- mice showed significantly reduced transcript and protein levels of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, collagen I and V, and lysyl oxidase (by reverse transcriptase-polymerase chain reaction and immunohistochemistry). Recruitment of macrophages and leukocytes (Mac3- and CD45-positive staining) into the atherosclerotic lesion was significantly reduced in ApoE-/--IL-6-/- mice. The transcript and serum protein (ELISA) levels of IL-10 were significantly reduced.Thus, a lifetime IL-6 deficiency enhances atherosclerotic plaque formation in ApoEK-/--IL-6-/- mice and leads to maladaptive vascular developmental processes. These observations are consistent with the notion that baseline levels of IL-6 are required to modulate lipid homeostasis, vascular remodeling, and plaque inflammation in atherosclerosis.

To the Editor: Although progressive stenosis of the arterial lumen constitutes the basis for ischemic symptoms in atherosclerotic vascular disease, acute vascular events are for the most part assoc...

After radiotherapy treatment, there is an increased incidence of localized atherosclerosis in patients with Hodgkin's disease, breast cancer, and head and neck cancer. Here, we established a mouse model to study the development and progression of radiation-induced atherosclerosis and to compare the phenotype of these lesions with age-related atherosclerosis. Atherosclerosis-prone ApoE-/- mice fed a regular chow diet received single radiation doses of 14 Gy or sham treatments (0 Gy) to the neck, including both carotid arteries. At 22, 28, and 34 weeks after irradiation, blood samples were taken, and the arterial tree was removed for histological examination. Cholesterol levels in irradiated mice were not significantly different from age-matched controls, and markers of systemic inflammation (soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, and C-reactive protein) were not elevated. The lesions in irradiated arteries were macrophage rich, with a remarkable influx of inflammatory cells, predominantly granulocytes. Intraplaque hemorrhage and erythrocyte-containing macrophages were seen only in lesions of irradiated arteries. Based on these data, we propose that irradiation accelerates the development of macrophage-rich, inflammatory atherosclerotic lesions prone to intraplaque hemorrhage.

Atherosclerosis is an immunoinflammatory disease; however, the key factors responsible for the maintenance of immune regulation in a proinflammatory milieu are poorly understood.Here, we show that milk fat globule-EGF factor 8 (Mfge8, also known as lactadherin) is expressed in normal and atherosclerotic human arteries and is involved in phagocytic clearance of apoptotic cells by peritoneal macrophages. Disruption of bone marrow-derived Mfge8 in a murine model of atherosclerosis leads to substantial accumulation of apoptotic debris both systemically and within the developing lipid lesions. The accumulation of apoptotic material is associated with a reduction in interleukin-10 in the spleen but an increase in interferon-gamma production in both the spleen and the atherosclerotic arteries. In addition, we report a dendritic cell-dependent alteration of natural regulatory T-cell function in the absence of Mfge8. These events are associated with a marked acceleration of atherosclerosis.Lack of Mfge8 in bone marrow-derived cells enhances the accumulation of apoptotic cell corpses in atherosclerosis and alters the protective immune response, which leads to an acceleration of plaque development.

The CD40-CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways. Consequently, this dyad is involved in chronic inflammatory diseases, including atherosclerosis. Inhibition of CD40L in apolipoprotein E (Apoe)-deficient (Apoe(-/-)) mice not only reduced atherosclerosis but also conferred a clinically favorable plaque phenotype that was low in inflammation and high in fibrosis. Blockade of CD40L may not be therapeutically feasible, as long-term inhibition will compromise systemic immune responses. Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects. We report that deficiency in hematopoietic CD40 reduces atherosclerosis and induces features of plaque stability. To elucidate the role of CD40-tumor necrosis factor receptor-associated factor (TRAF) signaling in atherosclerosis, we examined disease progression in mice deficient in CD40 and its associated signaling intermediates. Absence of CD40-TRAF6 but not CD40-TRAF2/3/5 signaling abolishes atherosclerosis and confers plaque fibrosis in Apoe(-/-) mice. Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6C(high) monocytes, an impaired recruitment of Ly6C(+) monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature. These data unveil a role for CD40-TRAF6, but not CD40-TRAF2/3/5, interactions in atherosclerosis and establish that targeting specific components of the CD40-CD40L pathway harbors the potential to achieve therapeutic effects in atherosclerosis.

Cathepsin K (catK), a lysosomal cysteine protease, was identified in a gene-profiling experiment that compared human early plaques, advanced stable plaques, and advanced atherosclerotic plaques containing a thrombus, where it was highly upregulated in advanced stable plaques.To assess the function of catK in atherosclerosis, catK(-/-)/apolipoprotein (apo) E(-/-) mice were generated. At 26 weeks of age, plaque area in the catK(-/-)/apoE(-/-) mice was reduced (41.8%) owing to a decrease in the number of advanced lesions as well as a decrease in individual advanced plaque area. This suggests an important role for catK in atherosclerosis progression. Advanced plaques of catK(-/-)/apoE(-/-) mice showed an increase in collagen content. Medial elastin fibers were less prone to rupture than those of apoE(-/-) mice. Although the relative macrophage content did not differ, individual macrophage size increased. In vitro studies of bone marrow derived-macrophages confirmed this observation. Scavenger receptor-mediated uptake (particularly by CD36) of modified LDL increased in the absence of catK, resulting in an increased macrophage size because of increased cellular storage of cholesterol esters, thereby enlarging the lysosomes.A deficiency of catK reduces plaque progression and induces plaque fibrosis but aggravates macrophage foam cell formation in atherosclerosis.

Vascular remodeling is the result of a close interplay of changes in vascular tone and structure. In this review, the role of angiotension-converting enzyme (ACE) and the impact of ACE inhibition on vascular remodeling processes during vascular injury and restenosis, hypertension, atherosclerosis, and aneurysm formation are discussed. The role of ACE and angiotensin II (Ang II) in neointimal thickening has been firmly established by animal studies and is mediated by Ang II type 1 (AT(1)) receptor signaling events via monocyte chemoattractant protein-1 and NAD(P)H oxidase. ACE and Ang II are involved in the remodeling of large and resistance arteries during hypertension; here, cell proliferation and matrix remodeling are also regulated by signaling events downstream of the AT(1) receptor. In atherosclerosis, Ang II is involved in the inflammatory and tissue response, mediated by various signaling pathways downstream of the AT(1) receptor. Although ACE inhibition has been shown to inhibit atherosclerotic processes in experimental animal models, results of large clinical trials with ACE inhibitors were not conclusive. Remodeling of vessel dimensions and structure during aneurysm formation is counteracted by ACE inhibition. Here, a direct effect of ACE inhibitors on matrix metalloproteinase activity has to be considered as part of the working mechanism. The role of ACE2 in vascular remodeling has yet to be established; however, ACE2 has been shown to be associated with vascular changes in hypertension and atherosclerosis.

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

Background— Thrombin generation in vivo may be important in regulating atherosclerotic progression. In the present study, we examined for the first time the activity and presence of relevant coagulation proteins in relation to the progression of atherosclerosis. Methods and Results— Both early and stable advanced atherosclerotic lesions were collected pairwise from each individual (n=27) during autopsy. Tissue homogenates were prepared from both total plaques and isolated plaque layers, in which the activity of factors (F) II, X, and XII and tissue factor was determined. Microarray analysis was implemented to elucidate local messenger RNA synthesis of coagulation proteins. Part of each specimen was paraffin embedded, and histological sections were immunohistochemically stained for multiple coagulation markers with the use of commercial antibodies. Data are expressed as median (interquartile range [IQR]). Tissue factor, FII, FX, and FXII activities were significantly higher in early atherosclerotic lesions than in stable advanced atherosclerotic lesions. Endogenous thrombin potential and thrombin-antithrombin complex values consolidated a procoagulant profile of early atherosclerotic lesions (endogenous thrombin potential, 1240 nmol/L · min [IQR, 1173 to 1311]; thrombin-antithrombin complex, 1045 ng/mg [IQR, 842.6 to 1376]) versus stable advanced atherosclerotic lesions (endogenous thrombin potential, 782 nmol/L · min [IQR, 0 to 1151]; thrombin-antithrombin complex, 718.4 ng/mg [IQR, 508.6 to 1151]). Tissue factor, FVII, and FX colocalized with macrophages and smooth muscle cells. In addition, multiple procoagulant and anticoagulant proteases were immunohistochemically mapped to various locations throughout the atherosclerotic vessel wall in both early and advanced atherosclerotic stages. Conclusions— This study shows an enhanced procoagulant state of early-stage atherosclerotic plaques compared with advanced-stage plaques, which may provide novel insights into the role of coagulation during atherosclerotic plaque progression.

Cardiac hypertrophy can lead to heart failure (HF), but it is unpredictable which hypertrophied myocardium will progress to HF. We surmised that apart from hypertrophy-related genes, failure-related genes are expressed before the onset of failure, permitting molecular prediction of HF. Hearts from hypertensive homozygous renin-overexpressing (Ren-2) rats that had progressed to early HF were compared by microarray analysis to Ren-2 rats that had remained compensated. To identify which HF-related genes preceded failure, cardiac biopsy specimens were taken during compensated hypertrophy and we then monitored whether the rat progressed to HF or remained compensated. Among 48 genes overexpressed in failing hearts, we focused on thrombospondin-2 (TSP2). TSP2 was selectively overexpressed only in biopsy specimens from rats that later progressed to HF. Moreover, expression of TSP2 was increased in human hypertrophied hearts with decreased (0.19+/-0.01) versus normal ejection fraction (0.11+/-0.03 [arbitrary units]; P<0.05). Angiotensin II induced fatal cardiac rupture in 70% of TSP2 knockout mice, with cardiac failure in the surviving mice; this was not seen in wild-type mice. In TSP2 knockout mice, angiotensin II increased matrix metalloproteinase (MMP)-2 and MMP-9 activity by 120% and 390% compared with wild-type mice (P<0.05). In conclusion, we identify TSP2 as a crucial regulator of the integrity of the cardiac matrix that is necessary for the myocardium to cope with increased loading and that may function by its regulation of MMP activity. This suggests that expression of TSP2 marks an early-stage molecular program that is activated uniquely in hypertrophied hearts that are prone to fail.

The purpose of this study was to investigate whether the use of a semiautomated plaque quantification algorithm (reporting volumetric and geometric plaque properties) provides additional prognostic value for the development of acute coronary syndromes (ACS) as compared with conventional reading from cardiac computed tomography angiography (CCTA). CCTA enables the visualization of coronary plaque characteristics, of which some have been shown to predict ACS. A total of 1,650 patients underwent 64-slice CCTA and were followed up for ACS for a mean 26 ± 10 months. In 25 patients who had ACS and 101 random controls (selected from 993 patients with coronary artery disease but without coronary event), coronary artery disease was evaluated using conventional reading (calcium score, luminal stenosis, morphology), and then independently quantified using semiautomated software (plaque volume, burden area [plaque area divided by vessel area times 100%], noncalcified percentage, attenuation, remodeling). Clinical risk profile was calculated with Framingham risk score (FRS). There were no significant differences in conventional reading parameters between controls and patients who had ACS. Semiautomated plaque quantification showed that compared to controls, ACS patients had higher total plaque volume (median: 94 mm3 vs. 29 mm3) and total noncalcified volume (28 mm3 vs. 4 mm3, p ≤ 0.001 for both). In addition, per-plaque maximal volume (median: 56 mm3 vs. 24 mm3), noncalcified percentage (62% vs. 26%), and plaque burden (57% vs. 36%) in ACS patients were significantly higher (p &lt; 0.01 for all). A receiver-operating characteristic model predicting for ACS incorporating FRS and conventional CCTA reading had an area under the curve of 0.64; a second model also incorporating semiautomated plaque quantification had an area under the curve of 0.79 (p &lt; 0.05). The semiautomated plaque quantification algorithm identified several parameters predictive for ACS and provided incremental prognostic value over clinical risk profile and conventional CT reading. The application of this tool may improve risk stratification in patients undergoing CCTA.

Abstract The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.

AimsOur objective was to study the effect of the genetic background on the wound healing process after myocardial infarction (MI) in mice.

Abstract Absence of shear stress due to disturbed blood flow at arterial bifurcations and curvatures leads to endothelial dysfunction and proinflammatory gene expression, ultimately resulting in atherogenesis. KLF2 has recently been implicated as a transcription factor involved in mediating the anti-inflammatory effects of flow. We investigated the effect of shear on basal and TNF-α–induced genomewide expression profiles of human umbilical vein endothelial cells (HUVECs). Cluster analysis confirmed that shear stress induces expression of protective genes including KLF2, eNOS, and thrombomodulin, whereas basal expression of TNF-α–responsive genes was moderately decreased. Promoter analysis of these genes showed enrichment of binding sites for ATF transcription factors, whereas TNF-α–induced gene expression was mostly NF-κB dependent. Furthermore, human endothelial cells overlying atherosclerotic plaques had increased amounts of phosphorylated nuclear ATF2 compared with endothelium at unaffected sites. In HUVECs, a dramatic reduction of nuclear binding activity of ATF2 was observed under shear and appeared to be KLF2 dependent. Reduction of ATF2 with siRNA potently suppressed basal proinflammatory gene expression under no-flow conditions. In conclusion, we demonstrate that shear stress and KLF2 inhibit nuclear activity of ATF2, providing a potential mechanism by which endothelial cells exposed to laminar flow are protected from basal proinflammatory, atherogenic gene expression.

The purpose of this study was to evaluate interstitial alterations in myocardial remodeling using a radiolabeled Cy5.5-RGD imaging peptide (CRIP) that targets myofibroblasts.Collagen deposition and interstitial fibrosis contribute to cardiac remodeling and heart failure after myocardial infarction (MI). Evaluation of myofibroblastic proliferation should provide indirect evidence of the extent of fibrosis.Of 46 Swiss-Webster mice, MI was induced in 41 by coronary artery occlusion, and 5 were unmanipulated. Of the 41 mice, 6, 6, and 5 received intravenous technetium-99m labeled CRIP for micro-single-photon emission computed tomography imaging 2, 4, and 12 weeks after MI, respectively; 8 received captopril or captopril with losartan up to 4 weeks after MI. Scrambled CRIP was used 4 weeks after MI in 6 mice; the remaining 10 of 46 mice received unradiolabeled CRIP for histologic characterization.Maximum CRIP uptake was observed in the infarct area; quantitative uptake (percent injected dose/g) was highest at 2 weeks (2.75 +/- 0.46%), followed by 4 (2.26 +/- 0.09%) and 12 (1.74 +/- 0.24%) weeks compared with that in unmanipulated mice (0.59 +/- 0.19%). Uptake was higher at 12 weeks in the remote areas. CRIP uptake was histologically traced to myofibroblasts. Captopril alone (1.78 +/- 0.31%) and with losartan (1.13 +/- 0.28%) significantly reduced tracer uptake; scrambled CRIP uptake in infarct area (0.74 +/- 0.17%) was similar to CRIP uptake in normal myocardium.Radiolabeled CRIP allows for noninvasive visualization of interstitial alterations during cardiac remodeling, and is responsive to antiangiotensin treatment. If proven clinically feasible, such a strategy would help identify post-MI patients likely to develop heart failure.

Rupture-prone atherosclerotic plaques are characterized by inflammation and a large necrotic core. Inflammation is linked to high metabolic activity. Advanced glycation endproducts (AGEs) and their major precursor methylglyoxal are formed during high metabolic activity and can have detrimental effects on cellular function and may induce cell death. Therefore, we investigated whether plaque AGEs are increased in human carotid rupture-prone plaques and are associated with plaque inflammation and necrotic core formation. The protein-bound major methylglyoxal-derived AGE 5-hydro-5-methylimidazolone (MG-H1) and Nɛ-(carboxymethyl)lysine (CML) were measured in human carotid endarterectomy specimens (n = 75) with tandem mass spectrometry. MG-H1 and CML levels were associated with rupture-prone plaques, increased protein levels of the inflammatory mediators IL-8 and MCP-1 and with higher MMP-9 activity. Immunohistochemistry showed that AGEs accumulated predominantly in macrophages surrounding the necrotic core and co-localized with cleaved caspase-3. Intra-plaque comparison revealed that glyoxalase-1 (GLO-1), the major methylglyoxal-detoxifying enzyme, mRNA was decreased (−13%, P < 0.05) in ruptured compared with stable plaque segments. In line, in U937 monoctyes, we found reduced (GLO-1) activity (−38%, P < 0.05) and increased MGO (346%, P < 0.05) production after stimulation with the inflammatory mediator TNF. Direct incubation with methylglyoxal increased apoptosis up to two-fold. This is the first study showing that AGEs are associated with human rupture-prone plaques. Furthermore, this study suggests a cascade linking inflammation, reduced GLO-1, methylglyoxal- and AGE-accumulation, and subsequent apoptosis. Thereby, AGEs may act as mediators of the progression of stable to rupture-prone plaques, opening a window towards novel treatments and biomarkers to treat cardiovascular diseases.

Macrophages are key immune cells found in atherosclerotic plaques and critically shape atherosclerotic disease development. Targeting the functional repertoire of macrophages may hold novel approaches for future atherosclerosis management. Here, we describe a previously unrecognized role of the epigenomic enzyme Histone deacetylase 3 (Hdac3) in regulating the atherosclerotic phenotype of macrophages. Using conditional knockout mice, we found that myeloid Hdac3 deficiency promotes collagen deposition in atherosclerotic lesions and thus induces a stable plaque phenotype. Also, macrophages presented a switch to anti-inflammatory wound healing characteristics and showed improved lipid handling. The pro-fibrotic phenotype was directly linked to epigenetic regulation of the Tgfb1 locus upon Hdac3 deletion, driving smooth muscle cells to increased collagen production. Moreover, in humans, HDAC3 was the sole Hdac upregulated in ruptured atherosclerotic lesions, Hdac3 associated with inflammatory macrophages, and HDAC3 expression inversely correlated with pro-fibrotic TGFB1 expression. Collectively, we show that targeting the macrophage epigenome can improve atherosclerosis outcome and we identify Hdac3 as a potential novel therapeutic target in cardiovascular disease.

Purpose Increased risk of atherosclerosis and stroke has been demonstrated in patients receiving radiotherapy for Hodgkin's lymphoma and head-and-neck cancer. We previously showed that 14 Gy to the carotid arteries of hypercholesterolemic ApoE−/− mice resulted in accelerated development of macrophage-rich, inflammatory atherosclerotic lesions. Here we investigate whether clinically relevant fractionated irradiation schedules and lower single doses also predispose to an inflammatory plaque phenotype. Methods and Materials ApoE−/− mice were given 8 or 14 Gy, or 20 × 2.0 Gy in 4 weeks to the neck, and the carotid arteries were subsequently examinated for presence of atherosclerotic lesions, plaque size, and phenotype. Results At 4 weeks, early atherosclerotic lesions were found in 44% of the mice after single doses of 14 Gy but not in age-matched controls. At 22 to 30 weeks after irradiation there was a twofold increase in the mean number of carotid lesions (8–14 Gy and 20 × 2.0 Gy) and total plaque burden (single doses only), compared with age-matched controls. The majority of lesions seen at 30 to 34 weeks after fractionated irradiation or 14-Gy single doses were granulocyte rich (100% and 63%, respectively), with thrombotic features (90% and 88%), whereas these phenotypes were much less common in age-matched controls or after a single dose of 8 Gy. Conclusions We showed that fractionated irradiation accelerated the development of atherosclerosis in ApoE−/− mice and predisposed to the formation of an inflammatory, thrombotic plaque phenotype. Increased risk of atherosclerosis and stroke has been demonstrated in patients receiving radiotherapy for Hodgkin's lymphoma and head-and-neck cancer. We previously showed that 14 Gy to the carotid arteries of hypercholesterolemic ApoE−/− mice resulted in accelerated development of macrophage-rich, inflammatory atherosclerotic lesions. Here we investigate whether clinically relevant fractionated irradiation schedules and lower single doses also predispose to an inflammatory plaque phenotype. ApoE−/− mice were given 8 or 14 Gy, or 20 × 2.0 Gy in 4 weeks to the neck, and the carotid arteries were subsequently examinated for presence of atherosclerotic lesions, plaque size, and phenotype. At 4 weeks, early atherosclerotic lesions were found in 44% of the mice after single doses of 14 Gy but not in age-matched controls. At 22 to 30 weeks after irradiation there was a twofold increase in the mean number of carotid lesions (8–14 Gy and 20 × 2.0 Gy) and total plaque burden (single doses only), compared with age-matched controls. The majority of lesions seen at 30 to 34 weeks after fractionated irradiation or 14-Gy single doses were granulocyte rich (100% and 63%, respectively), with thrombotic features (90% and 88%), whereas these phenotypes were much less common in age-matched controls or after a single dose of 8 Gy. We showed that fractionated irradiation accelerated the development of atherosclerosis in ApoE−/− mice and predisposed to the formation of an inflammatory, thrombotic plaque phenotype.

Plaque rupture and subsequent thrombotic occlusion of the coronary artery account for as many as three quarters of myocardial infarctions. The concept of plaque stabilisation emerged about 20 years ago to explain the discrepancy between the reduction of cardiovascular events in patients receiving lipid lowering therapy and the small decrease seen in angiographic evaluation of atherosclerosis. Since then, the concept of a vulnerable plaque has received a lot of attention in basic and clinical research leading to a better understanding of the pathophysiology of the vulnerable plaque and acute coronary syndromes. From pathological and clinical observations, plaques that have recently ruptured have thin fibrous caps, large lipid cores, exhibit outward remodelling and invasion by vasa vasorum. Ruptured plaques are also focally inflamed and this may be a common denominator of the other pathological features. Plaques with similar characteristics, but which have not yet ruptured, are believed to be vulnerable to rupture. Experimental studies strongly support the validity of anti-inflammatory approaches to promote plaque stability. Unfortunately, reliable non-invasive methods for imaging and detection of such plaques are not yet readily available. There is a strong biological basis and supportive clinical evidence that low-density lipoprotein lowering with statins is useful for the stabilisation of vulnerable plaques. There is also some clinical evidence for the usefulness of antiplatelet agents, beta blockers and renin-angiotensin-aldosterone system inhibitors for plaque stabilisation. Determining the causes of plaque rupture and designing diagnostics and interventions to prevent them are urgent priorities for current basic and clinical research in cardiovascular area.

Radiotherapy of thoracic and chest wall tumors increases the long-term risk of cardiotoxicity, but the underlying mechanisms are unclear.Single doses of 2, 8, or 16 Gy were delivered to the hearts of mice and damage was evaluated at 20, 40, and 60 weeks, relative to age matched controls. Single photon emission computed tomography (SPECT/CT) and ultrasound were used to measure cardiac geometry and function, which was related to histo-morphology and microvascular damage.Gated SPECT/CT and ultrasound demonstrated decreases in end diastolic and systolic volumes, while the ejection fraction was increased at 20 and 40 weeks after 2, 8, and 16 Gy. Cardiac blood volume was decreased at 20 and 60 weeks after irradiation. Histological examination revealed inflammatory changes at 20 and 40 weeks after 8 and 16 Gy. Microvascular density in the left ventricle was decreased at 40 and 60 weeks after 8 and 16 Gy, with functional damage to remaining microvasculature manifest as decreased alkaline phosphatase (2, 8, and 16 Gy), increased von Willebrand Factor and albumin leakage from vessels (8 and 16 Gy), and amyloidosis (16 Gy). 16 Gy lead to sudden death between 30 and 40 weeks in 38% of mice.Irradiation with 2 and 8 Gy induced modest changes in murine cardiac function within 20 weeks but this did not deteriorate further, despite progressive structural and microvascular damage. This indicates that heart function can compensate for significant structural damage, although higher doses, eventually lead to sudden death.

Unlike conventional dendritic cells, plasmacytoid DCs (PDC) are poor in antigen presentation and critical for type I interferon response. Though proposed to be present in human atherosclerotic lesions, their role in atherosclerosis remains elusive.To investigate the role of PDC in atherosclerosis.We show that PDC are scarcely present in human atherosclerotic lesions and almost absent in mouse plaques. Surprisingly, PDC depletion by 120G8 mAb administration was seen to promote plaque T-cell accumulation and exacerbate lesion development and progression in LDLr⁻/⁻ mice. PDC depletion was accompanied by increased CD4⁺ T-cell proliferation, interferon-γ expression by splenic T cells, and plasma interferon-γ levels. Lymphoid tissue PDC from atherosclerotic mice showed increased indoleamine 2,3-dioxygenase (IDO) expression and IDO blockage abrogated the PDC suppressive effect on T-cell proliferation.Our data reveal a protective role for PDC in atherosclerosis, possibly by dampening T-cell proliferation and activity in peripheral lymphoid tissue, rendering PDC an interesting target for future therapeutic interventions.

Purpose of review Hypoxia triggers various cellular processes, both in physiological and pathological conditions, and has recently also been implicated in atherosclerosis. This review summarizes the recent evidence for the presence and the role of hypoxia in atherosclerosis. Additionally, it will elucidate on hypoxic signaling, which is interlinked with inflammatory signaling, and discuss recent advances in imaging of hypoxia in atherosclerosis. Recent findings Hypoxia is present in atherosclerotic plaques in humans and animal models, and systemic hypoxia promotes atherosclerosis. Hypoxia stimulates proatherosclerotic processes, like deficient lipid efflux, inflammation, interference with macrophage polarization and glucose metabolism. However, the molecular mechanism of hypoxia-mediated atherogenesis remains unclear. Noninvasive imaging directly targeting plaque hypoxia has been applied in animal models of atherosclerosis, but remains to be validated in humans. Meanwhile, the metabolic marker 18F-fluorodeoxyglucose, used to detect human atherosclerosis in vivo, may serve as an indirect marker of plaque hypoxia due to enhanced glucose uptake in anaerobic metabolism. Summary Recent studies underscore the proatherogenic role of hypoxia in macrophage lipid and glucose metabolism, inflammation and polarization. These studies provide new insights into the pathogenesis of atherosclerosis and unravel novel therapeutic targets and new options for noninvasive imaging of human atherosclerotic plaques.

Variations in the blood coagulation activity, determined genetically or by medication, may alter atherosclerotic plaque progression, by influencing pleiotropic effects of coagulation proteases. Published experimental studies have yielded contradictory findings on the role of hypercoagulability in atherogenesis. We therefore sought to address this matter by extensively investigating the in vivo significance of genetic alterations and pharmacologic inhibition of thrombin formation for the onset and progression of atherosclerosis, and plaque phenotype determination.We generated transgenic atherosclerosis-prone mice with diminished coagulant or hypercoagulable phenotype and employed two distinct models of atherosclerosis. Gene-targeted 50% reduction in prothrombin (FII(-/WT):ApoE(-/-)) was remarkably effective in limiting disease compared to control ApoE(-/-) mice, associated with significant qualitative benefits, including diminished leukocyte infiltration, altered collagen and vascular smooth muscle cell content. Genetically-imposed hypercoagulability in TM(Pro/Pro):ApoE(-/-) mice resulted in severe atherosclerosis, plaque vulnerability and spontaneous atherothrombosis. Hypercoagulability was associated with a pronounced neutrophilia, neutrophil hyper-reactivity, markedly increased oxidative stress, neutrophil intraplaque infiltration and apoptosis. Administration of either the synthetic specific thrombin inhibitor Dabigatran etexilate, or recombinant activated protein C (APC), counteracted the pro-inflammatory and pro-atherogenic phenotype of pro-thrombotic TM(Pro/Pro):ApoE(-/-) mice.We provide new evidence highlighting the importance of neutrophils in the coagulation-inflammation interplay during atherogenesis. Our findings reveal that thrombin-mediated proteolysis is an unexpectedly powerful determinant of atherosclerosis in multiple distinct settings. These studies suggest that selective anticoagulants employed to prevent thrombotic events may also be remarkably effective in clinically impeding the onset and progression of cardiovascular disease.

The majority of coronary thrombi (∼75%) is caused by plaque rupture .1,2 Prototype of the rupture-prone plaque contains a large, soft, lipid-rich necrotic core with a thin and inflamed fibrous cap, so-called thin-cap fibroatheroma (TCFA) ( Figure 1 ).3,4 Other common features include expansive remodelling, large plaque size, plaque haemorrhage, neovascularization, adventitial inflammation, and ‘spotty’ calcifications.4 Thin-cap fibroatheroma caps are usually <65 µm thick.4 Figure 2 summarizes factors contributing to the formation of vulnerable plaques. No distinct morphological features have been identified for the erosion-prone plaques, but they are usually rarely associated with expansive remodelling, scarcely calcified, and contain only limited inflammation.2,5 Figure 1 Cross-sectioned coronary artery showing a ruptured thin-cap fibroatheroma. The fibrous cap is very thin near the rupture site (between arrows) and a non-obstructive mural thrombus (asterisks) is bordering the gap in the disrupted cap. A haemorrhage has penetrated from the lumen through the gap into the lipid-rich necrotic core in which the characteristic cholesterol clefts are clearly seen. The lumen contains contrast medium injected postmortem. Overview in inset. Trichrome, staining collagen blue, and thrombus and haemorrhage red. Figure 2 Factors contributing to the formation of vulnerable plaques. MCP-1, monocyte chemotactic protein-1; MIF, migration inhibitory factor; TNFα, tumour necrosis factor-α; ILs, interleukins; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitors of metalloproteinases; PDGFs, platelet-derived growth factors; VEGFs, vascular endothelial growth factors; FGFs, fibroblast growth factors; Mφ, macrophages. ### Inflammatory cells, cytokines, chemokines, and growth factors Vulnerable plaques contain monocytes, macrophages, and T-cells. Of the T-cells, CD4+ T-helper (Th) cells are the most prominent.6 T-cells can differentiate into a Th1 phenotype, which secretes and responds to IFN-γ or a Th2 phenotype, which secretes and responds to IL-4, IL-10, and IL-13 ( Figure 3 ). T-cells promote the vulnerability of plaques through their effects on macrophages. Similarly, there are two main plaque macrophage phenotypes: …

Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte β2-integrins bind to endothelial ICAM-1 (InterCellular Adhesion Molecule-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular endothelium for permissive sites to transmigrate suggestive for apical membrane heterogeneity within the endothelium. However, the molecular basis for this heterogeneity is unknown. Leukocyte adhesion induces ICAM-1 clustering which promotes its association to the actin-binding proteins FilaminB, α-Actinin-4 and Cortactin. We show that these endothelial proteins differentially control adhesion, spreading and transmigration of neutrophils. Loss of FilaminB, α-Actinin-4 and Cortactin revealed adapter-specific effects on a nuclear-to-peripheral gradient of endothelial cell stiffness. Conversely, increasing endothelial cell stiffness stimulates ICAM-1 function. We identify endothelial α-Actinin-4 as a key regulator of endothelial cell stiffness and of ICAM-1-mediated neutrophil transmigration. Finally, we found that the endothelial lining of human and murine atherosclerotic plaques shows elevated levels of α-Actinin-4. These results identify endothelial cell stiffness as an important regulator of endothelial surface heterogeneity and of ICAM-1 function which in turn controls adhesion and transmigration of neutrophils.

Haemodynamic factors influence all forms of vascular growth (vasculogenesis, angiogenesis, arteriogenesis). Because of its prominent role in atherosclerosis, shear stress has gained particular attention, but other factors such as circumferential stretch are equally important to maintain the integrity and to (re)model the vascular network. While these haemodynamic forces are crucial determinants of the appearance and the structure of the vasculature, they are in turn subjected to structural changes in the blood vessels, such as an increased arterial stiffness in chronic arterial hypertension and ageing. This results in an interplay between the various forces (biomechanical forces) and the involved vascular elements. Although many molecular mediators of biomechanical forces still need to be identified, there is plenty of evidence for the causal role of these forces in vascular growth processes, which will be summarized in this review. In addition, we will discuss the effects of concomitant diseases and disorders on these processes by altering either the biomechanics or their transduction into biological signals. Particularly endothelial dysfunction, diabetes, hypercholesterolaemia, and age affect mechanosensing and -transduction of flow signals, thereby underpinning their influence on cardiovascular health. Finally, current approaches to modify biomechanical forces to therapeutically modulate vascular growth in humans will be described.

Transcranial Doppler (TCD) sonography is a frequently employed technique for quantifying cerebral blood flow by assuming a constant arterial diameter. Given that exercise increases arterial pressure by sympathetic activation, we hypothesized that exercise might induce a change in the diameter of large cerebral arteries. Middle cerebral artery (MCA) cross-sectional area was assessed in response to handgrip exercise by direct magnetic resonance imaging (MRI) observations. Twenty healthy subjects (11 female) performed three 5 min bouts of rhythmic handgrip exercise at 60% maximum voluntary contraction, alternated with 5 min of rest. High-resolution 7 T MRI scans were acquired perpendicular to the MCA. Two blinded observers manually determined the MCA cross-sectional area. Sufficient image quality was obtained in 101 MCA-scans of 19 subjects (age-range 20-59 years). Mixed effects modelling showed that the MCA cross-sectional area decreased by 2.1 ± 0.8% (p = 0.01) during handgrip, while the heart rate increased by 11 ± 2% (p < 0.001) at constant end-tidal CO2 (p = 0.10). In conclusion, the present study showed a 2% decrease in MCA cross-sectional area during rhythmic handgrip exercise. This further strengthens the current concept of sympathetic control of large cerebral arteries, showing in vivo vasoconstriction during exercise-induced sympathetic activation. Moreover, care must be taken when interpreting TCD exercise studies as diameter constancy cannot be assumed.

Dysregulated lipid metabolism induces an inflammatory and immune response leading to atherosclerosis. Conversely, inflammation may alter lipid metabolism. Recent treatment strategies in secondary prevention of atherosclerosis support beneficial effects of both anti-inflammatory and lipid-lowering therapies beyond current targets. There is a controversy about the possibility that anti-inflammatory effects of lipid-lowering therapy may be either independent or not of a decrease in low-density lipoprotein cholesterol. In this Position Paper, we critically interpret and integrate the results obtained in both experimental and clinical studies on anti-inflammatory actions of lipid-lowering therapy and the mechanisms involved. We highlight that: (i) besides decreasing cholesterol through different mechanisms, most lipid-lowering therapies share anti-inflammatory and immunomodulatory properties, and the anti-inflammatory response to lipid-lowering may be relevant to predict the effect of treatment, (ii) using surrogates for both lipid metabolism and inflammation as biomarkers or vascular inflammation imaging in future studies may contribute to a better understanding of the relative importance of different mechanisms of action, and (iii) comparative studies of further lipid lowering, anti-inflammation and a combination of both are crucial to identify effects that are specific or shared for each treatment strategy.

Alzheimer disease (AD) is marked by profound neurodegeneration, neuroinflammation, and cognitive decline. Pathologically, AD is characterized by the accumulation of extracellular amyloid and intraneuronal tangles, consisting of hyperphosphorylated tau. To date, factors leading to disease onset and progression are still an important topic of investigation. Various epidemiological studies revealed cardiovascular disease as an important contributor to the development and progression of AD, leading to the so-called vascular hypothesis. Vascular risk factors, such as hypertension, diabetes, and hyperhomocysteinemia, are associated with a significantly increased chance of developing AD, suggesting an additive or even synergistic effect. These vascular risk factors are often linked to a reduction in cerebral blood flow and the resulting chronic cerebral hypoperfusion is suggested to play a key role in the onset of AD. However, the causal effects of such vascular risk factors for AD onset remain largely unknown. Evidence from animal studies support that chronic cerebral hypoperfusion induction causes a strong aggravation of AD-related pathology, but a comprehensive overview of how the various cardiovascular disease risk factors contribute to disease is lacking. Therefore, we here critically review current literature, to unravel the existing evidence derived from in vivo mouse studies and define the role of cardiovascular disease and chronic cerebral hypoperfusion in AD development. We conclude that, although many aspects of the vascular hypothesis are well supported by observational studies, in-depth mechanistic studies and well-designed randomized controlled trials are highly needed to establish temporal and causal relationships. Described new insights can have major prospective potential for therapeutic interventions.

To prospectively determine, by using a stepwise logistic regression model, the optimal magnetic resonance (MR) weighting (ie, pulse sequence) combinations for plaque assessment and corresponding cutoff values of relative signal intensities (rSIs).Institutional review board approval and patient consent were obtained. Eleven patients (seven men, four women; mean age +/- standard deviation, 68 years +/- 4) with symptomatic carotid disease and stenosis of more than 70% were investigated at MR imaging before carotid endarterectomy. The MR images were matched with histologic features of the endarterectomy specimens (reference standard). The rSIs (compared with that of muscle tissue) from regions of interest were assessed qualitatively and semiquantitatively. For all major components (calcification, lipid core, intraplaque hemorrhage, and fibrous tissue), optimal cutoff points for the rSIs were determined for five MR weightings by means of receiver operating characteristic curves. The best predicting combinations of these five dichotomized MR weightings were selected by means of stepwise logistic regression analysis. The potential sensitivity and specificity of MR imaging for vulnerable plaque with hemorrhage and/or lipid core were determined.The same optimal MR weighting combinations for identifying the four plaque components were found with qualitative and semiquantitative analysis. Sensitivity and specificity for vulnerable plaque were 93% (95% confidence interval: 77%, 99%) and 96% (95% confidence interval: 86%, 100%), respectively, for the qualitative analysis and 76% (95% confidence interval: 56%, 90%) and 100% (95% confidence interval: 93%, 100%) for the semiquantitative analysis.This study demonstrates the potential of a systematic approach of atherosclerotic plaque assessment with multisequence MR imaging by using the information provided from five different MR weightings in a stepwise logistic regression model.

It is generally established that the unstable plaque is the major cause of acute clinical sequelae of atherosclerosis. Unfortunately, terms indicating lesions prone to plaque instability, such as "vulnerable plaque," and the different phenotypes of unstable plaques, such as plaque rupture, plaque fissuring, intraplaque hemorrhage, and erosion, are often used interchangeably. Moreover, the different phenotypes of the unstable plaque are mostly referred to as plaque rupture. In the first part of this review, we will focus on the definition of true plaque rupture and the definitions of other phenotypes of plaque instability, especially on intraplaque hemorrhage, and discuss the phenotypes of available animal models of plaque instability. The second part of this review will address the pathogenesis of plaque rupture from a local and a systemic perspective. Plaque rupture is thought to occur because of changes in the plaque itself or systemic changes in the patient. Interestingly, contributing factors seem to overlap to a great extent and might even be interrelated. Finally, we will propose an integrative view on the pathogenesis of plaque rupture.

The extracellular matrix is no longer seen as the static embedding in which cells reside; it has been shown to be involved in cell proliferation, migration and cell-cell interactions. Turnover of the different extracellular matrix components is an active process with multiple levels of regulation. Collagen, a major extracellular matrix constituent of the myocardium and the arterial vascular wall, is synthesized by (myo)fibroblasts in the myocardium and smooth muscle cells in the medial arterial vascular wall. Its degradation is controlled by proteinases, which include matrix metalloproteinases. This review will focus on the impact of fibrosis and especially collagen turnover on the progression of heart failure and atherosclerosis, two of the main cardiovascular pathologies. We will discuss data from human studies and animal models, with an emphasis on the effects of interventions on collagen synthesis and degradation. We conclude that there is a dynamic (dis)balance in the rate of collagen synthesis and degradation during heart failure and atherosclerosis, which makes the outcome of interventions not always predictable. Alternative approaches for intervening in collagen metabolism will be discussed as possible therapeutic intervention strategies.

CD40-CD40 ligand (CD40L) interactions play a central role in the development and progression of atherosclerosis. In the late 1990s, we and others have shown that complete inhibition of the CD40L signaling pathway resulted in a decrease in atherosclerosis and in the induction of a stable atherosclerotic plaque phenotype. These stable plaques contained high amounts of collagen and vascular smooth muscle cells, whereas the amount of macrophages and T lymphocytes was low. Because clinical complications of atherosclerosis are mostly the result of plaque rupture, induction of plaque stability would significantly reduce the morbidity and mortality of atherosclerosis and thus validates inhibition of the CD40L system as a therapeutic target for atherosclerosis. However, long-term inhibition of this system probably compromises the immune system of the patient. Therefore, it is desirable to target either the downstream signaling modulators of the CD40-CD40L system that are associated with atherosclerosis, or target the CD40-CD40L system in a local, cell type-specific way. This is likely to induce plaque stabilization with limited systemic side effects, and a significant reduction of cardiovascular disease.

Disrupting the CD40-CD40L co-stimulatory pathway reduces atherosclerosis and induces a stable atherosclerotic plaque phenotype that is low in inflammation and high in fibrosis. Therefore, inhibition of the CD40-CD40L pathway is an attractive therapeutic target to reduce clinical complications of atherosclerosis. The CD40-CD40L dyad is known to interact with other co-stimulatory molecules, to activate antigen-presenting cells (APC) and to contribute to T-cell priming and B-cell isotype switching. Besides their presence on T-cells and APCs, CD40 and CD40L are also present on macrophages, endothelial cells and vascular smooth muscle cells in the plaque, where they can exert pro-atherogenic functions. Moreover, recent progress indicates the involvement of neutrophil CD40, platelet CD40L and dendritic cell CD40 in atherogenesis. Since systemic CD40-CD40L modulation compromises host defense, more targeted interventions are needed to develop superior treatment strategies for atherosclerosis. We believe that by unravelling the cell-cell CD40-CD40L interactions, inhibition of cell-type specific (signalling components of) CD40(L) that do not compromise the patient's immune system, will become possible. In this review, we highlight the cell-type specific multi-functionality of CD40-CD40L signalling in atherosclerosis.

Understanding atherogenesis will benefit significantly from simultaneous imaging, both ex vivo and in vivo, of structural and functional information at the (sub)cellular level within intact arteries. Due to limited penetration depth and loss of resolution with depth, intravital and confocal fluorescence microscopy are not suitable to study (sub)cellular details in arteries with wall thicknesses above 50 microm.Using two-photon laser scanning microscopy (TPLSM), which combines 3D resolution and large penetration depth, we imaged mouse carotid arteries.In thin slices, (sub)cellular structures identified using histochemical techniques could also be identified using TPLSM. Ex vivo, structural experiments on intact atherosclerotic arteries of Apo-E(-/-) mice demonstrated that in contrast to confocal or wide-field microscopy, TPLSM can be used to visualize (sub) cellular structural details of atherosclerotic plaques. In vivo, pilot experiments were carried out on healthy arteries of wild-type C57BL6 and atherosclerotic arteries of Apo-E(-/-) mice. As an example of functional measurements, we visualized fluorescently labeled leukocytes in vivo in the lumen. Additionally, detailed morphological information of vessel wall and atherosclerotic plaque was obtained after topical staining.Thus, TPLSM potentially allows combined functional and structural studies and can therefore be eminently suitable for investigating structure-function relationships at the cellular level in atherogenesis in the mouse.

Abstract Purpose To investigate the performance of high‐resolution T1‐weighted (T1w) turbo field echo (TFE) magnetic resonance imaging (MRI) for the identification of the high‐risk component intraplaque hemorrhage, which is described in the literature as a troublesome component to detect. Materials and Methods An MRI scan was performed preoperatively on 11 patients who underwent carotid endarterectomy because of symptomatic carotid disease with a stenosis larger than 70%. A commonly used double inversion recovery (DIR) T1w turbo spin echo (TSE) served as the T1w control for the T1w TFE pulse sequence. The MR images were matched slice by slice with histology, and the signal‐to‐noise ratio (SNR) and contrast‐to‐noise ratio (CNR) of the MR images were calculated. Additionally, two readers, who were blinded for the histological results, independently assessed the MR slices concerning the presence of intraplaque hemorrhage. Results More than 80% of the histological proven intraplaque hemorrhage could be detected using the TFE sequence with a high interobserver agreement (Kappa = 0.73). The TFE sequence proved to be superior to the TSE sequence concerning SNR and CNR, but also in the qualitative detection of intraplaque hemorrhage. The false positive TFE results contained fibrous tissue and were all located outside the main plaque area. Conclusion The present study shows that in vivo high‐resolution T1w TFE MRI can identify the high‐risk component intraplaque hemorrhage with a high detection rate in patients with symptomatic carotid disease. Larger clinical trials are warranted to investigate whether this technique can identify patients at risk for an ischemic attack. J. Magn. Reson. Imaging 2004;20:105–110. © 2004 Wiley‐Liss, Inc.

Angiotensin-converting enzyme (ACE)2 is a recently identified homologue of ACE. As ACE2 inactivates the pro-atherogenic angiotensin II, we hypothesize that ACE2 may play a protective role in atherogenesis. The spatiotemporal localization of ACE2 mRNA and protein in human vasculature and a possible association with atherogenesis were investigated using molecular histology (in situ hybridization, immunohistochemistry). Also, the ACE : ACE2 balance was investigated using enzymatic assays. ACE2 mRNA was expressed in early and advanced human carotid atherosclerotic lesions. In addition, ACE2 protein was present in human veins, non-diseased mammary arteries and atherosclerotic carotid arteries and expressed in endothelial cells, smooth muscle cells and macrophages. Quantitative analysis of immunoreactivity showed that total vessel wall expression of ACE and ACE2 was similar during all stages of atherosclerosis. The observed ACE2 protein was enzymatically active and activity was lower in the stable advanced atherosclerotic lesions, compared to early and ruptured atherosclerotic lesions. These results suggest a differential regulation of ACE2 activity during the progression of atherosclerosis and suggest that this novel molecule of the renin-angiotensin system may play a role in the pathogenesis of atherosclerosis.

Atherosclerotic cardiovascular disease is a major burden to health care. Because atherosclerosis is considered a systemic disease, we hypothesized that one single atherosclerotic plaque contains ample molecular information that predicts future cardiovascular events in all vascular territories.AtheroExpress is a biobank collecting atherosclerotic lesions during surgery, with a 3-year follow-up. The composite primary outcome encompasses all cardiovascular events and interventions, eg, cardiovascular death, myocardial infarction, stroke, and endovascular interventions. A proteomics search identified osteopontin as a potential plaque biomarker. Patients undergoing carotid surgery (n=574) served as the cohort in which plaque osteopontin levels were examined in relation to their outcome during follow-up and was validated in a cohort of patients undergoing femoral endarterectomy (n=151). Comparing the highest quartile of carotid plaque osteopontin levels with quartile 1 showed a hazard ratio for the primary outcome of 3.8 (95% confidence interval, 2.6-5.9). The outcome did not change after adjustment for plaque characteristics and traditional risk factors (hazard ratio, 3.5; 95% confidence interval, 2.0-5.9). The femoral validation cohort showed a hazard ratio of 3.8 (95% confidence interval 2.0 to 7.4) comparing osteopontin levels in quartile 4 with quartile 1.Plaque osteopontin levels in single lesions are predictive for cardiovascular events in other vascular territories. Local atherosclerotic plaques are a source of prognostic biomarkers with a high predictive value for secondary manifestations of atherosclerotic disease.

Background— Pathological aspects of atherosclerosis are well described, but gene profiles during atherosclerotic plaque progression are largely unidentified. Methods and Results— Microarray analysis was performed on mRNA of aortic arches of ApoE −/− mice fed normal chow (NC group) or Western-type diet (WD group) for 3, 4.5, and 6 months. Of 10 176 reporters, 387 were differentially (&gt;2×) expressed in at least 1 group compared with a common reference (ApoE −/− , 3- month NC group). The number of differentially expressed genes increased during plaque progression. Time-related expression clustering and functional grouping of differentially expressed genes suggested important functions for genes involved in inflammation (especially the small inducible cytokines monocyte chemoattractant protein [MCP]-1, MCP-5, macrophage inflammatory protein [MIP]-1α, MIP-1β, MIP-2, and fractalkine) and matrix degradation (cathepsin-S, matrix metalloproteinase-2/12). Validation experiments focused on the gene cluster of small inducible cytokines. Real-time polymerase chain reaction revealed a plaque progression–dependent increase in mRNA levels of MCP-1, MCP-5, MIP-1α, and MIP-1β. ELISA for MCP-1 and MCP-5 showed similar results. Immunohistochemistry for MCP-1, MCP-5, and MIP-1α located their expression to plaque macrophages. An inhibiting antibody for MCP-1 and MCP-5 (11K2) was designed and administered to ApoE −/− mice for 12 weeks starting at the age of 5 or 17 weeks. 11K2 treatment reduced plaque area and macrophage and CD45 + cell content and increased collagen content, thereby inducing a stable plaque phenotype. Conclusions— Gene profiling of atherosclerotic plaque progression in ApoE −/− mice revealed upregulation of the gene cluster of small inducible cytokines. Further expression and in vivo validation studies showed that this gene cluster mediates plaque progression and stability.

Vascular disease is increased after radiotherapy and is an important determinant of late treatment-induced morbidity and excess mortality. This study evaluates the nature of underlying pathologic changes occurring in medium-sized muscular arteries following irradiation.Biopsies of irradiated medium-sized arteries and unirradiated control arteries were taken from 147 patients undergoing reconstructive surgery with a vascularised free flap following treatment for head and neck (H&N) or breast cancer (BC). Relative intimal thickening was derived from the ratio of the thickness of the intima to the thickness of the media (IMR) on histological sections. Proteoglycan, collagen and inflammatory cell content were also scored.Intimal thickness was significantly increased in irradiated vessels: in the H&N group the IMR was 1.5-fold greater without correction for the control artery (p=0.018); in the BC group the IMR increased 1.4-fold after correction for the control artery (p=0.056) at a mean of 4 years following irradiation. There was an increase in the proteoglycan content of the intima of the irradiated IMA vessels, from 65% to 73% (p=0.024). Inflammatory cell content was increased in the intima of the irradiated H&N vessels (p=0.014).Radiation-induced vascular pathology differs quantitatively and qualitatively from age-related atherosclerosis.

Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molecular mechanism has been described in humans. Experimental evidence in mouse models suggests that the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays an important role in infarct rupture. The present study was designed to study the role of MMP-2, MMP-8, and MMP-9 in human infarct rupture.Heart samples were obtained from patients who died from infarct rupture and control MI patients. The MMP activity was determined by zymography and quantitative immunocapture activity assay. TIMP-1 levels were measured and immunohistochemistry for MMP-2 and MMP-9 was performed.The amounts of both total and active MMP-8 and MMP-9 were significantly higher in ruptured infarct tissue than in control MI tissue, but no differences in MMP-2 activity were observed. Furthermore, the number of inflammatory cells was significantly higher in the ruptured infarcts than in control infarcts.These data suggest that increased MMP-8 and MMP-9 activity in the infarct area, caused by a more prominent infiltration of inflammatory cells, contribute to infarct rupture in humans.

Recent clinical data indicates that hemodynamic changes caused by cardiovascular diseases such as atherosclerosis, heart failure, and hypertension affect cognition. Yet, the underlying mechanisms of the resulting vascular cognitive impairment (VCI) are poorly understood. One reason for the lack of mechanistic insights in VCI is that research in dementia primarily focused on Alzheimer's disease models. To fill in this gap, we critically reviewed the published data and various models of VCI. Typical findings in VCI include reduced cerebral perfusion, blood-brain barrier alterations, white matter lesions, and cognitive deficits, which have also been reported in different cardiovascular mouse models. However, the tests performed are incomplete and differ between models, hampering a direct comparison between models and studies. Nevertheless, from the currently available data we conclude that a few existing surgical animal models show the key features of vascular cognitive decline, with the bilateral common carotid artery stenosis hypoperfusion mouse model as the most promising model. The transverse aortic constriction and myocardial infarction models may be good alternatives, but these models are as yet less characterized regarding the possible cerebral changes. Mixed models could be used to study the combined effects of different cardiovascular diseases on the deterioration of cognition during aging.

Background Patients with symptomatic carotid artery stenosis are at high risk for recurrent stroke. To date, the decision to perform carotid endarterectomy in patients with a recent cerebrovascular event is mainly based on degree of stenosis of the ipsilateral carotid artery. However, additional atherosclerotic plaque characteristics might be better predictors of stroke, allowing for more precise selection of patients for carotid endarterectomy. Aims and hypothesis We investigate the hypothesis that the assessment of carotid plaque characteristics with magnetic resonance imaging, multidetector-row computed tomography angiography, ultrasonography, and transcranial Doppler, either alone or in combination, may improve identification of a subgroup of patients with &lt;70% carotid artery stenosis with an increased risk of recurrent stroke. Methods The Plaque At RISK (PARISK) study is a prospective multicenter cohort study of patients with recent (&lt;3 months) neurological symptoms due to ischemia in the territory of the carotid artery and &lt; 70% ipsilateral carotid artery stenosis who are not scheduled for carotid endarterectomy or stenting. At baseline, 300 patients will undergo magnetic resonance imaging, multidetector-row computed tomography angiography, and ultrasonography examination of the carotid arteries. In addition, magnetic resonance imaging of the brain, ambulatory transcranial Doppler recording of the middle cerebral artery and blood withdrawal will be performed. After two-years, imaging will be repeated in 150 patients. All patients undergo a follow-up brain magnetic resonance imaging, and there will be regular clinical follow-up until the end of the study. Study outcomes The combined primary end-point contains ipsilateral recurrent ischemic stroke or transient ischemic attack or new ipsilateral ischemic brain lesions on follow-up brain magnetic resonance imaging.

Radiotherapy of thoracic and chest-wall tumors increases the long-term risk of radiation-induced heart disease, like a myocardial infarct. Cancer patients commonly have additional risk factors for cardiovascular disease, such as hypercholesterolemia. The goal of this study is to define the interaction of irradiation with such cardiovascular risk factors in radiation-induced damage to the heart and coronary arteries.Hypercholesterolemic and atherosclerosis-prone ApoE(-/-) mice received local heart irradiation with a single dose of 0, 2, 8 or 16 Gy. Histopathological changes, microvascular damage and functional alterations were assessed after 20 and 40 weeks.Inflammatory cells were significantly increased in the left ventricular myocardium at 20 and 40 weeks after 8 and 16 Gy. Microvascular density decreased at both follow-up time-points after 8 and 16 Gy. Remaining vessels had decreased alkaline phosphatase activity (2-16 Gy) and increased von Willebrand Factor expression (16 Gy), indicative of endothelial cell damage. The endocardium was extensively damaged after 16 Gy, with foam cell accumulations at 20 weeks, and fibrosis and protein leakage at 40 weeks. Despite an accelerated coronary atherosclerotic lesion development at 20 weeks after 16 Gy, gated SPECT and ultrasound measurements showed only minor changes in functional cardiac parameters at 20 weeks.The combination of hypercholesterolemia and local cardiac irradiation induced an inflammatory response, microvascular and endocardial damage, and accelerated the development of coronary atherosclerosis. Despite these pronounced effects, cardiac function of ApoE(-/-) mice was maintained.

<h3>BACKGROUND AND PURPOSE:</h3> Several studies have attempted to characterize intracranial atherosclerotic plaques by using MR imaging sequences. However, dedicated validation of these sequences with histology has not yet been performed. The current study assessed the ability of ultra-high-resolution 7T MR imaging sequences with different image contrast weightings to image plaque components, by using histology as criterion standard. <h3>MATERIALS AND METHODS:</h3> Five specimens of the circle of Wills were imaged at 7T with 0.11 × 0.11 mm in-plane-resolution proton attenuation–, T1-, T2-, and T2*-weighted sequences (through-plane resolution, 0.11–1 mm). Tissue samples from 13 fiducial-marked locations (per specimen) on MR imaging underwent histologic processing and atherosclerotic plaque classification. Reconstructed MR images were matched with histologic sections at corresponding locations. <h3>RESULTS:</h3> Forty-four samples were available for subsequent evaluation of agreement or disagreement between plaque components and image contrast differences. Of samples, 52.3% (<i>n</i> = 23) showed no image contrast heterogeneity; this group comprised solely no lesions or early lesions. Of samples, 25.0% (<i>n</i> = 11, mostly advanced lesions) showed good correlation between the spatial organization of MR imaging heterogeneities and plaque components. Areas of foamy macrophages were generally seen as proton attenuation–, T2-, and T2*- hypointense areas, while areas of increased collagen content showed more ambiguous signal intensities. Five samples showed image-contrast heterogeneity without corresponding plaque components on histology; 5 other samples showed contrast heterogeneity based on intima-media artifacts. <h3>CONCLUSIONS:</h3> MR imaging at 7T has the image contrast capable of identifying both focal intracranial vessel wall thickening and distinguishing areas of different signal intensities spatially corresponding to plaque components within more advanced atherosclerotic plaques.

In this article, we describe a novel straightforward method for the specific identification of viable cells (macrophages and cancer cell lines MCF-7 and Jurkat) in a buffer solution. The detection of the various cell types is based on changes of the heat transfer resistance at the solid-liquid interface of a thermal sensor device induced by binding of the cells to a surface-imprinted polymer layer covering an aluminum chip. We observed that the binding of cells to the polymer layer results in a measurable increase of heat transfer resistance, meaning that the cells act as a thermally insulating layer. The detection limit was found to be on the order of 10(4) cells/mL, and mutual cross-selectivity effects between the cells and different types of imprints were carefully characterized. Finally, a rinsing method was applied, allowing for the specific detection of cancer cells with their respective imprints while the cross-selectivity toward peripheral blood mononuclear cells was negligible. The concept of the sensor platform is fast and low-cost while allowing also for repetitive measurements.

Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions.

Hallmarks of vulnerable atherosclerotic plaques are inflammation that can be assessed with 18fluorine-fluorodeoxyglucose positron emission tomography/computed tomography, and increased neovascularization that can be evaluated by dynamic contrast-enhanced-MRI. It remains unclear whether these parameters are correlated or represent independent imaging parameters. This study determines whether there is a correlation between inflammation and neovascularization in atherosclerotic carotid plaques.A total of 58 patients with transient ischemic attack or minor stroke in the carotid territory and ipsilateral carotid artery stenosis of 30% to 69% were included. All patients underwent positron emission tomography/computed tomography and dynamic contrast-enhanced-MRI of the carotid plaque. 18Fluorine-fluorodeoxyglucose standard uptake values with target/background ratio were determined. Neovascularization was quantified by the mean (leakage) volume transfer constant Ktrans. Spearman rank correlation coefficients between target/background ratio and Ktrans were calculated.Images suitable for further analysis were obtained in 49 patients. A weak but significant positive correlation between target/background ratio and mean Ktrans (Spearman ρ=0.30 [P=0.035]) and 75th percentile Ktrans (Spearman ρ=0.29 [P=0.041]) was found.There is a weak but significant positive correlation between inflammation on positron emission tomography/computed tomography and neovascularization as assessed with dynamic contrast-enhanced-MRI. Future studies should investigate which imaging modality has the highest predictive value for recurrent stroke, as these are not interchangeable.http://www.clinicaltrials.gov. Unique identifier: NCT00451529.

<h3>BACKGROUND AND PURPOSE:</h3> An important characteristic of vulnerable plaque, intraplaque hemorrhage, may predict plaque rupture. Plaque rupture can be visible on noninvasive imaging as a disruption of the plaque surface. We investigated the association between intraplaque hemorrhage and disruption of the plaque surface. <h3>MATERIALS AND METHODS:</h3> We selected the first 100 patients of the Plaque At RISK study, an ongoing prospective noninvasive plaque imaging study in patients with mild-to-moderate atherosclerotic lesions in the carotid artery. In carotid artery plaques, disruption of the plaque surface (defined as ulcerated plaques and/or fissured fibrous cap) and intraplaque hemorrhage were assessed by using MDCTA and 3T MR imaging, respectively. We used a χ² test and multivariable logistic regression to assess the association between intraplaque hemorrhage and disrupted plaque surface. <h3>RESULTS:</h3> One hundred forty-nine carotid arteries in 78 patients could be used for the current analyses. Intraplaque hemorrhage and plaque ulcerations were more prevalent in symptomatic compared with contralateral vessels (hemorrhage, 38% versus 11%; <i>P</i> &lt; .001; and ulcerations, 27% versus 7%; <i>P</i> = .001). Fissured fibrous cap was more prevalent in symptomatic compared with contralateral vessels (13% versus 4%; <i>P</i> = .06). After adjustment for age, sex, diabetes mellitus, and degree of stenosis, intraplaque hemorrhage was associated with disrupted plaque surface (OR, 3.13; 95% CI, 1.25–7.84) in all vessels. <h3>CONCLUSIONS:</h3> Intraplaque hemorrhage is associated with disruption of the plaque surface in patients with a carotid artery stenosis of &lt;70%. Serial studies are needed to investigate whether intraplaque hemorrhage indeed increases the risk of plaque rupture and subsequent ischemic stroke during follow-up.

Although it has been demonstrated that carcinogenic environmental polycyclic aromatic hydrocarbons (PAHs) cause progression of atherosclerosis, the underlying mechanism remains unclear. In the present study, we aimed to investigate whether DNA binding events are critically involved in the progression of PAH-mediated atherogenesis. Apolipoprotein E knockout mice were orally (24 wk, once/wk) exposed to 5 mg/kg benzo[a]pyrene (B[a]P), or its nonmutagenic, noncarcinogenic structural isoform benzo[e]pyrene (B[e]P). 32P-postlabeling of lung tissue confirmed the presence of promutagenic PAH-DNA adducts in B[a]P-exposed animals, whereas in B[e]P-exposed and vehicle control animals, these adducts were undetectable. Morphometrical analysis showed that both B[a]P and B[e]P caused an increase in plaque size, whereas location or number of plaques was unaffected. Immunohistochemistry revealed no differences in oxidative DNA damage (8-OHdG) or apoptosis in the plaques. Also plasma lipoprotein levels remained unchanged after PAH-exposure. However, T lymphocytes were increased ≥2-fold in the plaques of B[a]P- and B[e]P-exposed animals. Additionally, B[a]P and to a lesser extent B[e]P exposure resulted in increased TGFβ protein levels in the plaques, that was mainly localized in the plaque macrophages. In vitro studies using the murine macrophage like RAW264.7 cells showed that inhibition of TGFβ resulted in decreased tumor necrosis factor (TNF) α release, suggesting that enhanced TGFβ expression in the plaque macrophages contributes to the proinflammatory effects in the vessel wall. In general, this inflammatory reaction in the plaques appeared to be a local response since peripheral blood cell composition (T cells, B cells, granulocytes, and macrophages) was not changed upon PAH exposure. In conclusion, we showed that both B[a]P and B[e]P cause progression of atherosclerosis, irrespective of their DNA binding properties. Moreover, our data revealed a possible novel mechanism of PAH-mediated atherogenesis, which likely involves a TGFβ-mediated local inflammatory reaction in the vessel wall.

Spondyloarthritides are associated with increased cardiovascular risks, which can only partly be explained by traditional risk factors. It is likely that the chronic inflammatory state is involved. In this review, novel findings regarding cardiac and vascular pathologies and potential overlapping mechanisms will be discussed.Cardiac pathologies in spondyloarthritides are conduction disturbances and valvular heart diseases. Recent studies have also focused on vascular pathologies and showed impaired endothelial function, suggesting that atherosclerotic alterations could also be involved in increased cardiovascular mortality. Novel findings suggest that chronic systemic inflammation is involved in these cardiac and vascular pathologies. Thus, spondyloarthritides and ankylosing spondylitis are associated with increased levels of circulating inflammatory mediators such as C-reactive protein. Interestingly, ankylosing spondylitis patients may also have an atherogenic lipid profile and disturbances in their T-helper lymphocyte subsets, which may be involved in cardiovascular disease development. The beneficial effects of statin treatment on circulating inflammatory mediators and atherogenic lipid profiles may reveal new therapeutic options for patients with spondyloarthritides.Recent studies have highlighted that the chronic, systemic inflammatory condition of patients with spondyloarthritides may be involved in the development of cardiac and vascular pathologies.

Abstract We investigated the role of CD40 and CD40L in neointima formation and identified the downstream CD40-signaling intermediates (tumor necrosis factor [TNF]–receptor associated factors [TRAF]) involved. Neointima formation was induced in wild-type, CD40−/−, CD40L−/−, and in CD40−/− mice that contained a CD40 transgene with or without mutations at the CD40-TRAF2,3&amp;5, TRAF6, or TRAF2,3,5&amp;6 binding sites. Compared with wild-type mice, CD40−/− mice showed a significant decrease in neointima formation with increased collagen deposition and decreased inflammatory cell infiltration. Neointima formation was also impaired in wild-type mice reconstituted with CD40−/− bone marrow. In vitro, the capacity of CD40−/− leukocytes to adhere to the endothelium was reduced. Ligated carotid arteries of CD40−/− mice showed a smaller total vessel volume and an impaired remodeling capacity, reflected by decreased gelatinolytic/collagenolytic activity. Comparable results were found in mice with defects in CD40-TRAF6 and CD40-TRAF 2/3/5&amp;6 binding, but not in mice with defects in CD40-TRAF2/3&amp;5 binding. Neointima formation and vascular remodeling in CD40-receptor–deficient mice is impaired, due to a decreased inflammatory cell infiltration and matrix-degrading protease activity, with CD40-TRAF6 signaling as the key regulator. This identifies the CD40-TRAF6 axis as a potential therapeutic target in vascular disease.

Inflammation, interstitial fibrosis (IF), and tubular atrophy (TA) precede chronic transplant dysfunction, which is a major cause of renal allograft loss. There is an association between IF/TA and loss of peritubular capillaries (PTCs) in advanced renal disease, but whether PTC loss occurs in an early stage of chronic transplant dysfunction is unknown. Here, we studied PTC number, IF/TA, inflammation, and renal function in 48 patients who underwent protocol biopsies. Compared with before transplantation, there was a statistically significant loss of PTCs by 3 months after transplantation. Fewer PTCs in the 3-month biopsy correlated with high IF/TA and inflammation scores and predicted lower renal function at 1 year. Predictors of PTC loss during the first 3 months after transplantation included donor type, rejection, donor age, and the number of PTCs at the time of implantation. In conclusion, PTC loss occurs during the first 3 months after renal transplantation, associates with increased IF and TA, and predicts reduced renal function.

The importance of transforming growth factor beta (TGFβ) as an immune regulatory cytokine in atherosclerosis has been established. However, the role of TGFβ signalling in dendritic cells (DCs) and in DC-mediated T cell proliferation and differentiation in atherosclerosis is unknown.Here, we investigated the effect of disrupted TGFβ signalling in DCs on atherosclerosis by using mice carrying a transgene resulting in functional inactivation of TGFβ receptor II (TGFβRII) signalling in CD11c(+) cells (Apoe(-/-)CD11cDNR). Apoe(-/-)CD11cDNR mice exhibited an over two-fold increase in the plaque area compared with Apoe(-/-) mice. Plaques of Apoe(-/-)CD11cDNR mice showed an increase in CD45(+) leucocyte content, and specifically in CD3(+), CD4(+) and CD8(+) cells, whereas macrophage content was not affected. In lymphoid organs, Apoe(-/-)CD11cDNR mice had equal amounts of CD11c(+) cells, and CD11c(+)CD8(+) and CD11c(+)CD8(-) subsets, but showed a subtle shift in the CD11c(+)CD8(-) population towards the more inflammatory CD11c(+)CD8(-)CD4(-) DC subset. In addition, the number of plasmacytoid-DCs decreased. Maturation markers such as MHCII, CD86 and CD40 on CD11c(hi) cells did not change, but the CD11cDNR DCs produced more TNFα and IL-12. CD11c(+) cells from CD11cDNR mice strongly induced T-cell proliferation and activation, resulting in increased amounts of effector T cells producing high amounts of Th1 (IFN-γ), Th2 (IL-4, IL-10), Th17 (IL-17), and Treg (IL-10) cytokines.Here, we show that loss of TGFβRII signalling in CD11c(+) cells induces subtle changes in DC subsets, which provoke uncontrolled T cell activation and maturation. This results in increased atherosclerosis and an inflammatory plaque phenotype during hypercholesterolaemia.

We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal alpha(2)-antiplasmin-based contrast agent (CA).Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links alpha(2)-antiplasmin to fibrin, indicating the potential of alpha(2)-antiplasmin-based CAs in the detection of early thrombus formation.A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an alpha(2)-antiplasmin-based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of alpha(2)-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI.In vitro-generated thrombi exposed to the alpha(2)-antiplasmin-based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the alpha(2)-antiplasmin-based CA in the presence of FXIII inhibitor dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the alpha(2)-antiplasmin-based CA bound to fibrin. Immunohistochemistry demonstrated substantial alpha(2)-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 +/- 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs -0.14 +/- 0.55, p = 0.003, n = 6) and alpha(2)-antiplasmin-based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 +/- 0.23, p = 0.006, n = 6).A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation.

Neovascularization of human atherosclerotic plaques is implicated in plaque progression and destabilization, although its functional implications are yet unresolved. Here, we aimed to elucidate functional and morphological properties of plaque microvessels in mice in vivo.Atherosclerotic carotid arteries from aged (>40 weeks) apolipoprotein E-deficient mice were imaged in vivo using multiphoton laser scanning microscopy. Two distinct groups of vasa vasorum microvessels were observed at sites of atherosclerosis development (median diameters of 18.5 and 5.9 μm, respectively), whereas microvessels within the plaque could only rarely be found. In vivo imaging showed ongoing angiogenic activity and injection of fluorescein isothiocyanate-dextran confirmed active perfusion. Plaque vasa vasorum showed increased microvascular leakage, combined with a loss of endothelial glycocalyx. Mean blood flow velocity in plaque-associated vasa vasorum was reduced by ±50% compared with diameter-matched control capillaries, whereas mean blood flow was reduced 8-fold. Leukocyte adhesion and extravasation were increased 6-fold in vasa vasorum versus control capillaries.Using a novel in vivo functional imaging strategy, we showed that plaque-associated vasa vasorum were angiogenically active and, albeit poorly, perfused. Moreover, plaque-associated vasa vasorum showed increased permeability, reduced blood flow, and increased leukocyte adhesion and extravasation (ie, characteristics that could contribute to plaque progression and destabilization).

Advanced murine and human plaques are hypoxic, but it remains unclear whether plaque hypoxia is causally related to atherogenesis. Here, we test the hypothesis that reversal of hypoxia in atherosclerotic plaques by breathing hyperoxic carbogen gas will prevent atherosclerosis.Low-density lipoprotein receptor-deficient mice (LDLR(-/-)) were fed a Western-type diet, exposed to carbogen (95% O2, 5% CO2) or air, and the effect on plaque hypoxia, size, and phenotype was studied. First, the hypoxic marker pimonidazole was detected in murine LDLR(-/-) plaque macrophages from plaque initiation onwards. Second, the efficacy of breathing carbogen (90 minutes, single exposure) was studied. Compared with air, carbogen increased arterial blood pO2 5-fold in LDLR(-/-) mice and reduced plaque hypoxia in advanced plaques of the aortic root (-32%) and arch (-84%). Finally, the effect of repeated carbogen exposure on progression of atherosclerosis was studied in LDLR(-/-) mice fed a Western-type diet for an initial 4 weeks, followed by 4 weeks of diet and carbogen or air (both 90 min/d). Carbogen reduced plaque hypoxia (-40%), necrotic core size (-37%), and TUNEL(+) (terminal uridine nick-end labeling positive) apoptotic cell content (-50%) and increased efferocytosis of apoptotic cells by cluster of differentiation 107b(+) (CD107b, MAC3) macrophages (+36%) in advanced plaques of the aortic root. Plaque size, plasma cholesterol, hematopoiesis, and systemic inflammation were unchanged. In vitro, hypoxia hampered efferocytosis by bone marrow-derived macrophages, which was dependent on the receptor Mer tyrosine kinase.Carbogen restored murine plaque oxygenation and prevented necrotic core expansion by enhancing efferocytosis, likely via Mer tyrosine kinase. Thus, plaque hypoxia is causally related to necrotic core expansion.

Vascular endothelial-cadherin- and integrin-based cell adhesions are crucial for endothelial barrier function. Formation and disassembly of these adhesions controls endothelial remodeling during vascular repair, angiogenesis, and inflammation. In vitro studies indicate that vascular cytokines control adhesion through regulation of the actin cytoskeleton, but it remains unknown whether such regulation occurs in human vessels. We aimed to investigate regulation of the actin cytoskeleton and cell adhesions within the endothelium of human arteries and veins.We used an ex vivo protocol for immunofluorescence in human vessels, allowing detailed en face microscopy of endothelial monolayers. We compared arteries and veins of the umbilical cord and mesenteric, epigastric, and breast tissues and find that the presence of central F-actin fibers distinguishes the endothelial phenotype of adult arteries from veins. F-actin in endothelium of adult veins as well as in umbilical vasculature predominantly localizes cortically at the cell boundaries. By contrast, prominent endothelial F-actin fibers in adult arteries anchor mostly to focal adhesions containing integrin-binding proteins paxillin and focal adhesion kinase and follow the orientation of the extracellular matrix protein fibronectin. Other arterial F-actin fibers end in vascular endothelial-cadherin-based endothelial focal adherens junctions. In vitro adhesion experiments on compliant substrates demonstrate that formation of focal adhesions is strongly induced by extracellular matrix rigidity, irrespective of arterial or venous origin of endothelial cells.Our data show that F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries from veins. We conclude that the biomechanical properties of the vascular extracellular matrix determine this endothelial characteristic.

Dyslipidaemia and inflammation are closely interconnected in their contribution to atherosclerosis. In fact, low-density lipoprotein (LDL)-lowering drugs have anti-inflammatory effects. The Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) has shown that interleukin (IL)-1β blockade reduces the incidence of cardiovascular events in patients with previous myocardial infarction and C-reactive protein levels >2 mg/L. These data confirm the connection between lipids and inflammation, as lipids activate the Nod-like receptor protein 3 inflammasome that leads to IL-1β activation. LDL-lowering drugs are the foundation of cardiovascular prevention. Now, the CANTOS trial demonstrates that combining them with IL-1β blockade further decreases the incidence of cardiovascular events. However, both therapies are not at the same level, given the large evidence showing that LDL-lowering drugs reduce cardiovascular risk as opposed to only one randomized trial of IL-1β blockade. In addition, IL-1β blockade has only been studied in patients with C-reactive protein >2 mg/L, while the benefit of LDL-lowering is not restricted to these patients. Also, lipid-lowering drugs are not harmful even at very low ranges of LDL, while anti-inflammatory therapies may confer a higher risk of developing fatal infections and sepsis. In the future, more clinical trials are needed to explore whether targeting other inflammatory molecules, both related and unrelated to the IL-1β pathway, reduces the cardiovascular risk. In this regard, the ongoing trials with methotrexate and colchicine may clarify whether the cardiovascular benefit of IL-1β blockade extends to other anti-inflammatory mechanisms. A positive result would represent a major change in the future treatment of atherosclerosis.

While both cardiac dysfunction and progressive loss of cognitive functioning are prominent features of an aging population, surprisingly few studies have addressed the link between heart and brain function. This is probably due to the monodisciplinary approach to these problems by cardiologists, neurologists, and geriatricians. Recent data indicate that autoregulation of cerebral flow cannot always protect the brain from hypoperfusion when cardiac output is reduced or atherosclerosis is prominent. This suggests a close link between cardiac function and large vessel atherosclerosis on the one hand and brain perfusion and cognitive functioning on the other. In a national research program, we will test the hypothesis that impaired hemodynamic status of both heart and brain is an important and potentially reversible cause of vascular cognitive impairment (VCI) offering promising opportunities for treatment. Using a multidisciplinary approach, we will address the following questions: 1) To what extent do hemodynamic changes contribute to VCI? 2) What are the mechanisms involved? 3) Does improvement of the hemodynamic status lead to improvement of cognitive dysfunction? To this end we will perform clinical studies in elderly patients with clinically manifest VCI, carotid occlusive disease, or heart failure and evaluate their cardiac and large vascular function, atherosclerotic load, and cerebral perfusion with a comprehensive magnetic resonance imaging protocol and thoroughly test their cognitive function. We will also analyze epidemiological data from the Rotterdam Study.

Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)-mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1α in APCs in atherosclerosis.We found upregulated HIF1α expression in CD11c(+) APCs within atherosclerotic plaques of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Conditional deletion of Hif1a in CD11c(+) APCs in high-fat diet-fed Ldlr(-/-) mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1α directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1α-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr(-/-) mice. Notably, deletion of Hif1a in LysM(+) bone marrow cells in Ldlr(-/-) mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1α, STAT3, and interleukin-12 protein were found to colocalize with APCs.Our findings identify HIF1α to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr(-/-) mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.

Normalization of hypercholesterolaemia, inflammation, hyperglycaemia, and obesity are main desired targets to prevent cardiovascular clinical events. Here we present a novel regulator of cholesterol metabolism, which simultaneously impacts on glucose intolerance and inflammation.Mice deficient for oxygen sensor HIF-prolyl hydroxylase 1 (PHD1) were backcrossed onto an atherogenic low-density lipoprotein receptor (LDLR) knockout background and atherosclerosis was studied upon 8 weeks of western-type diet. PHD1-/-LDLR-/- mice presented a sharp reduction in VLDL and LDL plasma cholesterol levels. In line, atherosclerotic plaque development, as measured by plaque area, necrotic core expansion and plaque stage was hampered in PHD1-/-LDLR-/- mice. Mechanistically, cholesterol-lowering in PHD1 deficient mice was a result of enhanced cholesterol excretion from blood to intestines and ultimately faeces. Additionally, flow cytometry of whole blood of these mice revealed significantly reduced counts of leucocytes and particularly of Ly6Chigh pro-inflammatory monocytes. In addition, when studying PHD1-/- in diet-induced obesity (14 weeks high-fat diet) mice were less glucose intolerant when compared with WT littermate controls.Overall, PHD1 knockout mice display a metabolic phenotype that generally is deemed protective for cardiovascular disease. Future studies should focus on the efficacy, safety, and gender-specific effects of PHD1 inhibition in humans, and unravel the molecular actors responsible for PHD1-driven, likely intestinal, and regulation of cholesterol metabolism.

Plaque fissuring, a phenomenon morphologically distinct from the classical rupture of a thinned fibrous cap, has not been well characterized in carotid atherosclerosis. The aim of this study was to establish the prevalence of plaque fissures in advanced carotid plaques with an otherwise intact luminal surface, and to determine whether they might be a source of intraplaque hemorrhage (IPH).We evaluated 244 surgically intact, 'en bloc' embedded, serially sectioned carotid endarterectomy specimens and included only those plaques with a grossly intact luminal surface.Among the 67 plaques with grossly intact luminal surface, cap fissure was present in 39 (58%) plaques. A total of 60 individual fissures were present, and longitudinally mean fissure length was 1.3 mm. Most fissures were found distal to the bifurcation (63%), proximal to the stenosis (88%), and in the posterior (opposite the flow divider) or lateral quadrants (80%). 36% of the fissures remained in the superficial third of the plaque. 52% extended from the lumen surface to the middle third of the plaque and 12% reached the outer third of the plaque on cross section. Fissures often occurred between two tissue planes and were connected to IPH (fresh: 63%; any type: 92%) and calcifications (43%). No correlation was found with patient characteristics such as symptom status, carotid stenosis, hypertension, diabetes, smoking and medications (statins or antiplatelet agents).Plaque fissures are common in advanced carotid plaques with an otherwise grossly intact luminal surface and are associated with fresh intraplaque hemorrhage. As they occur on the interface between plaque components with different mechanical properties, further biomechanical studies are needed to unravel the underlying failure mechanisms.

Background and aimsLipoprotein(a) is an independent risk factor for cardiovascular disease and recurrent ischemic stroke. Lipoprotein(a) levels are known to be associated with carotid artery stenosis, but the relation of lipoprotein(a) levels to carotid atherosclerotic plaque composition and morphology is less known. We hypothesize that higher lipoprotein(a) levels and lipoprotein(a)-related SNPs are associated with a more vulnerable carotid plaque and that this effect is sex-specific.MethodsIn 182 patients of the Plaque At RISK study we determined lipoprotein(a) concentrations, apo(a) KIV-2 repeats and LPA SNPs. Imaging characteristics of carotid atherosclerosis were determined by MDCTA (n = 161) and/or MRI (n = 171). Regressions analyses were used to investigate sex-stratified associations between lipoprotein(a) levels, apo(a) KIV-2 repeats, and LPA SNPs and imaging characteristics.ResultsLipoprotein(a) was associated with presence of lipid-rich necrotic core (LRNC) (aOR = 1.07, 95% CI: 1.00; 1.15), thin-or-ruptured fibrous cap (TRFC) (aOR = 1.07, 95% CI: 1.01; 1.14), and degree of stenosis (β = 0.44, 95% CI: 0.00; 0.88). In women, lipoprotein(a) was associated with presence of intraplaque hemorrhage (IPH) (aOR = 1.25, 95% CI: 1.06; 1.61). In men, lipoprotein(a) was associated with degree of stenosis (β = 0.58, 95% CI: 0.04; 1.12). Rs10455872 was significantly associated with increased calcification volume (β = 1.07, 95% CI: 0.25; 1.89) and absence of plaque ulceration (aOR = 0.25, 95% CI: 0.04; 0.93). T3888P was associated with absence of LRNC (aOR = 0.36, 95% CI: 0.16; 0.78) and smaller maximum vessel wall area (β = -10.24, 95%CI: -19.03; -1.44).ConclusionsIn patients with symptomatic carotid artery stenosis, increased lipoprotein(a) levels were associated with degree of stenosis, and IPH, LRNC, and TRFC, known as vulnerable plaque characteristics, in the carotid artery. T3888P was associated with lower LRNC prevalence and smaller maximum vessel wall area. Further research in larger study populations is needed to confirm these results.

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon with atherogenic and carcinogenic properties. The role of B[a]P in carcinogenesis is well established, and thought to exert via enzymatic activation into reactive metabolites that are capable of binding to the DNA leading to uncontrolled proliferation. However, the mechanism underlying the atherogenic properties of B[a]P is still unclear. Therefore, the effects of chronic B[a]P exposure on atherosclerotic plaque development in apolipoprotein E knockout (apoE-KO) mice were studied. ApoE-KO mice were orally treated with 5 mg/kg/bw B[a]P once per week for 12 or 24 consecutive weeks. Levels of reactive B[a]P metabolites in the arterial tree (from the aortic arch until the iliac artery bifurcations) were high as shown by the level of B[a]P DNA-binding products measured in DNA isolated from the entire aorta (38.9 ± 4.8 adducts/108 nucleotides). Analysis of atherosclerotic lesions in the aortic arch showed no influence of B[a]P on location or number of lesions. Moreover, no increased levels of p53 nuclear protein accumulation or cell proliferation, as detected by immunohistochemistry, were seen in the plaques of the B[a]P-exposed animals. However, the effects of B[a]P on advanced lesions were obvious: advanced plaques were larger and more prone to lipid core development and plaque layering at both 12 and 24 weeks (P < 0.05). In the B[a]P-exposed animals advanced plaques contained more T-lymphocytes and macrophages than in the control animals at both end points (P < 0.05). These data suggest that B[a]P does not initiate atherosclerosis in apoE-KO mice, but accelerates the progression of atherosclerotic plaques via a local inflammatory response. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon with atherogenic and carcinogenic properties. The role of B[a]P in carcinogenesis is well established, and thought to exert via enzymatic activation into reactive metabolites that are capable of binding to the DNA leading to uncontrolled proliferation. However, the mechanism underlying the atherogenic properties of B[a]P is still unclear. Therefore, the effects of chronic B[a]P exposure on atherosclerotic plaque development in apolipoprotein E knockout (apoE-KO) mice were studied. ApoE-KO mice were orally treated with 5 mg/kg/bw B[a]P once per week for 12 or 24 consecutive weeks. Levels of reactive B[a]P metabolites in the arterial tree (from the aortic arch until the iliac artery bifurcations) were high as shown by the level of B[a]P DNA-binding products measured in DNA isolated from the entire aorta (38.9 ± 4.8 adducts/108 nucleotides). Analysis of atherosclerotic lesions in the aortic arch showed no influence of B[a]P on location or number of lesions. Moreover, no increased levels of p53 nuclear protein accumulation or cell proliferation, as detected by immunohistochemistry, were seen in the plaques of the B[a]P-exposed animals. However, the effects of B[a]P on advanced lesions were obvious: advanced plaques were larger and more prone to lipid core development and plaque layering at both 12 and 24 weeks (P < 0.05). In the B[a]P-exposed animals advanced plaques contained more T-lymphocytes and macrophages than in the control animals at both end points (P < 0.05). These data suggest that B[a]P does not initiate atherosclerosis in apoE-KO mice, but accelerates the progression of atherosclerotic plaques via a local inflammatory response. It is demonstrated that chemicals such as polycyclic aromatic hydrocarbons (PAHs) play a role in both cancer and cardiovascular diseases.1Ross JS Stagliano NE Donovan MJ Breitbart RE Ginsburg GS Atherosclerosis and cancer: common molecular pathways of disease development and progression.Ann NY Acad Sci. 2001; 947: 271-292Crossref PubMed Scopus (178) Google Scholar PAHs, products of the incomplete combustion of organic materials, are a large group of structurally related lipophilic compounds with two or more condensed benzene rings. They are abundantly present in, for example, cigarette smoke and charcoal-broiled and smoked foods. The general population is exposed to PAHs on a daily basis, mainly via ingestion of contaminated foods and inhalation of polluted air.2Waldman JM Lioy PJ Greenberg A Butler JP Analysis of human exposure to benzo(a)pyrene via inhalation and food ingestion in the Total Human Environmental Exposure Study (THEES).J Expo Anal Environ Epidemiol. 1991; 1: 193-225PubMed Google Scholar Benzo[a]pyrene (B[a]P), a model PAH, is metabolized via cytochrome P450s into reactive dihydrodiol epoxide derivates, eg, B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), which are capable of binding covalently to the DNA.3International Agency for Research on Cancer Polynuclear Aromatic Compounds, Part 1, Chemical, Environmental and Experimental data.IARC Monogr Eval Carcinog Risk Chem Hum. 1983; 32: 1-453PubMed Google Scholar In the carcinogenic process, formation of these so-called BPDE-DNA adducts is considered to be a crucial initial step leading to mutations and subsequently to uncontrolled cell growth and tumor formation.4De Flora S Izzotti A Randerath K Randerath E Bartsch H Nair J Balansky R van Schooten F Degan P Fronza G Walsh D Lewtas J DNA adducts and chronic degenerative disease. Pathogenetic relevance and implications in preventive medicine.Mutat Res. 1996; 366: 197-238Crossref PubMed Scopus (160) Google Scholar Although B[a]P has been shown to influence atherosclerosis in animal models,5Wakabayashi K International Commission for Protection Against Environmental Mutagens and Carcinogens. ICPEMC Working Paper 7/1/3. Animal studies suggesting involvement of mutagen/carcinogen exposure in atherosclerosis.Mutat Res. 1990; 239: 181-187Crossref PubMed Scopus (2) Google Scholar the exact underlying mechanism of chemical atherogenesis is still not elucidated. In the 1970s initial studies from Benditt and Benditt6Benditt EP Benditt JM Evidence for a monoclonal origin of human atherosclerotic plaques.Proc Natl Acad Sci USA. 1973; 70: 1753-1756Crossref PubMed Scopus (659) Google Scholar showed that human atherosclerotic plaques had a monoclonal origin. At the same time, the first animal experiments proved the involvement of chemical carcinogens in atherosclerotic plaque development.7Penn A Snyder C Arteriosclerotic plaque development is ‘promoted’ by polynuclear aromatic hydrocarbons.Carcinogenesis. 1988; 9: 2185-2189Crossref PubMed Scopus (104) Google Scholar Finally, in 1986 it was shown that DNA extracted from human coronary artery plaques were capable of transforming NIH3T3 cells.8Penn A Garte SJ Warren L Nesta D Mindich B Transforming gene in human atherosclerotic plaque DNA.Proc Natl Acad Sci USA. 1986; 83: 7951-7955Crossref PubMed Scopus (122) Google Scholar These consecutive observations have lead to the suggestion that atherosclerotic plaques are presumably benign smooth muscle cell tumors that develop according to an initiation-promotion-progression protocol. More recently, animal studies have shown that the aorta is a target for carcinogen-induced DNA damage.9Izzotti A Camoirano A Cariglia C Tampa E De Flora S Formation of DNA adducts in the aorta of smoke-exposed rats, and modulation by chemopreventive agents.Mutat Res. 2001; 494: 97-106Crossref PubMed Scopus (26) Google Scholar Similarly in humans exposed to environmental carcinogens, arterial DNA damage is high and related to atherogenic risk factors.10Izzotti A De Flora S Petrilli GL Gallagher J Rojas M Alexandrov K Bartsch H Lewtas J Cancer biomarkers in human atherosclerotic lesions: detection of DNA adducts.Cancer Epidemiol Biomarkers Prev. 1995; 4: 105-110PubMed Google Scholar, 11Binkova B Strejc P Boubelik O Stavkova Z Chvatalova I Sram RJ DNA adducts and human atherosclerotic lesions.Int J Hyg Environ Health. 2001; 204: 49-54Crossref PubMed Scopus (18) Google Scholar, 12De Flora S Izzotti A Walsh D Degan P Petrilli GL Lewtas J Molecular epidemiology of atherosclerosis.EMBO J. 1997; 11: 1021-1031Google Scholar Moreover, several human studies showed that DNA damage and repair seem to be associated with atherosclerosis.13Van Schooten FJ Hirvonen A Maas LM De Mol BA Kleinjans JC Bell DA Durrer JD Putative susceptibility markers of coronary artery disease: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue.EMBO J. 1998; 12: 1409-1417Google Scholar, 14Martinet W Knaapen MW De Meyer GR Herman AG Kockx MM Elevated levels of oxidative DNA damage and DNA repair enzymes in human atherosclerotic plaques.Circulation. 2002; 106: 927-932Crossref PubMed Scopus (380) Google Scholar However, although it is clear that carcinogens cause substantial DNA damage in the vessel wall and are able to promote atherosclerotic plaque growth, carcinogen-induced initiation of new plaques has hardly ever been observed. This suggests that the processes involved in chemical carcinogenesis (ie, DNA damage, mutagenesis, proliferation) cannot simply explain the mechanisms underlying chemical atherogenesis. Still, research into chemical atherogenesis has kept its focus mainly on arterial DNA damage as an initiating step for subsequent smooth muscle cell proliferation, akin a benign tumor.10Izzotti A De Flora S Petrilli GL Gallagher J Rojas M Alexandrov K Bartsch H Lewtas J Cancer biomarkers in human atherosclerotic lesions: detection of DNA adducts.Cancer Epidemiol Biomarkers Prev. 1995; 4: 105-110PubMed Google Scholar, 11Binkova B Strejc P Boubelik O Stavkova Z Chvatalova I Sram RJ DNA adducts and human atherosclerotic lesions.Int J Hyg Environ Health. 2001; 204: 49-54Crossref PubMed Scopus (18) Google Scholar With the development of the apolipoprotein E-knockout (apoE-KO) mouse, a transgenic animal model was created in which diet-independent atherosclerotic lesions develop that have striking similarities to the human disease.15Nakashima Y Plump AS Raines EW Breslow JL Ross R ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree.Arterioscler Thromb. 1994; 14: 133-140Crossref PubMed Google Scholar This model proved very useful in studying biochemical and cellular events leading to several aspects of atherosclerosis such as initiation, progression, growth arrest, and regression.16Fazio S Linton MF Mouse models of hyperlipidemia and atherosclerosis.Front Biosci. 2001; 6: D515-D525Crossref PubMed Google Scholar In the present study this mouse model was applied to gain more knowledge on the effects of chronic B[a]P exposure on plaque formation and differentiation. The specific aim of this study was to obtain new insights in the pathways involved in chemical atherogenesis. Therefore, rather than focusing only on DNA damage and plaque area, extensive immunohistochemistry was used to explore differences in plaque phenotype and composition more closely. Male apoE-KO mice were purchased from IFFA CREDO S.A. (Charles River Co., Lyon, France) and fed a normal mouse chow (SRM-A; Hope Farms, Woerden, the Netherlands). B[a]P (B1760; Sigma, St. Louis, MO, USA) was initially dissolved in acetone and added to tricaprylin (103104; ICN, Costa Mesa, CA, USA). Evaporation of acetone resulted in a homogenous solution of 0.5 mg of B[a]P/ml tricaprylin. At 5 weeks of age, animals (17.2 ± 1.8 g) were orally treated with 5 mg/kg/bw B[a]P or vehicle, after an overnight fasting period. This procedure was repeated once per week for 12 (n = 31) and 24 (n = 19) consecutive weeks. By weighing the animals weekly, growth was monitored. To confirm that B[a]P was capable of inducing vascular DNA damage before plaque formation, four wild-type C57BL/6 mice were treated once orally with 25 mg/kg/bw B[a]P. Animals were killed after 4 days and DNA adducts were measured by 32P-postlabeling. On euthanasia, ∼0.5 ml of blood was drawn from the inferior caval vein. The arterial tree was perfused in situ for 3 minutes with 0.9% NaCl containing 20% nitroprusside (3 minutes) and subsequently with 1% phosphate-buffered paraformaldehyde (pH 7.4; 3 minutes) via a catheter in the left ventricular apex. The arterial tree was excised and fixed overnight in 1% phosphate-buffered paraformaldehyde (pH 7.4). The aortic arch (including the brachiocephalic trunk, left carotid artery, and left subclavian artery), thoracic and abdominal aorta (TA and AA, respectively) were longitudinally embedded in paraffin, and subsequently serial 4-μm sections were cut. Pieces of lung and liver tissue of all animals (n = 50) as well as the entire aorta (aortic arch and the descending aorta until the iliac artery bifurcations) of 22 animals from the 12-week group (11 B[a]P versus 11 controls) were used for DNA adduct measurement. Furthermore, >20 organs were investigated macroscopically for abnormalities. Hematoxylin and eosin (H&E)-stained sections of lung, liver, and spleen were also microscopically evaluated. Lipid levels were measured using standard enzymatic techniques, automated on the Cobas Fara centrifugal analyzer (Hoffmann-La Roche, Basel, Switzerland). Total plasma cholesterol and high-density lipoprotein were measured using kit no. 0736635 and no. 543004 (Hoffmann-La Roche), total glycerol using kit no. 337-40A/337-10B (Sigma) and free glycerol using kit no. 0148270 (Hoffmann-La Roche). Precipath (standardized serum) was used as an internal standard. Low-density lipoprotein was calculated using the formula: low-density lipoprotein cholesterol (mmol/L) = total cholesterol − (triglycerides/2.2) − high-density lipoprotein cholesterol. DNA was isolated from lung, liver, and aorta using a traditional phenol extraction procedure and dissolved in 2 mmol/L of Tris (2 mg/ml).17Godschalk RW Maas LM Van Zandwijk N van't Veer LJ Breedijk A Borm PJ Verhaert J Kleinjans JC van Schooten FJ Differences in aromatic-DNA adduct levels between alveolar macrophages and subpopulations of white blood cells from smokers.Carcinogenesis. 1998; 19: 819-825Crossref PubMed Scopus (83) Google Scholar Subsequently, 10 μg of DNA was digested into nucleotide monophosphates and treated with nuclease P1 for 40 minutes at 37°C. Then, nucleotides were 5′-labeled with 32P using T4-polynucleotide kinase (5.0 U) for 30 minutes at 37°C. Radiolabeled adduct nucleotide biphosphates were separated by chromatography on polyethyleneimine (PEI)-cellulose sheets (Macherey Nagel, Düren, Germany) using solvents as described previously.17Godschalk RW Maas LM Van Zandwijk N van't Veer LJ Breedijk A Borm PJ Verhaert J Kleinjans JC van Schooten FJ Differences in aromatic-DNA adduct levels between alveolar macrophages and subpopulations of white blood cells from smokers.Carcinogenesis. 1998; 19: 819-825Crossref PubMed Scopus (83) Google Scholar In each experiment, two standards of [3H]BPDE-modified DNA with known modification levels (1 per 107, 108 nucleotides) were included. Adduct quantification, by means of phosphor-imaging technology (Molecular Dynamics, Sunnyvale, CA, USA), was performed by calibrating toward the BPDE-DNA adduct standards. Of the aortic arch, the thoracic aorta, and the abdominal aorta, four H&E-stained sections, each separated by 20 μm, were used for histological analysis. Lesions were classified as initial or advanced based on the guidelines given by the American Heart Association.18Stary H Blankenhorn D Chandler A Glagov S Insull W Richardson M Rosenfeld M Schaffer S Schwartz C Wagner W Wissler R A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis.Arterioscler Thromb Vasc Biol. 1995; 15: 1513-1531Crossref Scopus (1001) Google Scholar The absence or presence of a lipid core and the cell density (number of cells per μm2) of the plaques were also determined. For morphometrical analysis, four sections consecutive to the H&E-stained sections, were stained according to Lawson (a modified elastica von Giesson staining). Sirius Red staining was performed for the detection of collagen. The relative collagen content was calculated by dividing the Sirius Red-positive plaque area by the total plaque area. Morphometrical parameters such as plaque area, lipid core area, and plaque layering were measured using a computerized morphometry system (Quantimet 570; Leica).19Lutgens E de Muinck ED Heeneman S Daemen MJ Compensatory enlargement and stenosis develop in apoE(−/−) and apoE*3-Leiden transgenic mice.Arterioscler Thromb Vasc Biol. 2001; 21: 1359-1365Crossref PubMed Scopus (21) Google Scholar Several immunohistochemical markers (cell proliferation, DNA repair, and apoptosis) generally used in cancer biology were applied on aortic sections. Cell proliferation was detected with the Zymed PCNA staining kit (Zymed Laboratories Inc., San Francisco, CA, USA) and p53 nuclear protein accumulation by using the antibody CM5 (1:500; Novocastra Laboratories Ltd., Newcastle-upon-Tyne, UK). terminal dUTP nick-end labeling staining (using the ApopTag kit; Oncor, Gaithersburg, MD, USA) for the detection of apoptotic cells was performed in the laboratory of Dr. M. Kockx (Antwerp, Belgium). Moreover, immunohistochemical techniques were applied to classify specific cell types in the atherosclerotic plaques; smooth muscle cells were stained using an α-smooth muscle actin antibody (ASMAFITC, 1:3000, Sigma F-3777); T-cells were labeled with anti-CD3 (1:200, A0452; DAKO, Glostrup, Denmark) and macrophages with Mac 3 (1:30; Pharmingen, San Jose, CA, USA). Factor VIII (1:2000; Eurodiagnostics, Malmö, Sweden) was used for the detection of endothelial cells. Quantification of all immunohistochemical stainings was performed by counting the respective positive stained cells per plaque divided by the total number of cells in the plaque. Results are presented as mean ± SEM. All plaque parameters as well as DNA adduct levels and plasma lipid levels were compared by a nonparametric Mann-Whitney U-test. P < 0.05 was considered statistically significant. Two independent observers who were blinded for the exposure treatment performed the analyses. Inter- and intra-observer variation was <10%. Treatment with B[a]P resulted in growth retardation of the mice at both 12 and 24 weeks (P < 0.01, n = 31, and P < 0.01, n = 19, respectively; Table 1). Macroscopic and microscopic evaluations of internal organs showed no tumor development or signs of inflammation in B[a]P-exposed and control mice (data not shown).Table 1Weight Gain, DNA Adducts, and Lipoprotein Levels12 weeks24 weeksControlB[a]PControlB[a]PWeight gain (g)(n = 15)(n = 16)(n = 9)(n = 10)11.3 ± 0.69.8 ± 0.6†P < 0.01 versus control group;13.9 ± 0.310.7 ± 0.4†P < 0.01 versus control group;DNA adducts (per 108 nucleotides)(n = 15)(n = 16)(n = 9)(n = 10) Aorta<0.01∥n = 11.34.6 ± 2.8‡P ≤ 0.05 versus control group;<0.01—*No arterial tissue available for DNA-adduct measurement. Lung<0.0115.8 ± 1.6‡P ≤ 0.05 versus control group;<0.0116.1 ± 1.0‡P ≤ 0.05 versus control group; Liver<0.01 5.2 ± 0.3‡P ≤ 0.05 versus control group;<0.015.5 ± 0.5Lipoprotein levels (mmol/L)(n = 9)(n = 9)(n = 9)(n = 8) Total cholesterol9.5 ± 0.7¶P ≤ 0.05 versus control 24 weeks;11.3 ± 0.6*No arterial tissue available for DNA-adduct measurement.‡P ≤ 0.05 versus control group;§P ≤ 0.05 versus B[a]P group 24 weeks;11.4 ± 0.59.5 ± 0.5†P < 0.01 versus control group;§P ≤ 0.05 versus B[a]P group 24 weeks; Total glycerol0.8 ± 0.04¶P ≤ 0.05 versus control 24 weeks;0.7 ± 0.06§P ≤ 0.05 versus B[a]P group 24 weeks;1.4 ± 0.11.5 ± 0.08 HDL cholesterol0.2 ± 0.060.2 ± 0.010.3 ± 0.030.1 ± 0.03 Free glycerol0.4 ± 0.020.3 ± 0.020.3 ± 0.060.2 ± 0.05 LDL cholesterol9.0 ± 0.7¶P ≤ 0.05 versus control 24 weeks;10.9 ± 0.6‡P ≤ 0.05 versus control group;§P ≤ 0.05 versus B[a]P group 24 weeks;10.6 ± 0.58.8 ± 0.5‡P ≤ 0.05 versus control group;Values are mean ± SEM.* No arterial tissue available for DNA-adduct measurement.† P < 0.01 versus control group;‡ P ≤ 0.05 versus control group;§ P ≤ 0.05 versus B[a]P group 24 weeks;¶ P ≤ 0.05 versus control 24 weeks;∥ n = 11. Open table in a new tab Values are mean ± SEM. B[a]P treatment did not affect total glycerol, high-density lipoprotein cholesterol, and free glycerol levels at both time points (Table 1). The influence of B[a]P on total cholesterol and low-density lipoprotein cholesterol was not consistent: total cholesterol and concomitant low-density lipoprotein cholesterol levels were raised after 12 weeks of B[a]P exposure (P < 0.05 versus control), but lowered after 24 weeks of B[a]P exposure (P < 0.05 versus controls). Total glycerol levels increased with age regardless of B[a]P treatment. In aorta, lung, and liver of the B[a]P-exposed animals the predominant adduct spot co-migrated with the BPDE-DNA-adduct standard (Figure 1). In lung and liver minor additional adduct spots were observed. DNA adduct quantification was performed based on the major adduct spot that co-migrated with the BPDE-DNA-adduct standards. Inclusion of the minor additional adduct spots did not change the results significantly. After 12 weeks of exposure, adduct levels were ranked aorta > lung > liver (Table 1). At 24 weeks, no DNA adducts were measured in the aorta because all aortic segments were used for histological purposes. However, DNA adduct levels were determined in lung and liver and were comparable with the adduct levels found after 12 weeks of exposure. In the pilot study in which wild-type C57BL6 mice were treated acutely with B[a]P, DNA adduct distribution was comparable to the 12 and 24 weeks study in ApoE-KO mouse. High adduct levels were found in aorta (16.0 ± 1.1 adducts/108 nucleotides) and lung (14.5 ± 0.8 adducts/108 nucleotides) and lower levels were found in the liver (6.0 ± 0.6 adducts/108 nucleotides). After 12 weeks, 24 lesions in the aortic arch of B[a]P mice (n = 5) and 14 lesions in controls (n = 4) were analyzed. After 24 weeks, analysis included 45 lesions in the aortic arch of B[a]P-treated mice (n = 10) and 41 lesions of the control group (n = 9). In both B[a]P-exposed and control animals lesions were mainly present in the inner curve of the aortic arch and its main branch points the brachiocephalic trunk, the left common carotid artery, and left subclavian artery (Figure 2, a and b). After 12 weeks, all mice had developed mild atherosclerosis with predominantly initial lesions, but after 24 weeks all animals exhibited severe atherosclerosis characterized by a high number of advanced lesions and large lesion areas. There were no differences in location and number of lesions per arch between groups at both time points (Figure 3a). Initial lesion area per arch was significantly smaller than the advanced lesion area within B[a]P and control animals (Figure 3b; P < 0.01). Advanced lesions occupied a significantly larger area per aortic arch in B[a]P animals than in controls at both end points (P < 0.05; Figure 3b).Figure 3Plaque characteristics of the apoE-KO mice after 12 and 24 weeks of B[a]P exposure. a: Number of lesions per aortic arch; b: mean plaque area; c: cell density of the plaque layers; d: number of lesions with a lipid core; e: T-lymphocyte content; f: macrophage content. *, P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because TA and AA are less sensitive to spontaneous plaque development, we used them to further investigate whether B[a]P treatment had any effect on plaque initiation. At 12 weeks, TA and AA showed no or few plaques and were not further used for analysis. At 24 weeks a total of 42 initial lesions in the TA of the B[a]P group and 35 initial lesions in the controls were analyzed. In the AA a total of 42 and 35 initial lesions were analyzed, respectively. Both TA and AA showed no significant differences between B[a]P and control animals in the mean number of initial lesions (4.2 ± 0.8 versus 4.8 ± 0.6 and 4.2 ± 0.8 versus 5.8 ± 0.7, respectively) or the mean initial lesion size (59,784 ± 16,711 μm2Waldman JM Lioy PJ Greenberg A Butler JP Analysis of human exposure to benzo(a)pyrene via inhalation and food ingestion in the Total Human Environmental Exposure Study (THEES).J Expo Anal Environ Epidemiol. 1991; 1: 193-225PubMed Google Scholar versus 124,895 ± 49,996 μm2Waldman JM Lioy PJ Greenberg A Butler JP Analysis of human exposure to benzo(a)pyrene via inhalation and food ingestion in the Total Human Environmental Exposure Study (THEES).J Expo Anal Environ Epidemiol. 1991; 1: 193-225PubMed Google Scholar and 122,368 ± 35,687 μm2Waldman JM Lioy PJ Greenberg A Butler JP Analysis of human exposure to benzo(a)pyrene via inhalation and food ingestion in the Total Human Environmental Exposure Study (THEES).J Expo Anal Environ Epidemiol. 1991; 1: 193-225PubMed Google Scholar versus 152,676 ± 48,039 μm2, respectively), indicating no effects of B[a]P treatment on initiation of lesions. Because B[a]P is a carcinogen capable of inducing and promoting tumor formation, we were interested in its effect on the p53 protein, a very important cell-cycle regulatory protein. Very low staining of p53 nuclear protein was observed in the normal vessel wall (< 2%). In the plaques, however, percentages of cells positive for the p53 nuclear protein staining were high (69.9 ± 3.1%), but these levels were not further induced by the 24 weeks of B[a]P treatment (68.2 ± 3.2%). Moreover, B[a]P exposure did not result in enhanced proliferation as measured by the number of PCNA-positive cells in both initial and advanced plaques after 24 weeks (1.9 ± 0.8% versus 3.9 ± 2.0% and 4.0 ± 0.7% versus 2.7 ± 0.6%). Finally, the number of terminal dUTP nick-end labeling positive cells in plaques of the B[a]P-exposed animals was not significantly raised after 24 weeks (1.25 ± 0.17% versus 0.74 ± 0.12%; P = 0.13). To investigate the effects of B[a]P exposure on plaque formation, we studied atherosclerotic plaque composition in more depth. After 24 weeks of exposure, advanced lesions in B[a]P-treated animals exhibited more often plaque layering than advanced lesions of the control group (Figure 2, e and f; 30 of 36 lesions versus 13 of 30 lesions, respectively). The cell density of these layers, which consisted mainly of macrophages, was significantly higher in the B[a]P-exposed animals (P < 0.05; Figure 3c). Furthermore, in the B[a]P-exposed group advanced lesions were more prone to lipid core development (P < 0.05; Figure 3d). Both lipid core area and layering area did not differ between B[a]P and control animals (lipid core, 20.0% versus 21.3%; layers, 19.3% versus 21.9%, respectively). Total cell density of initial and advanced lesions was not influenced by B[a]P exposure at 12 and 24 weeks (data not shown). T-lymphocyte content, measured as the number of CD3-positive cells, was higher in lesions of the B[a]P-treated animals at both time points compared to the control animals (Figure 2, c and d). Analysis of lesions in TA and AA showed no difference in T-lymphocyte content between plaques of exposed and control animals (3.0 ± 0.8% versus 2.1 ± 0.5% and 1.9 ± 0.5% versus 1.7 ± 0.5%; Figure 3e). Initial atherosclerotic lesions showed no differences in macrophage content, but a significant increase in macrophages was seen in advanced atherosclerotic lesions of the B[a]P groups at both 12 and 24 weeks (P < 0.05; Figure 3f). Levels of collagen in initial and advanced lesions were not changed on B[a]P treatment (12 weeks, 4.2 ± 2.8% versus 4.1 ± 2.1% and 29.4 ± 4.7% versus 34.5 ± 1.0%; 24 weeks, 13.3 ± 4.8% versus 16.3 ± 9.4% and 38.1 ± 2.2% versus 37.5 ± 2.3%, respectively). Finally, no differences were observed in α-smooth muscle actin-positive SMCs in initial and advanced lesions of B[a]P and control animals after 12 (2.1 ± 1.7% versus 2.3 ± 2.3% and 7.7 ± 2.2% versus 8.8 ± 0.1%) and 24 weeks (8.2 ± 3.9% versus 2.8 ± 1.6% and 7.4 ± 1.5% versus 6.0 ± 1.0%). The present study has focused on atherosclerotic plaque morphology and phenotype after chronic B[a]P exposure using genetic identically apoE-KO mice. First, we confirmed that B[a]P causes very high levels of DNA damage in the vessel wall. Because these levels were even higher than in lung, which is the normal target organ, this indicates that the vessel wall is extra vulnerable for this chemical. Furthermore, we showed that the observed effects of B[a]P on the atherosclerotic plaques could not be because of the induction of plasma lipids, because there was no consistent effect of B[a]P on these levels. In B[a]P-treated ApoE−/− mice larger advanced plaque development was observed. These plaques were more prone to lipid core development and high cell-densed plaque layering. Interestingly, B[a]P treatment did not result in the induction of p53 nuclear protein or proliferation, but in a higher plaque macrophage and T-lymphocyte content, whereas no signs of systemic inflammation were present in spleen or liver. This suggests a local, plaque-specific, inflammatory response. The effects of chemicals on atherogenesis were studied as early as the 1970s when Albert and colleagues20Albert RE Vanderlaan M Burns FJ Nishizumi M Effect of carcinogens on chicken atherosclerosis.Cancer Res. 1977; 37: 2232-2235PubMed Google Scholar demonstrated that chronic exposure to the PAH 7,12-dimethylbenz(a)anthracene (DMBA) resulted in large, focal, fibromuscular lesions in the abdominal aorta of chickens. They showed not only an increased induction, but also larger lesions in the carcinogen-treated animals as compared to the controls. Although later studies confirmed the dose-dependent increase of the plaque size, no further studies have described the induction of new plaques.21Bond JA Gown AM Yang HL Benditt EP Juchau MR Further investigations of the capacity of polynuclear aromatic hydrocarbons to elicit atherosclerotic lesions.J Toxicol Environ Health. 1981; 7: 327-335Cross

Incidence of atherosclerosis and atherosclerosis-related complications will increase significantly in the coming decennia. Research identified many serum and plasma markers that are associated with cardiovascular disease. However, little is known about the prognostic value of these markers to identify patients at risk for future cardiovascular events. Therefore, we aimed to investigate the prognostic value of three of these markers (soluble CD40 ligand (sCD40L), interleukin-6 (IL-6) and oxidized low-density lipoprotein (oxLDL)) with respect to coronary vascular disease and stroke. For this reason the Medline database was searched for the period January 1999-January 2005. To be selected in our study, concentration of the marker had to be determined at baseline, follow-up period had to be longer than 3 months and an estimate of relative risk had to be available. Based on these criteria, 4 studies for sCD40L, 10 for IL-6 and 2 for oxLDL were selected. Relative risk estimates adjusted for potential confounders varied between 1.9 and 2.8 for sCD40L, between 1.1 and 3.1 for IL-6 and between 1.9 and 3.2 for oxLDL. In conclusion, this systematic review shows that sCD40L, IL-6 and oxLDL are associated with an increased relative risk of developing cardiovascular disease.

Exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs) has been implicated in the aetiology of atherosclerosis. Previously we showed that chronic exposure of ApoE-/- mice to the prototype PAH benzo[a]pyrene (B[a]P) causes enhanced progression of atherosclerosis, which was characterised by an increased inflammatory cell content in the atherosclerotic plaques. The aim of the present study was to evaluate the effect of B[a]P on vascular expression of monocyte-chemoattractant protein 1 (MCP-1), which is a crucial molecule promoting the recruitment of monocytes into atherosclerotic lesions. We hypothesised that B[a]P-induced expression of MCP-1 is mediated through aryl hydrocarbon receptor (AhR) activation. Initially we performed in vivo studies showing that acute treatment with B[a]P induces MCP-1 gene expression in aortic tissue of ApoE-/- mice. These observations could be confirmed by in vitro studies with human endothelial cells (RF24 cell line and primary HUVEC), showing a dose- and time-dependent increase in MCP-1 expression upon exposure to B[a]P. This was paralleled by an induction of cytochrome P450 1A1 and 1B1, indicating Ah receptor activation. No increased gene expression (MCP-1, CYP1A1 and 1B1) was found upon incubation with the structural isomer benzo[e]pyrene, which is a weak AhR agonist. Moreover, B[a]P-induced MCP-1 gene and protein expression was inhibited by co-treatment with the AhR antagonist alpha-naphthoflavone. In addition to its effect on basal gene expression, we showed that B[a]P significantly enhanced TNFalpha-induced expression of MCP-1. We were unable to block B[a]P-induced MCP-1 expression by antioxidant treatment. In contrast, we found that addition of N-acetylcysteine or vitamin C enhanced transcription of MCP-1 by B[a]P. In conclusion, our studies revealed potent vascular pro-inflammatory effects of B[a]P, as evidenced by AhR-mediated induction of MCP-1. These observations further contribute to the concept that induction of inflammation is a crucial process in PAH-enhanced atherogenesis.

Knowledge about the in vivo role of endothelium in chronic human atherosclerosis has mostly been derived by insights from mouse models. Therefore, we set out to establish by microarray analyses the gene expression profiles of endothelium from human large arteries, as isolated by laser microbeam microdissection, having focal atherosclerosis of the early or the advanced stage. Within individual arteries, the endothelial transcriptomes of the lesional and unaffected sides were compared pairwise, thus limiting genetic and environmental confounders. Specific endothelial signature gene sets were identified with changed expression levels in either early (n = 718) or advanced atherosclerosis (n = 403), relative to their paired plaque-free controls. Gene set enrichment analysis identified distinct sets of chemokines and differential enrichments of nuclear factor-κB-, p53-, and transforming growth factor-β-related genes in advanced plaques. Immunohistochemistry validated the discriminative value of corresponding endothelial protein expression between early (fractalkine/CX3CL1, IP10/CCL10, TBX18) or advanced (BAX, NFKB2) stages of atherosclerosis and versus their plaque-free controls. The functional involvement of transforming growth factor-β signaling in directing its downstream gene repertoire was substantiated by a consistent detection of activated SMAD2 in advanced lesions. Thus, we identified truly common, local molecular denominators of pathological changes to vascular endothelium, with a marked distinction of endothelial phenotype between early and advanced plaques. Knowledge about the in vivo role of endothelium in chronic human atherosclerosis has mostly been derived by insights from mouse models. Therefore, we set out to establish by microarray analyses the gene expression profiles of endothelium from human large arteries, as isolated by laser microbeam microdissection, having focal atherosclerosis of the early or the advanced stage. Within individual arteries, the endothelial transcriptomes of the lesional and unaffected sides were compared pairwise, thus limiting genetic and environmental confounders. Specific endothelial signature gene sets were identified with changed expression levels in either early (n = 718) or advanced atherosclerosis (n = 403), relative to their paired plaque-free controls. Gene set enrichment analysis identified distinct sets of chemokines and differential enrichments of nuclear factor-κB-, p53-, and transforming growth factor-β-related genes in advanced plaques. Immunohistochemistry validated the discriminative value of corresponding endothelial protein expression between early (fractalkine/CX3CL1, IP10/CCL10, TBX18) or advanced (BAX, NFKB2) stages of atherosclerosis and versus their plaque-free controls. The functional involvement of transforming growth factor-β signaling in directing its downstream gene repertoire was substantiated by a consistent detection of activated SMAD2 in advanced lesions. Thus, we identified truly common, local molecular denominators of pathological changes to vascular endothelium, with a marked distinction of endothelial phenotype between early and advanced plaques. Inflammatory activation of vascular endothelium plays a central role in the development of atherosclerosis.1Hansson GK Inflammation, atherosclerosis, coronary artery disease.N Engl J Med. 2005; 352: 1685-1695Crossref PubMed Scopus (6910) Google Scholar This occurs via trafficking and retention of leukocytes into the vessel wall by an enhanced expression of chemokines and adhesion molecules, such as CCR2, E-selection, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1).2Charo IF Taubman MB Chemokines in the pathogenesis of vascular disease.Circ Res. 2004; 95: 858-866Crossref PubMed Scopus (643) Google Scholar, 3Muller WA Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response.Trends Immunol. 2003; 24: 327-334PubMed Google Scholar The inflammatory status of the endothelium can be enhanced by a diverse set of local and systemic stimuli, such as oxidized low-density lipoprotein (oxLDL), interleukin-1, tumor necrosis factor-α, and C-reactive protein.4Ross R Atherosclerosis—an inflammatory disease.N Engl J Med. 1999; 340: 115-126Crossref PubMed Scopus (19128) Google Scholar, 5Libby P Sukhova G Lee RT Galis ZS Cytokines regulate vascular functions related to stability of the atherosclerotic plaque.J Cardiovasc Pharmacol. 1995; 25: S9-S12Crossref PubMed Scopus (345) Google Scholar, 6Venugopal SK Devaraj S Jialal I Effect of C-reactive protein on vascular cells: evidence for a proinflammatory, proatherogenic role.Curr Opin Nephrol Hypertens. 2005; 14: 33-37Crossref PubMed Scopus (164) Google Scholar Nuclear factor (NF)-κB, which can be activated by several of these stimuli, including cytokines and oxidized lipids,7Matsunaga T Hokari S Koyama I Harada T Komoda T NF-kappa B activation in endothelial cells treated with oxidized high-density lipoprotein.Biochem Biophys Res Commun. 2003; 303: 313-319Crossref PubMed Scopus (74) Google Scholar, 8Yeh M Leitinger N de Martin R Onai N Matsushima K Vora DK Berliner JA Reddy ST Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.Arterioscler Thromb Vasc Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (99) Google Scholar has shown to be an important transcription factor responsible for modulating this enhanced proinflammatory status.9Brand K Page S Walli AK Neumeier D Baeuerle PA Role of nuclear factor-kappa B in atherogenesis.Exp Physiol. 1997; 82: 297-304PubMed Google Scholar In addition, hemodynamic forces have been shown to modulate endothelial inflammation and the development of atherosclerotic plaques.10Lehoux S Castier Y Tedgui A Molecular mechanisms of the vascular responses to haemodynamic forces.J Intern Med. 2006; 259: 381-392Crossref PubMed Scopus (353) Google Scholar, 11Cheng C Tempel D van Haperen R van der Baan A Grosveld F Daemen MJ Krams R de Crom R Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.Circulation. 2006; 113: 2744-2753Crossref PubMed Scopus (785) Google Scholar Throughout the past decade, inbred mouse models of accelerated atherosclerosis have also significantly contributed to the large body of pathophysiological knowledge on the development of atherosclerosis.12Lusis AJ Fogelman AM Fonarow GC Genetic basis of atherosclerosis: part I: new genes and pathways.Circulation. 2004; 110: 1868-1873Crossref PubMed Scopus (152) Google Scholar In addition to these mouse studies and single-gene approaches, a number of recent studies have assessed the role of transcriptome changes in the development of human atherosclerosis.13Bijnens AP Lutgens E Ayoubi T Kuiper J Horrevoets AJ Daemen MJ Genome-wide expression studies of atherosclerosis. Critical issues in methodology, analysis, interpretation of transcriptomics data.Arterioscler Thromb Vasc Biol. 2006; 26: 1226-1235Crossref PubMed Scopus (44) Google Scholar However, there is little direct knowledge about the in vivo role of the endothelium in the development of chronic atherosclerosis in humans. Previous human gene expression profiling studies, in which atherosclerotic tissues were compared with plaque-free tissues from different individuals, have been complicated by confounding effects of interindividual genetic variation, differences in vessel-specific transcriptomes, and differences in systemic parameters.13Bijnens AP Lutgens E Ayoubi T Kuiper J Horrevoets AJ Daemen MJ Genome-wide expression studies of atherosclerosis. Critical issues in methodology, analysis, interpretation of transcriptomics data.Arterioscler Thromb Vasc Biol. 2006; 26: 1226-1235Crossref PubMed Scopus (44) Google Scholar One of the distinctive properties of human atherosclerosis is, however, the focality of its lesions, probably related to differences in local hemodynamics.10Lehoux S Castier Y Tedgui A Molecular mechanisms of the vascular responses to haemodynamic forces.J Intern Med. 2006; 259: 381-392Crossref PubMed Scopus (353) Google Scholar, 11Cheng C Tempel D van Haperen R van der Baan A Grosveld F Daemen MJ Krams R de Crom R Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.Circulation. 2006; 113: 2744-2753Crossref PubMed Scopus (785) Google Scholar Hence, within a single artery, distinct regions with early or advanced plaques coexist with morphologically normal looking vascular wall, despite the fact that both individual genetic background as well as exposure to systemic, circulating risk factors such as high low-density lipoprotein (LDL) levels or proinflammatory cytokines are identical. Apparently, because this is observed in all major arteries affected by the disease, there must be common functional denominators that lie at the basis of pathological changes of the vessel wall, irrespective of genetic background, vascular origin, or systemic factors. We chose to study this by applying laser microbeam microdissection (LMM) for the isolation of the arterial endothelial cells. Since the introduction of LMM, it has become possible to obtain cell type-enriched isolates from vascular material.14Tuomisto TT Yla-Herttuala S What have we learnt about microarray analyses of atherogenesis?.Curr Opin Lipidol. 2005; 16: 201-205Crossref PubMed Scopus (14) Google Scholar In addition, T7 RNA polymerase-mediated mRNA amplification has enabled reliable microarray gene expression profiling from small quantities of input mRNA, both in vitro and ex vivo.15Van Gelder RN von Zastrow ME Yool A Dement WC Barchas JD Eberwine JH Amplified RNA synthesized from limited quantities of heterogeneous cDNA.Proc Natl Acad Sci USA. 1990; 87: 1663-1667Crossref PubMed Scopus (1032) Google Scholar, 16Polacek DC Passerini AG Shi C Francesco NM Manduchi E Grant GR Powell S Bischof H Winkler H Stoeckert Jr, CJ Davies PF Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA.Physiol Genomics. 2003; 13: 147-156PubMed Google Scholar, 17Passerini AG Polacek DC Shi C Francesco NM Manduchi E Grant GR Pritchard WF Powell S Chang GY Stoeckert Jr, CJ Davies PF Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta.Proc Natl Acad Sci USA. 2004; 101: 2482-2487Crossref PubMed Scopus (312) Google Scholar, 18Tuomisto TT Korkeela A Rutanen J Viita H Brasen JH Riekkinen MS Rissanen TT Karkola K Kiraly Z Kolble K Yla-Herttuala S Gene expression in macrophage-rich inflammatory cell infiltrates in human atherosclerotic lesions as studied by laser microdissection and DNA array: overexpression of HMG-CoA reductase, colony stimulating factor receptors, CD11A/CD18 integrins, interleukin receptors.Arterioscler Thromb Vasc Biol. 2003; 23: 2235-2240Crossref PubMed Scopus (55) Google Scholar, 19Trogan E Choudhury RP Dansky HM Rong JX Breslow JL Fisher EA Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice.Proc Natl Acad Sci USA. 2002; 99: 2234-2239Crossref PubMed Scopus (142) Google Scholar, 20Scheidl SJ Nilsson S Kalen M Hellstrom M Takemoto M Hakansson J Lindahl P mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.Am J Pathol. 2002; 160: 801-813Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar To identify only the genes that are potentially functionally involved in lesion formation, irrespective of arterial location and individual differences, we used a diverse set of human large arteries of which the types as well as the locations were randomly selected. To minimize the individual or local confounders, we envisioned a paired comparative approach thereby enabling us to study gene expression differences in a limited set of arteries. Thus, we compared within individual arterial sections with focal atherosclerosis, the endothelial microarray expression profiles of endothelium from plaque-free regions with endothelium overlying atherosclerotic plaques of either the early or the advanced stage. Strikingly, we observed that there was little overlap between the genes with changed microarray intensities in the early and advanced stages of atherosclerosis, as compared with the plaque-free controls. At the molecular pathway level, these plaque stage-specific differences in the microarray data, as assessed by gene set enrichment analysis (GSEA),21Subramanian A Tamayo P Mootha VK Mukherjee S Ebert BL Gillette MA Paulovich A Pomeroy SL Golub TR Lander ES Mesirov JP Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (25801) Google Scholar revealed important roles for transforming growth factor (TGF)-β-signaling in endothelium during advanced atherosclerosis. In addition, the present study confirmed the involvement of NF-κB and p53 signaling and of chemokines during plaque progression in human patients. Human large arteries were collected postmortem after disease in compliance with institutional guidelines, being an informed consent as approved by the medical ethical committees of the Academic Medical Center (Amsterdam, The Netherlands) or the Academic Hospital Maastricht (Maastricht, The Netherlands), in full compliance with the principles and ethical considerations of the Declaration of Helsinki (1989). Samples collected in the Department of Pathology were stored in the Maastricht Pathology Tissue Collection, and use of tissue and patient data was performed in agreement with the “Code for Proper Secondary Use of Human Tissue in The Netherlands” (). The atherosclerotic plaques were classified according to Virmani and colleagues.22Virmani R Kolodgie FD Burke AP Farb A Schwartz SM Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.Arterioscler Thromb Vasc Biol. 2000; 20: 1262-1275Crossref PubMed Scopus (3198) Google Scholar The main criterion for discriminating between early and advanced plaques was the presence of a fibrous cap. Human arteries were fixed in Zinkfix (24 hours at 4°C). Zinkfix was prepared as described by Scheidl and colleagues.20Scheidl SJ Nilsson S Kalen M Hellstrom M Takemoto M Hakansson J Lindahl P mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.Am J Pathol. 2002; 160: 801-813Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar For the isolation of endothelial cells, 10-μm-thick cross sections were cut by using a microtome (no. 340E; Microm International, Walldorf, Germany) and attached to UV-crosslinked PET membrane frame slides (no. 11505151; Leica, Wetzlar, Germany) by using 0.001% (w/v) poly-l-lysine (P8920; Sigma-Aldrich, St. Louis, MO). After deparaffinization (100% xylene, 1 minute; 100% ethanol, 1 minute) and subsequent air-drying for 2 hours at room temperature, LMM was applied for isolation of the endothelial monolayer from four serial cross sections (P.A.L.M. Systems, Bernried, Germany). CD31 immunopositivity was used as a marker for endothelial integrity. RNA was isolated after proteinase K digestion (2 hours at 45°C) by using a paraffin block kit (no. 1902; Ambion, Austin, TX), including the digestion with DNase I. The RNAs were then linearly amplified for two rounds (RiboAmp kit, kit0102; Arcturus, Mountain View, CA), followed by a third-round amplification (MessageAmp aRNA kit, no. 1750; Ambion), synthesizing anti-sense cRNAs with an average base length of 500 nucleotides (see Supplemental Figure 1 at ). Within these cRNAs the molar ratio of incorporated aminoallyl-rUTP (A5660; Sigma-Aldrich) to rUTP was 1:1. Cy3 or Cy5 monoreactive dyes (PA23001, PA25001; Amersham Biosciences, Piscataway, NJ) were coupled according to the manufacturer's instructions. Labeled cRNA was purified using the RNeasy purification kit (Qiagen GmbH, Hilden, Germany). Each endothelial cRNA sample (1.5 μg; Cy5-labeled) was hybridized in duplicate against a common reference cRNA sample (1.5 μg; Cy3-labeled) on glass-based microarrays representing 18,649 unique oligonucleotide sequences (Micro Array Department, University of Amsterdam, Amsterdam, The Netherlands; ). The common reference cRNA sample comprised equal quantities of the following three components 1) human umbilical cord endothelial cells, stimulated with tumor necrosis factor-α (50 μg/ml, 6 hours); 2) THP-1 cell line incubated for 24 hours with phorbyl myristate acetate (100 μg/ml), subsequently for 6 hours with lipopolysaccharide (1 μg/ml); and 3) a mix of whole-mount human aorta and iliac artery. The microarray intensity data were acquired and imported in the Rosetta Resolver database after Loess normalization (limma package; Bioconductor; ) and were statistically analyzed by a paired Cyber-T test, essentially as previously described,23Dekker RJ Boon RA Rondaij MG Kragt A Volger OL Elderkamp YW Meijers JC Voorberg J Pannekoek H Horrevoets AJ KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium.Blood. 2006; 107: 4354-4363Crossref PubMed Scopus (275) Google Scholar and analyzed at the functional group level by using GSEA, of which the methodology has been described.21Subramanian A Tamayo P Mootha VK Mukherjee S Ebert BL Gillette MA Paulovich A Pomeroy SL Golub TR Lander ES Mesirov JP Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (25801) Google Scholar After combining the duplicate microarray data per sample, a paired Cyber-T test was applied to the microarray intensity data, as previously described.23Dekker RJ Boon RA Rondaij MG Kragt A Volger OL Elderkamp YW Meijers JC Voorberg J Pannekoek H Horrevoets AJ KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium.Blood. 2006; 107: 4354-4363Crossref PubMed Scopus (275) Google Scholar Criteria for significant differences: Bayesian P value <0.05 and an average microarray intensity >20. The genome-wide microarray expression profiles were analyzed at the functional group level, by using GSEA, of which the methodology has been described.21Subramanian A Tamayo P Mootha VK Mukherjee S Ebert BL Gillette MA Paulovich A Pomeroy SL Golub TR Lander ES Mesirov JP Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (25801) Google Scholar The dataset that was used for GSEA comprised 8574 unique gene symbols. Filtering criteria for the microarray dataset gene set were the presence of a gene symbol and an average microarray intensity >20. The molecular signature database that was used for the GSEA was a modified version of the original set named c2.symbols.gmt (available on-line at ) that was extended with the most relevant KEGG pathways and consisted of 594 gene sets. Analysis settings were 100 permutations on phenotype; gene sets with more than 50 genes were included in the analysis. The gene sets named NF-κB-induced, p53-signaling, and TGF-β-induced consisted of 61, 77, and 89 genes, respectively, that were present in the microarray dataset. A separate set of human arteries from different individuals with focal atherosclerosis was used for immunohistochemical validation (nos. 11 to 20, Table 1). Individual cross sections were incubated with the listed antibodies (Table 2). All antibodies were diluted in Tris-buffered saline containing bovine serum albumin [1% (w/v); Sigma] and Tween 20 [0.01% (w/v)]. The incubations with the listed secondary biotin-conjugated antibodies were followed by amplification with a streptavidin-horseradish peroxidase complex (no. K0377; DAKO, Glostrup, Denmark) and a peroxidase-substrate staining (SK-4800, Nova Red kit; Vector Laboratories, Burlingame, CA).Table 1Human Donor Arteries Used in LMM and ImmunohistochemistryNo.TypeAtherosclerosisClassificationSexAgeCause of death1IPlaque-free, earlyIXFemale43SAH2CPlaque-free, earlyIXFemale70Pneumonia and AML3CPlaque-free, earlyIXFemale30OC4CEarly (GSEA)IXMale58Aortic rupture5CPlaque-free, early, advancedIT, IX, FCAMale85CH (trauma)6CPlaque-free, early, advancedPIT, FCPMale85Fatal MI, liver failure7APlaque-free, early, advancedIX, FCPMale43CH (trauma)8IPlaque-free, advancedFCA9CPlaque-free, advancedFCAFemale62VC10IPlaque-free, advancedFCAFemale51CH (trauma)11EIPlaque-free, earlyITMale30Acute trauma12CPlaque-free, earlyIXMale36Unknown13CPlaque-free, earlyIXFemale77Unknown14APlaque-free, earlyIXFemale41SAH15CIPlaque-free, earlyIXMale41Sepsis16CIPlaque-free, earlyIXMale55Fatal MI17CPlaque-free, advancedFCAMale60Unknown18CPlaque-free, advancedFCAMale70Unknown19CIPlaque-free, advancedFCAMale49Alcohol intoxication20ABPlaque-free, advancedFCPMale48Brain infarction21CPlaque-free, advancedFCPFemale62UnknownAML, myeloid leukemia; CH, cerebral hemorrhage; OC, ovarium carcinoma; MI, myocardial infarction; SAH, subarachnoidal hemorrhage; VC, vulva carcinoma; Virmani-classified22Virmani R Kolodgie FD Burke AP Farb A Schwartz SM Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.Arterioscler Thromb Vasc Biol. 2000; 20: 1262-1275Crossref PubMed Scopus (3198) Google Scholar lesions: early plaques: IT, intimal thickening; PIT, pathological intimal thickening; IX, intimal xanthoma; advanced plaques: FCA, fibrous cap atheroma; FCP, fibro-calcific plaques; arteries: A, abdominal aorta; AB, aortic bifurcation; C, carotid; CI, common iliac; EI, external iliac. Open table in a new tab Table 2Antibodies Used for ImmunohistochemistryAntibody reactivityHost speciesCodeCloneManufacturerIncubationCD31Mouse mAbM0823JC70ADakoCytomation Denmark A/SOvernight at 4°CSMC α-actinM08511A41 hour at room temperatureMonocyte/macrophageM063201HAM562 hours at room temperatureBAXMouse mAb18–02182D2Zymed, South San Francisco, CAOvernight at 4°CCD81Mouse mAbMCA18471D6Serotec, Oxford, UKCX3CL1Mouse mAbmAB365181513R&D Systems, Minneapolis, MNCYP1B1Rabbit pAbCYP1B11-AAlpha Diagnostics, San Antonio, TXICAM-1Mouse mAb18-0173My13ZymedIP10Goat pAbAF266R&D SystemsNF-κB2Rabbit pAbARP32043Aviva Systems Biology, San Diego, CAPhosphorylated-SMAD2*For details about the antibodies raised against the active phosphorylated forms of SMAD1 and SMAD2, respectively, see Persson et al.57Rabbit pAbPersson et alTBX18Rabbit pAbPAB-11145Orbigen, San Diego, CAMouse IgG-biotinGoat pAbE0433DakoCytomation Denmark A/S30 minutes at room temperatureRabbit IgG-biotinGoat pAbE0432DakoCytomation Denmark A/SGoat IgG-biotinDonkey pAbBAF109R&D SystemsmAb, monoclonal antibody; pAb, polyclonal antibody.* For details about the antibodies raised against the active phosphorylated forms of SMAD1 and SMAD2, respectively, see Persson et al.57Persson U Izumi H Souchelnytskyi S Itoh S Grimsby S Engstrom U Heldin CH Funa K ten Dijke P The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation.FEBS Lett. 1998; 434: 83-87Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar Open table in a new tab AML, myeloid leukemia; CH, cerebral hemorrhage; OC, ovarium carcinoma; MI, myocardial infarction; SAH, subarachnoidal hemorrhage; VC, vulva carcinoma; Virmani-classified22Virmani R Kolodgie FD Burke AP Farb A Schwartz SM Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.Arterioscler Thromb Vasc Biol. 2000; 20: 1262-1275Crossref PubMed Scopus (3198) Google Scholar lesions: early plaques: IT, intimal thickening; PIT, pathological intimal thickening; IX, intimal xanthoma; advanced plaques: FCA, fibrous cap atheroma; FCP, fibro-calcific plaques; arteries: A, abdominal aorta; AB, aortic bifurcation; C, carotid; CI, common iliac; EI, external iliac. mAb, monoclonal antibody; pAb, polyclonal antibody. The atherosclerotic plaques were classified according to Virmani and colleagues.22Virmani R Kolodgie FD Burke AP Farb A Schwartz SM Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions.Arterioscler Thromb Vasc Biol. 2000; 20: 1262-1275Crossref PubMed Scopus (3198) Google Scholar The main criterion for discriminating between early and advanced plaques was the presence of a fibrous cap. The transcriptome differences between the endothelium of plaque-free sections and sections having initial plaques or plaques of the advanced stage were probed by microarray gene expression profiling. LMM was applied for the isolation of arterial endothelium (Figure 1, A and B). To find the truly common local molecular denominators of pathological changes to the vascular wall, a pairwise comparison was made within individual arteries. A representative example of this approach in an individual human carotid artery with a lateral plaque of the advanced stage is shown (Figure 1C). Furthermore, within this artery the endothelial microarray intensity data of the sections without and with atherosclerosis can be directly compared, using Rosetta Resolver statistics as shown in the scatterplot (Figure 1D). Currently, it is technically impossible to isolate 100% pure endothelium by LMM from human arterial walls because, in contrast to mice, the intimal layers of human large arteries without histological signs of atherosclerosis often contain smooth muscle cells (SMCs). To verify whether the level of SMC contamination was comparable in our LMM endothelium samples, we checked these samples for whether there were differences between the microarray intensities of 13 SMC-specific genes, based on a publication of Nelander et al24Nelander S Mostad P Lindahl P Prediction of cell type-specific gene modules: identification and initial characterization of a core set of smooth muscle-specific genes.Genome Res. 2003; 13: 1838-1854PubMed Google Scholar(Figure 1, E–H). Paired statistical Cyber-T tests, comparing the plaque-free situation with either the early or the advanced stage of atherosclerosis, respectively, showed that there were no differences between the microarray intensities (see Supplemental Table 1 at ). The individual paired analyses for a diverse set of large human arteries (Table 1) were collectively analyzed by paired Cyber-T statistical analysis, corrected for false discovery rate by the Benjamini-Hochberg method.25Reiner A Yekutieli D Benjamini Y Identifying differentially expressed genes using false discovery rate controlling procedures.Bioinformatics. 2003; 19: 368-375Crossref PubMed Scopus (1327) Google Scholar A large set of reproducibly differential genes were detected at high statistical significance, which were commonly and reproducibly differential between lesional endothelium versus the endothelium overlying the apparently normal sites of the same arteries. Intriguingly, these signature sets revealed a clear distinction between the sets of genes showing differential microarray intensities in endothelium overlying plaques of the early or the advanced stage relative to their plaque-free controls, with a very limited overlap (Figure 2A). Heat maps show the reproducibility of these findings between the different indi-vidual arteries that constitute the two distinct groups (Figure 2, B and C). The corresponding lists of genes are given in Supplemental Table 2 (available at ). Manual inspection revealed that the absence in these sets of a number of established marker genes derived from murine models was caused by a lack of statistical significance mainly attributable to low-intensity levels on microarray, the relatively small sample size, or lack of consistency between all of the samples analyzed. Thus, increased levels of ICAM-1 and VCAM-1 and decreased levels of KLF2 were detected, although not at statistically significant levels. Still, we could confirm increases at the level of ICAM-1 protein expression, for example, in atherosclerotic plaques of the early and the advanced stage by paired semiquantitative immunohistochemical analysis (see below, Table 3). These data corroborate earlier findings that microarray mRNA expression profiling analyses are not suitable to detect all expected known genes, especially in the low-intensity range in which background noise can be an issue. Rather, microarray expression profiling is typically suited for detection of large numbers of novel (panels of) genes and of genome-wide signatures.Table 3Paired Evaluation of the Microarray Data by Immunohistochemistry in Endothelium from Large Human Arteries with Focal Atherosclerosis of the Early or the Advanced StageEarly versus plaque-freeAdvanced versus plaque-freeAntibodyPlaque-freeEarlyPlaque-freeAdvancedCX3CL10/65/60/53/5CYP1B11/63/62/52/5ICAM-10/65/60/54/5IP103/66/64/54/5TBX180/63/61/51/5BAX0/61/60/53/5NFKB20/61/60/55/5pSMAD20/61/60/55/5Immunohistochemistry was performed on cross sections from human arteries (nos. 11 to 21 in Table 1) with focal atherosclerosis of the early (n = 6) or the advanced stage (n = 5). Ratio scores: the number of artery sections with clusters of immunopositive endothelial cells over the total number of evaluated sections. A cluster is defined as more than three adjacent cells with immunopositivity (Figure 5, Figure 6). Open table in a new tab Immunohistochemistry was performed on cross sections from human arteries (nos. 11 to 21 in Table 1) with focal atheroscleros

Using molecular imaging techniques, we examined interstitial alterations during postmyocardial infarction (MI) remodeling and assessed the efficacy of antiangiotensin and antimineralocorticoid intervention, alone and in combination.The antagonists of the renin-angiotensin-aldosterone axis restrict myocardial fibrosis and cardiac remodeling after MI and contribute to improved survival. Radionuclide imaging with technetium-99m-labeled Cy5.5 RGD imaging peptide (CRIP) targets myofibroblasts and indirectly allows monitoring of the extent of collagen deposition post-MI.CRIP was intravenously administered for gamma imaging after 4 weeks of MI in 63 Swiss-Webster mice and in 6 unmanipulated mice. Of 63 animals, 50 were treated with captopril (C), losartan (L), spironolactone (S) alone, or in combination (CL, SC, SL, and SCL), 8 mice received no treatment. Echocardiography was performed for assessment of cardiac remodeling. Hearts were characterized histopathologically for the presence of myofibroblasts and thick and thin collagen fiber deposition.Acute MI size was similar in all groups. The quantitative CRIP percent injected dose per gram uptake was greatest in the infarct area of untreated control mice (2.30 +/- 0.14%) and decreased significantly in animals treated with 1 agent (C, L, or S; 1.71 +/- 0.35%; p = 0.0002). The addition of 2 (CL, SC, or SL 1.31 +/- 0.40%; p < 0.0001) or 3 agents (SCL; 1.16 +/- 0.26%; p < 0.0001) demonstrated further reduction in tracer uptake. The decrease in echocardiographic left ventricular function, strain and rotation parameters, as well as histologically verified deposition of thin collagen fibers, was significantly reduced in treatment groups and correlated with CRIP uptake.Radiolabeled CRIP allows for the evaluation of the efficacy of neurohumoral antagonists after MI and reconfirms superiority of combination therapy. If proven clinically, molecular imaging of the myocardial healing process may help plan an optimal treatment for patients susceptible to heart failure.

The growth arrest-specific gene 6 (Gas6) plays a role in pro-atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that Gas6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. Gas6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of Gas6 does not affect the number and size of initial and advanced plaques in ApoE(-/-) mice, but alters its plaque composition. Compared to Gas6(+/+): ApoE(-/-) mice, initial and advanced plaques of Gas6(-/-): ApoE(-/-) mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFbeta was increased. In addition, fewer macrophages were found in advanced plaques of Gas6(-/-): ApoE(-/-) mice. Hence, loss of Gas6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for Gas6 in plaque composition and stability.

Current developments in cardiovascular biology and imaging enable the noninvasive molecular evaluation of atherosclerotic vascular disease. Intraplaque neovascularization sprouting from the adventitial vasa vasorum has been identified as an independent predictor of intraplaque hemorrhage and plaque rupture. These intraplaque vasa vasorum result from angiogenesis, most likely under influence of hypoxic and inflammatory stimuli. Several molecular imaging techniques are currently available. Most experience has been obtained with molecular imaging using positron emission tomography and single photon emission computed tomography. Recently, the development of targeted contrast agents has allowed molecular imaging with magnetic resonance imaging, ultrasound and computed tomography. The present review discusses the use of these molecular imaging techniques to identify inflammation and intraplaque vasa vasorum to identify vulnerable atherosclerotic plaques at risk of rupture and thrombosis. The available literature on molecular imaging techniques and molecular targets associated with inflammation and angiogenesis is discussed, and the clinical applications of molecular cardiovascular imaging and the use of molecular techniques for local drug delivery are addressed.

TWEAK is a multifunctional cytokine belonging to the tumor necrosis factor superfamily and binds to the receptor Fn14. TWEAK and Fn14 are expressed in atherosclerotic plaques in areas rich in macrophages and foam cells. We investigated the role of TWEAK/Fn14 interactions in ApoE(-/-) mice and bone marrow-derived macrophages in vitro.ApoE(-/-) mice were treated with TWEAK-inhibiting fusion protein, Fn14-Fc, in an early (5 to 17 weeks of age) or delayed (17 to 29 weeks of age) setting. In the aortic arch, Fn14-Fc as compared to control treatment resulted in advanced plaques which were smaller (early treatment), fewer (delayed treatment), lower in fibrotic content (early and delayed treatment), and exhibited an increased macrophage content and smaller macrophage size (delayed treatment). There were no differences in apoptosis in atherosclerotic plaques after Fn14-Fc versus control Ab treatment. However, blocking TWEAK resulted in less macrophage uptake of modified lipids in vitro.Fn14-Fc fusion protein treatment did not prevent lesion initiation but inhibited some features of plaque progression and induced a unique advanced plaque phenotype with increased macrophage content and smaller macrophage size, which may be attributable to reduced lipid uptake. These findings indicate that TWEAK/Fn14 interactions regulate atherosclerosis and mediate lipid uptake in macrophages.

Objective: To investigate the potential of gadofosveset-enhanced MR imaging for the characterization of human carotid atherosclerotic plaques. Materials and Methods: Sixteen (9 symptomatic, 7 asymptomatic) patients with 70% to 99% carotid stenosis (according to NASCET criteria) were included (13 men, 3 women, mean age 67.6 years). All patients underwent baseline precontrast MR imaging of the carotid plaque. Immediately after completion of the baseline examination, 0.03 mmol/kg gadofosveset was administered. At 24 hours postinjection, the acquisition was repeated. Twelve patients were scheduled for carotid endarterectomy. Carotid endarterectomy specimens were HE-, CD31-, CD68-, and albumin-stained to correlate signal enhancement with plaque composition, intraplaque microvessel density, and macrophage and albumin content. A random intercept model was used to compare signal enhancement between symptomatic and asymptomatic patients, adjusting for size of various plaque components. This study was approved by the institutional medical ethics committee. All participants gave written informed consent. Results: Signal enhancement (SE) of the plaque was significantly higher in symptomatic patients compared with asymptomatic patients (median log SE 0.182 vs. −0.109, respectively, P < 0.001). A positive association (as expressed by a regression coefficient β = 0.0035) was found between signal enhancement on the log scale and intraplaque albumin content (P = 0.038). There was no association between signal enhancement and various other plaque components. Conclusion: In this study, the potential of gadofosveset-enhanced human carotid plaque MR imaging for identification of high-risk plaques was demonstrated. Signal enhancement of the plaque after administration of gadofosveset was associated with differences in intraplaque albumin content. Although promising, we emphasize that these results are based on a small patient population. Larger prospective studies are warranted.

Background and purpose We have previously shown that irradiation to the carotid arteries of hypercholesterolemic ApoE−/− mice accelerated the development of macrophage-rich, inflammatory atherosclerotic lesions. We now investigated the mechanism underlying the development of radiation-induced atherosclerosis. Materials and methods ApoE−/− and wildtype C57BL/6J mice received 0, 8 or 14 Gy to the neck and the carotid arteries were harvested 1 day, 1 or 4 weeks later. Immunohistochemical stainings were performed to evaluate well-known inflammatory and thrombotic molecules. A hypothesis-generating approach was used to compare gene expression profiles of irradiated and unirradiated carotid arteries. Results Basal levels of endothelial VCAM-1 and thrombomodulin immunoexpression were higher in ApoE−/− mice than in C57BL/6J mice. At 1 week after 14 Gy VCAM-1 immunoexpression was decreased in ApoE−/− mice, whereas ICAM-1 immunoexpression was decreased at 1 and 4 weeks after 14 Gy in C57BL/6J mice. Thrombomodulin and tissue factor immunoexpression were elevated at 4 weeks after 14 Gy in ApoE−/− mice and reduced in C57BL/6J mice. There were no changes in immunoexpression of eNOS, MCP-1 or endoglin. Several canonical pathways were differentially expressed after irradiation, including tight junction pathways, leukocyte extravasation signaling and PI3K/AKT signaling. Conclusion ApoE−/− and C57BL/6J mice respond differently to irradiation. The thrombotic pathways were activated after irradiation in ApoE−/− mice only. Genes involved in tight junction regulation were up-regulated in ApoE−/− mice and decreased in C57BL/6J mice. These factors may have contributed to fatty-streak formation in ApoE−/− mice.

Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE null mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, LXR/RXR pathway genes were significantly over-represented among the genes up-regulated in foam cell macrophages. Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-β action, was also over-represented. Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages. Increased expression of several of these genes was confirmed at the protein level in foam cell macrophages from subcutaneous granulomas and in atherosclerotic plaques. Moreover, phosphorylation and nuclear translocation of SMAD2, which is downstream of several transforming growth factor-β family members, was also detected in foam cell macrophages. We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.

Summary Atherosclerosis is a chronic inflammation of the arterial wall and the continuous infiltration of leukocytes into the plaque enhances the progression of the lesion. Because of the scarce detection of neutrophils in atherosclerotic plaques compared to other immune cells, their contribution was largely neglected. However, in the last years studies have accumulated pointing towards the contribution of neutrophils to atherogenesis. In addition, studies are emerging implying a role for neutrophils in advanced atherosclerosis and/or plaque destabilization. Thus, this brief review delivers an overview of the role of neutrophils during early and late stage atherosclerosis.

<h3>BACKGROUND AND PURPOSE:</h3> In recent years, several high-resolution vessel wall MR imaging techniques have emerged for the characterization of intracranial atherosclerotic vessel wall lesions in vivo. However, a thorough validation of MR imaging results of intracranial plaques with histopathology is still lacking. The aim of this study was to characterize atherosclerotic plaque components in a quantitative manner by obtaining the MR signal characteristics (<i>T1</i>, <i>T2</i>, <i>T2</i>*, and proton density) at 7T in ex vivo circle of Willis specimens and using histopathology for validation. <h3>MATERIALS AND METHODS:</h3> A multiparametric ultra-high-resolution quantitative MR imaging protocol was performed at 7T to identify the MR signal characteristics of different intracranial atherosclerotic plaque components, and using histopathology for validation. In total, 38 advanced plaques were matched between MR imaging and histology, and ROI analysis was performed on the identified tissue components. <h3>RESULTS:</h3> Mean <i>T1</i>, <i>T2</i>, and <i>T2</i>* relaxation times and proton density values were significantly different between different tissue components. The quantitative <i>T1</i> map showed the most differences among individual tissue components of intracranial plaques with significant differences in <i>T1</i> values between lipid accumulation (<i>T1</i> = 838 ± 167 ms), fibrous tissue (<i>T1</i> = 583 ± 161 ms), fibrous cap (<i>T1</i> = 481 ± 98 ms), calcifications (<i>T1</i> = 314 ± 39 ms), and the intracranial arterial vessel wall (<i>T1</i> = 436 ± 122 ms). <h3>CONCLUSIONS:</h3> Different tissue components of advanced intracranial plaques have distinguishable imaging characteristics with ultra-high-resolution quantitative MR imaging at 7T. Based on this study, the most promising method for distinguishing intracranial plaque components is T1-weighted imaging.

Purpose of review Neovascularization is a prominent feature in advanced human atherosclerotic plaques. This review surveys recent evidence for and remaining uncertainties regarding a role of neovascularization in atherosclerotic plaque progression. Specific emphasis is given to hypoxia, angiogenesis inhibition, and perivascular adipose tissue (PVAT). Recent findings Immunohistochemical and imaging studies showed a strong association between hypoxia, inflammation and neovascularization, and the progression of the atherosclerotic plaque both in humans and mice. Whereas in humans, a profound invasion of microvessels from the adventitia into the plaque occurs, neovascularization in mice is found mainly (peri)adventitially. Influencing neovascularization in mice affected plaque progression, possibly by improving vessel perfusion, but supportive clinical data are not available. Whereas plaque neovascularization contributes to monocyte/macrophage accumulation in the plaque, lymphangiogenesis may facilitate egress of cells and waste products. A specific role for PVAT and its secreted factors is anticipated and wait further clinical evaluation. Summary Hypoxia, inflammation, and plaque neovascularization are associated with plaque progression as underpinned by recent imaging data in humans. Recent studies provide new insights into modulation of adventitia-associated angiogenesis, PVAT, and plaque development in mice, but there is still a need for detailed information on modulating human plaque vascularization in patients.

The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis.The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells.Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.

Intracranial atherosclerosis is one of the main causes of ischemic stroke. However, the characteristics of intracranial arteries and atherosclerosis have rarely been studied. Therefore, we systematically investigated atherosclerotic changes in all arteries of the Circle of Willis (CoW).Sixty-seven CoWs obtained at autopsy from randomly chosen hospital patients (mean age, 67.3 ± 12.5 years), of which a total of 1220 segments were collected from 22 sites. Atherosclerotic plaques were classified according to the revised American Heart Association classification and were related to local vessel characteristics, such as the presence of an external and internal elastic lamina and the elastic fibre density of the media.181 out of the 1220 segments had advanced plaques (15%), which were mainly observed in large arteries such as the internal carotid, middle cerebral, basilar and vertebral artery. Only 11 out of 1220 segments (1%) showed complicated plaques (p < 0.001). Six of these were intraplaque hemorrhages (IPH) and observed only in patients who had cardiovascular-related events (p = 0.015). The frequency of characteristics such as the external elastic lamina and a high elastin fibre density in the media was most often associated with the vertebral artery. Only 3% (n = 33) of the CoW arteries contained calcification (p < 0.001), which were mostly observed in the vertebral artery (n = 13, 12%).Advanced atherosclerotic plaques in the CoW are relatively scarce and mainly located in the 4 large arteries, and mostly characterized by an early and stable phenotype, a low calcific burden, and a low frequency of IPH.