Manfred Boehm

The energy that sustains cancer cells is derived preferentially from glycolysis. This metabolic change, the Warburg effect, was one of the first alterations in cancer cells recognized as conferring a survival advantage. Here, we show that p53, one of the most frequently mutated genes in cancers, modulates the balance between the utilization of respiratory and glycolytic pathways. We identify Synthesis of Cytochrome c Oxidase 2 (SCO2) as the downstream mediator of this effect in mice and human cancer cell lines. SCO2 is critical for regulating the cytochrome c oxidase (COX) complex, the major site of oxygen utilization in the eukaryotic cell. Disruption of the SCO2 gene in human cancer cells with wild-type p53 recapitulated the metabolic switch toward glycolysis that is exhibited by p53-deficient cells. That SCO2 couples p53 to mitochondrial respiration provides a possible explanation for the Warburg effect and offers new clues as to how p53 might affect aging and metabolism.

The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation.

We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood.

Vascular calcifications (VCs) are actively regulated biological processes associated with crystallization of hydroxyapatite in the extracellular matrix and in cells of the media (VCm) or intima (VCi) of the arterial wall. Both patterns of VC often coincide and occur in patients with type II diabetes, chronic kidney disease, and other less frequent disorders; VCs are also typical in senile degeneration. In this article, we review the current state of knowledge about the pathology, molecular biology, and nosology of VCm, expand on potential mechanisms responsible for poor prognosis, and expose some of the directions for future research in this area.

The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2–4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide. Organoids derived from human stem cells recapitulate the structure and functions of human blood vessels, and can be used to model and identify regulators of diabetic vasculopathy.

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet's disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-κB signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB-mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB-dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.

Endothelial to mesenchymal transition (EndMT) plays a major role during development, and also contributes to several adult cardiovascular diseases. Importantly, mesenchymal cells including fibroblasts are prominent in atherosclerosis, with key functions including regulation of: inflammation, matrix and collagen production, and plaque structural integrity. However, little is known about the origins of atherosclerosis-associated fibroblasts. Here we show using endothelial-specific lineage-tracking that EndMT-derived fibroblast-like cells are common in atherosclerotic lesions, with EndMT-derived cells expressing a range of fibroblast-specific markers. In vitro modelling confirms that EndMT is driven by TGF-β signalling, oxidative stress and hypoxia; all hallmarks of atherosclerosis. 'Transitioning' cells are readily detected in human plaques co-expressing endothelial and fibroblast/mesenchymal proteins, indicative of EndMT. The extent of EndMT correlates with an unstable plaque phenotype, which appears driven by altered collagen-MMP production in EndMT-derived cells. We conclude that EndMT contributes to atherosclerotic patho-biology and is associated with complex plaques that may be related to clinical events.

Arterial calcifications are associated with increased cardiovascular risk, but the genetic basis of this association is unclear.We performed clinical, radiographic, and genetic studies in three families with symptomatic arterial calcifications. Single-nucleotide-polymorphism analysis, targeted gene sequencing, quantitative polymerase-chain-reaction assays, Western blotting, enzyme measurements, transduction rescue experiments, and in vitro calcification assays were performed.We identified nine persons with calcifications of the lower-extremity arteries and hand and foot joint capsules: all five siblings in one family, three siblings in another, and one patient in a third family. Serum calcium, phosphate, and vitamin D levels were normal. Affected members of Family 1 shared a single 22.4-Mb region of homozygosity on chromosome 6 and had a homozygous nonsense mutation (c.662C→A, p.S221X) in NT5E, encoding CD73, which converts AMP to adenosine. Affected members of Family 2 had a homozygous missense mutation (c.1073G→A, p.C358Y) in NT5E. The proband of Family 3 was a compound heterozygote for c.662C→A and c.1609dupA (p.V537fsX7). All mutations found in the three families result in nonfunctional CD73. Cultured fibroblasts from affected members of Family 1 showed markedly reduced expression of NT5E messenger RNA, CD73 protein, and enzyme activity, as well as increased alkaline phosphatase levels and accumulated calcium phosphate crystals. Genetic rescue experiments normalized the CD73 and alkaline phosphatase activity in patients' cells, and adenosine treatment reduced the levels of alkaline phosphatase and calcification.We identified mutations in NT5E in members of three families with symptomatic arterial and joint calcifications. This gene encodes CD73, which converts AMP to adenosine, supporting a role for this metabolic pathway in inhibiting ectopic tissue calcification. (Funded by the National Human Genome Research Institute and the National Heart, Lung, and Blood Institute of the National Institutes of Health.).

HomeCirculationVol. 125, No. 14Epithelial-to-Mesenchymal and Endothelial-to-Mesenchymal Transition Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBEpithelial-to-Mesenchymal and Endothelial-to-Mesenchymal TransitionFrom Cardiovascular Development to Disease Jason C. Kovacic, MD, PhD, Nadia Mercader, PhD, Miguel Torres, PhD, Manfred Boehm, MD and Valentin Fuster, MD, PhD Jason C. KovacicJason C. Kovacic From the Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (J.C.K., V.F.); the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain (N.M., M.T., V.F.); Center of Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda, MD (M.B.); Marie-Josée and Henry R. Kravis Cardiovascular Health Center, Mount Sinai School of Medicine, New York, NY (V.F.). Search for more papers by this author , Nadia MercaderNadia Mercader From the Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (J.C.K., V.F.); the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain (N.M., M.T., V.F.); Center of Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda, MD (M.B.); Marie-Josée and Henry R. Kravis Cardiovascular Health Center, Mount Sinai School of Medicine, New York, NY (V.F.). Search for more papers by this author , Miguel TorresMiguel Torres From the Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (J.C.K., V.F.); the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain (N.M., M.T., V.F.); Center of Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda, MD (M.B.); Marie-Josée and Henry R. Kravis Cardiovascular Health Center, Mount Sinai School of Medicine, New York, NY (V.F.). Search for more papers by this author , Manfred BoehmManfred Boehm From the Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (J.C.K., V.F.); the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain (N.M., M.T., V.F.); Center of Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda, MD (M.B.); Marie-Josée and Henry R. Kravis Cardiovascular Health Center, Mount Sinai School of Medicine, New York, NY (V.F.). Search for more papers by this author and Valentin FusterValentin Fuster From the Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY (J.C.K., V.F.); the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain (N.M., M.T., V.F.); Center of Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda, MD (M.B.); Marie-Josée and Henry R. Kravis Cardiovascular Health Center, Mount Sinai School of Medicine, New York, NY (V.F.). Search for more papers by this author Originally published10 Apr 2012https://doi.org/10.1161/CIRCULATIONAHA.111.040352Circulation. 2012;125:1795–1808IntroductionCellular switching from an epithelial-to-mesenchymal phenotype, and conversely from a mesenchymal-to-epithelial phenotype, are important biological programs that are operative from conception to death in mammalian organisms. Indeed, the capacity of cells to switch between these states has been fundamental to the generation of complex body patterns throughout evolution. Phenotypic switching from an epithelial to mesenchymal cell, termed epithelial-to-mesenchymal transition (EMT), was a paradigm that evolved from numerous observations on early embryonic development, the foundations of which date back to the 1920s and the pioneering work of Johannes Holtfreter on embryo formation and differentiation.1,2 By the late 1960s, seminal chick embryo studies by Elizabeth Hay3 led to the first formal description that epithelial cells can undergo a dramatic phenotypic transformation and give rise to embryonic mesoderm.4 Subsequent studies have revealed that this process is reversible (mesenchymal-to-epithelial transition [MET]), and gradually the term ‘transition” has come to replace ‘transformation.”Given that EMT/MET was initially identified and described by developmental biologists, it is perhaps not surprising that these processes are best understood during embryonic implantation and development. As explored in this review, it is now known that successive waves of cellular transition, from an epithelial to mesenchymal and then back to an epithelial state, are required for normal embryonic patterning and organ formation. In addition, numerous studies that span a broad spectrum of physiological and pathological conditions have expanded our knowledge of EMT/MET and now provide evidence for the important role played by these processes in various adult conditions including fibrosis, wound repair, inflammation, and malignancy. Indeed, our conceptual framework now also encompasses several variations and subcategories of cellular phenotypic switching, including endothelial-to-mesenchymal transition (EndMT).In this review, epithelial, endothelial, and mesenchymal phenotypic cellular switching will be explored in the cardiovascular system, spanning cardiovascular development through to adult end organ disease. Key areas of recent scientific progress will be examined, including recent developmental and pathological insights, which potentially may lead to novel therapeutic opportunities.EMT: A Key Role in Early DevelopmentWithin days of conception and during very early embryonic implantation, the process of EMT is already operative. Typically at the blastocyst stage, after initial adherence to the uterine lining (decidua), the outer trophoblast sends forward columns of epithelial cells to penetrate the uterine wall.5 At the leading edge of these embryonic cellular columns, epithelial trophoblast cells undergo EMT and invade the underlying maternal decidual interstitum and vessels. These invading embryonic cells ultimately go on to become mesenchymal placental giant cells, participating in the remodeling of the maternal uterine vasculature and securing a functional placental blood supply.5 This embryonic execution of the EMT program establishes cellular phenotypic switching as a key biological paradigm and, interestingly, sets an early precedent for the vascular involvement of this process.Soon after these events, EMT plays a pivotal role in germ layer specification (ectoderm, mesoderm, endoderm). Epithelial cells from the primitive epiblast layer migrate to the midline and undergo EMT to give rise to mesoderm and endoderm.6 This process is highly ordered in time and space and generates pools of primitive stem/progenitor cells at precise anatomic locations within the embryo, which constitute the primordia of the developing organs. For example, lateral plate mesoderm gives rise to the heart and hematopoietic cells, the paraxial mesoderm to the musculo-skeletal system, and the intermediate mesoderm to the urogenital tract. These events, occurring in tissues that have not previously undergone cellular phenotypic switching, are termed primary EMT (Figure 1).Download figureDownload PowerPointFigure 1. Organ formation occurs via EMT/MET. Embryonic cells from the epiblast layer (a single layer of epithelial cells) give rise to mesodermal cells via primary EMT. Definitive organ formation then ensues via secondary EMT and successive rounds of EMT/MET. Differing regions of the mesodermal layer give rise to differing organs/structures: The heart, circulatory system, and hematopoietic cells arise from lateral plate mesoderm, the urogenital system from intermediate mesoderm, the axial skeleton and skeletal muscle from paraxial mesoderm, and the primitive notochord (which becomes the nucleus pulposus of intervertebral discs in the adult human) from the axial (midline) mesoderm. This Figure depicts cardiac formation. EMT-MET is also involved with endoderm and ectoderm tissue/organ formation. EMT indicates epithelial-to-mesenchymal transition; MET, mesenchymal-to-epithelial transition; TGF-B, transforming growth factor β; BMPs, bone morphogenic proteins; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; and TB4, thymosin β4.Having established these primitive mesodermal populations, successive waves of EMT/MET then typically occur before final organ formation. In the case of the heart, this can involve recurrent waves of EMT/MET before the heart begins to assume a recognizable 4-chambered form (see section ‘Early Cardiac Formation”). For other organs, such as the kidney, successive waves of EMT/MET are required to ultimately give rise to epithelial structures such as nephrons and nephric ducts.7Genetic, Molecular, and Cellular Basis of EMT/METAt the core of EMT/MET, fundamental molecular and architectural rearrangements occur to bring about the dramatic cellular changes necessary to switch phenotypes. Underpinning this is a complex network of gene activation and repression programs that are required for the initiation, execution, and maintenance of EMT or MET (Figure 2).Download figureDownload PowerPointFigure 2. Key aspects of the molecular and cellular changes that occur with EMT. BM indicates basement membrane; MMPs, matrix metalloproteinases; EMT, epithelial-to-mesenchymal transition; αSMA, α–smooth muscle actin; DDR2, discoidin domain receptor 2; and FSP1, fibroblast-specific protein 1.At the cellular level, gross changes in polarity, morphology, functionality, and cell–cell interactions are requisite steps. Epithelial cells are arranged on a basement membrane, exhibiting apico-basal polarity and abundant expression of intercellular adhesion complexes such as E-Cadherin and integrins. In order to adopt a mesenchymal phenotype, these cells must lose cell adhesion by E-Cadherin downregulation and degradation.8,9 Other epithelial proteins such as zonula occludens, cytokeratin and desmoplakin must also be repressed.8–11 Transitioning cells then progressively lose polarity while eroding the basement membrane by matrix metalloproteinase production (matrix metalloproteinases 2, 3, and 9).12,13 Cytoskeletal changes mediated by Rho GTPases induce apical constriction and further structural rearrangements to permit passage through the degraded basement membrane, culminating in delamination from the epithelial layer.14 Completing the transition, cells activate the expression of additional mesenchymal genes and proteins, such as α–smooth muscle actin (αSMA), smooth muscle protein 22α, collagen I and III, vimentin, fibronectin, or fibroblast-specific protein 1 (FSP1; also known as S100A4).8,15–17Orchestrating these processes, the most widely described regulator of EMT/MET is the transforming growth factor β (TGFβ) superfamily of signaling molecules (TGFβ, Nodal, bone morphogenic proteins [BMPs], and growth and differentiation factors).18–21 Downstream of the receptors for the various TGFβ superfamily members, the Smad family of signal transducers is also of key importance, and in particular Smad2 and Smad3 appear to control EMT program activation.18 In turn, TGFβ stimulation with Smad activation leads to transcription of Snail 1 and 2 (the latter formerly known as Slug) and Twist, further triggering a cascade of signaling pathways that culminate in EMT.22 Snail 1 is considered a key organizer of EMT, with one of its pivotal functions being to downregulate E-Cadherin transcription.22,23 The downregulation of E-Cadherin and other tight junction components9 directly facilitates the loss of epithelial intercellular adhesions and cellular delamination from the epithelial layer. The TGFβ signaling network is a key mediator of early-development EMT, and although other non-EMT effects are also operative, the genetic mutation of any number of these TGFβ family members often results in a nonviable embryo with major tissue, cardiovascular, or other organ specification defects.24–28 Importantly, not all members of the TGFβ superfamily stimulate EMT, with BMP-7 antagonizing TGFβ signaling and inhibiting EndMT in cardiac (see section ‘EndMT Contributes to Cardiac Fibrosis in the Adult Heart”)19 and renal20 fibrosis models.Several other signaling pathways also modulate EMT and MET, such as Notch and Wnt. For example, the canonical Wnt signaling pathway is crucial for events in early embryonic development, with chick embryos deficient in Wnt unable to properly form the ectoderm, mesoderm, and endodermal layers.29 The Notch pathway is an evolutionarily conserved and complex signaling network that also plays an important role in embryonic development. Although not generally considered a master regulator of EMT, at least in certain scenarios it appears to act upstream of TGFβ signaling.30 As a whole, the cellular rearrangements and changes that occur with EMT are extensive, and it is estimated that EMT in human cells changes the expression of ≈4000 genes, representing ≈10% of the entire genome.31 Additional signaling pathways are discussed below as relevant to cardiovascular development and disease, and interested readers are referred to any of the several excellent reviews on the molecular basis of EMT.32–34Classification and Types of EMTEpithelial-to-mesenchymal transition is classified into 3 types depending on its biological (or pathological) role and the time window in which it occurs. The embryonic and developmental EMT programs described above are classified as type-1 EMT, being distinct in that they do not generally cause fibrosis or give rise to mesenchymal cells with an invasive phenotype. As shown in Figure 3, the remaining types (2 and 3) are operative after birth and are concerned with fibrosis and malignant cellular transformation respectively. Type-2 EMT is extensively described in the literature, and it appears that chronic inflammation may be the sovereign inciting injury that triggers this form of EMT and sets the stage for end organ disease. Although the hypothesis is not without controversy,35,36 evidence exists to support type-2 EMT in numerous adult conditions, including those affecting the kidney,17,20,37 liver,38 skin,39 intestines,40 lungs,41 eyes,42 and heart (the latter will be considered separately in the section ‘EndMT Contributes to Cardiac Fibrosis in a Adult Heart”).19 Type-3 EMT is an important step in malignant cell transformation. Of particular relevance with respect to carcinoma (epithelial cell tumors), EMT is proposed as a critical mechanism for the acquisition of malignant characteristics, with loss of E-Cadherin expression facilitating the delamination and metastasis of transformed epithelial cells.34,43Download figureDownload PowerPointFigure 3. Classification of EMT. Type-1 EMT is highly regulated and is associated with embryonic implantation and organ formation. Type-1 EMT may be subclassified into primary (cells giving rise to EMT for the first time), secondary, and tertiary, with these latter forms involved in the successive waves of EMT-MET leading to definitive organ formation. Type-2 EMT is associated with inflammation and fibrosis and is now increasingly recognized in adult pathological conditions. Type-3 EMT is involved with malignant cell transformation, including the acquisition of invasive metastatic cellular properties. As distinct from type-1, neither type-2 nor type-3 EMT adhere to any higher-order program of spatial or temporal restriction. EMT indicates epithelial-to-mesenchymal transition; MET, mesenchymal-to-epithelial transition.A further aspect of this classification system requiring clarification is the place of EndMT. The endothelium is a specialized form of squamous epithelial tissue, and as such, EndMT is a subcategory of EMT. Accordingly, EndMT may be observed in each of the 3 categories of EMT. For example, the endothelium gives rise to hematopoietic cells during embryonic development (type-1 EMT),44 and to both fibrosis and malignancy in the adult (types-2 and -3 EMT, respectively).19,45EMT During Cardiac Development: Valves, Cushions, Neural Crest, and Epicardium-Derived CellsEarly Cardiac FormationThe heart forms via a remarkable series of sequential waves of EMT/MET. As the definitive germ layers emerge in the developing embryo, cardiac progenitors are among the first epiblast cells to undergo EMT and to migrate out from the primitive streak.46,47 This population of mesenchymal cardiac precursors migrates bilaterally within the lateral plate toward the anterior pole of the embryo47–49 to coalesce in mammals into an anterior cardiac crescent.47 The formation of the celomic cavity divides the lateral plate to give rise to the somatic and splanchnic mesodermal layers. Within the splanchnic layer, primitive cardiac progenitor cells organize into a bilayered epithelium via MET.50 Next, either via another round of EMT/MET involving these mesodermal cardiac progenitor cells or from a separate cell population, the endocardial cells that will line the cardiac structures are formed.51–53 The primitive heart tube soon emerges by folding and remodeling of the cardiac crescent cells. Genetic and direct labeling of cardiac precursor cells at various stages of heart tube development has shown that the primary heart tube only contains the precursors of the left ventricle and that the remaining chambers are formed by the progressive infiltration and incorporation of new cardiac precursors into the outflow and inflow poles of the heart tube.54–56 The area of the cardiac crescent that gives rise to the initial heart tube is named the primary heart field whereas the area that remains behind in the pharyngeal region that is added later is called the secondary heart field. Primary and secondary heart field precursors occupy adjacent areas in the cardiac crescent but differ in the mechanism by which they are added to the heart tube (folding versus migration) and in the timing of addition. At the current time, the role of EMT in the migration and incorporation of second heart field precursors remains under investigation.EndMT Contributes to Valve Formation and Heart SeptationSoon after the primitive heart tube appears, endothelial cells from the region of the forming atrioventricular (A-V) canal and of the outflow tract (OFT) region undergo another round of EMT, or more specifically EndMT because the cells undergoing phenotypic switching are endothelial. At this time, the endocardium and the myocardium are separated by a thick acellular matrix termed the cardiac jelly. As the cardiac chambers start to form, the cardiac jelly gets thicker in the A-V canal and OFT regions, where endothelium-derived mesenchymal cells invade the adjacent cardiac jelly to form endocardial cushion tissue.57 The OFT cushions are the precursors of the semilunar valves whereas the A-V cushions give rise to the A-V septum, the membranous part of the ventricular septum and the mitral and tricuspid valves.58 The extent to which EndMT contributes to these 2 cushion areas differs significantly, and although most of the A-V cushion mesenchyme derives from EndMT,57 most of the OFT cushion derives from pharyngeal mesodermal cells. Subsequently, both cushions receive further specific mesenchymal cellular contributions involving EMT. The OFT region receives an important third mesenchymal population arising from the neural crest, which delaminates by EMT from the neural tube.59 This neural crest–derived population is essential for OFT septation into the aortic and pulmonary trunks; however, it represents a transient population that does not contribute significantly to the definitive heart structures. In the region of the A-V canal, a third mesenchymal population derives from EMT of the epicardium.60,61EMT and the EpicardiumIn parallel with endocardial EndMT and cushion formation, the outermost epicardial layer of the heart is also coming into existence.62 Like the primitive early cardiac progenitor cells, epicardial progenitor cells also arise from the splanchnic mesoderm (likely also via MET). Initially, the cells destined to form the epicardium assemble to create a transitory body of cells termed the proepicardial organ, consisting of an accumulation of pericardial progenitor cells lying adjacent to the sinus venosus (the venous pole of the heart; Figure 4A). These proepicardial cells migrate, or in some species float freely within the pericardial cavity, and attach to the myocardial surface.63–67 There, they proliferate and flatten to cover the embryonic heart as the epicardial sheet. Concomitantly, some epicardial cells undergo EMT and generate a mesenchymal population of epicardium-derived cells (EPDCs). Although a population of EPDCs remain to occupy the extracellular matrix–rich region between the epicardium and myocardium named the subepicardial space, some migrate further to invade the myocardium.Download figureDownload PowerPointFigure 4. Origin and fate of EPDCs. A, Messenger RNA in situ hybridization for tbx18 on zebrafish sagittal heart sections marking epicardial cells (blue staining). Additional immunostaining against myosin heavy chain was performed to identify myocardium (brown staining) and is shown in the lower panels (e–g). These panels show the progressive developmental sequence of epicardial formation in zebrafish, with vertically matched panels taken from the same developmental stage (a, 2 days postfertilization [dpf]; b and e, 3 dpf; c and f, 4 dpf; and d and g, 5 dpf). PE indicates proepicardium; Epi, epicardium; HT, heart tube; V, ventricle; and A, atrium. B, Epicardial EMT during zebrafish regeneration. a and c, Anti-GFP immunohistochemistry on adult zebrafish heart sections from the transgenic ET-27 line, expressing GFP constitutively in all epicardial cells (brown staining). b and d, In situ hybridization on sections of cryoinjured Tg(wt1b:GFP) zebrafish hearts, in which GFP is reactivated in the epicardium on damage (blue staining). Dorsal is to the top (a). The control heart reveals a single layer of GFP-positive cells on the ventricular surface. b, On cryoinjury, the epicardium thickens as a consequence of injury-induced EMT. c, Enlargement is 20× the scale of control heart. d, Enlargment is 20× the scale of injured heart. Images acquired at Centro Nacional de Investigaciones Cardiovasculares (Madrid, Spain) by the group of Nadia Mercader.The developmental cellular contributions of EPDCs are controversial and potentially species-dependent. An important contribution of EPDCs is made to the A-V canal cushion mesenchyme where EPDCs merge with endocardium-derived cells to form the mitral and tricuspid valves and cardiac septa.60,61,68 Interestingly, EPDCs do not colonize the OFT cushions, perhaps implying a specific role for these cells in the formation of the tricuspid and mitral but not the pulmonary and aortic valves. Epicardium-derived cells also play a role in coronary vascular formation. Studies in avian species, which included labeling the proepicardium with replication-deficient virus62,69 or the generation of quail-chick chimeras,60–62,70 indicated that EPDCs are the primary source of coronary endothelial cells, coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts. More recently, genetic fate mapping experiments have investigated the fate of EPDCs in the mouse. T-box transcription factor 18 (Tbx18) and Wilms tumor suppressor 1 (Wt1) are broadly expressed in the proepicardium and epicardium during development and represent appropriate markers to trace EPDCs.71–74 Using Cre-based technology, the fate of Tbx18+ and Wt1+ cells was analyzed,68,75 confirming that EPDCs differentiate into cVSMCs and cardiac fibroblasts. However, in mice, in contrast to the chick, no contribution to coronary endothelial cells was identified for Tbx18+-derived cells and only a minor contribution was found from Wt1+ cells. Very recently, Kikuchi et al have also demonstrated that in zebrafish, whereas EPDCs contribute to perivascular cells, they do not give rise to endothelial cell populations.76 Potentially, this discrepancy in the contribution of EPDCs to the endothelium in avian versus other species may be explained by species-specific differences or may reflect differing experimental approaches used to study EPDC fate. It is also possible that the initial observations performed in the chick were not correctly interpreted or that contamination by non-EPDC endothelial precursor cells migrating together with the proepicardium during grafting/labeling may have occurred.77,78Consistent with this, genetic evidence from the mouse supports the classic notion that the coronary endothelium derives from the sinus venosus.79 Using inducible vascular endothelial-cadherin-Cre mice to trace endothelial cell clones, it was determined that most coronary veins and arteries derive from sprouts arising at the sinus venosus, with a lesser proportion potentially arising from the endocardium. Importantly, no endothelial clones were linked to the proepicardium, suggesting that this structure does not contribute to endothelial progenitors, although it remains possible that epicardial cells may commit to the endothelial lineage at a later developmental stage, after colonization of the myocardial surface.Interestingly, analysis of Tbx18+ and Wt1+ epicardium-derived lineages also revealed an unexpected myocardial contribution. Thus, ≈4% of total cardiomyocytes were found to have arisen from Wt1+ cells, contributing mostly to the intraventricular septum (10%), atria (18%), and to a lesser extent the ventricular walls (7%).75 Similarly, the Tbx18+ lineage was found to contribute to cardiomyocytes in the ventricular septum and in scattered areas within the ventricular walls and atria.68 However, these findings remain under scrutiny because Tbx18 expression has been detected in maturing cardiomyocytes, potentially suggesting epicardium-independent Tbx18 activation in these cells.80 Furthermore, studies in zebrafish have also refuted the ability of EPDCs to give rise to cardiomyocytes.76Most recently, yet another contribution of EPDCs was revealed by Harvey and coworkers, who described a population of mesenchymal stem cell–like cells that occupy a perivascular adventitial niche in the adult mouse.81 Although the precise extent of their normal and pathological cellular contributions remains to be defined, these proepicardium/epicardium-derived stem cells exhibit transgerm layer potency in vitro and in vivo and appear distinct from previously described resident cardiac stem cell populations.In summary, it is clear that EMT-derived EPDCs support coronary artery development by supplying vascular pericytes and cVSMCs and that they make a major contribution to fibrous cardiac tissues/populations, including resident perivascular fibroblasts and the fibrous cardiac skeleton.60–62,68,69,75,77,78,82 Although the ability of EPDCs, other epicaridal cells, or both to give rise to cardiomyocytes and endothelium during development remains controversial, it is clear that the EMT and MET programs are of key importance during cardiac formation and for epicardial provisioning of specific cell populations.Signaling Pathways Governing EndMT/EMT During Cardiovascular DevelopmentSignaling via the TGFβ superfamily, as previously described in this review, is the major regulator of EMT during cardiac formation, including TGFβ2, TGFβ3, and BMP-2 and the downstream transcription factors Snail1 and Snail2.83 The precise role of these TGFβ isoforms differs between species, and at least in the chick TGFβ2 mediates EndMT via endothelial cell activation and separation whereas TGFβ3 mediates cell invasion into the extracellular matrix.84In addition to TGFβ, numerous other pathways modulate EMT during cardiac formation and development. Notch signaling is of particular significance during murine cardiac development, functioning to promote endocardial EndMT and with Notch deficient embryos displaying atrophic valve formation.85 Expanding our understanding of these pathways, Luna-Zurita et al86 recently showed that Notch1 is sufficient to activate a cell-autonomous promesenchymal gene-expression program in endocardial cells. Bone morphogenic protein 2 was found to drive endocardial EndMT and mesenchymal cell invasion into the cardiac jelly. Further, myocardial BMP-2 inactivation impaired Notch1 activity,86 suggesting a model in which the interplay between myocardial BMP-2 and endocardial Notch signaling restricts EndMT to prospective valve territory.86,87Endocardial EndMT is also dependent on receptor tyrosine kinase signaling via the phosphoinositide-3 kinase–phosphoinositide–dependent protein kinase 1–Akt/protein kinase B cascade, which is upstream of Snail. Genetic ablation of dependent protein kinase 1 in endothelial cells leads to embryonic lethality with abnormal vascular remodeling and a failure of endocardial cushion development because of defective EndMT.88 Gata4, an upstream regulator of an Erbb3-Erk pathway, is expressed in the endothelium and mesenchyme of the embryonic A-V valves and is also required for endocardial EndMT. Selective Gata4 inactivation in endothelium-derived cells results in a failure of EndMT and hypocellular endocardial cushions in animal models.89 This phenotype corresponds with that seen in humans, where heterozygous Gata4 mutation is associated with defects in the interatrial or interventricular septae.90,91 Endocardial expression of the protein tyrosine phosphatase SHP2, encoded by the gene PTPN11, also regulates EndMT and endocardial cushion formation. Gain of function PTPN11 mutations are responsible for a significant proportion of cases of Noonan syndrome and cause cardiac valve and septal defects by increasing Erk–mitogen-activated protein kinase activation, probably downstream of ErbB family–receptor tyrosine ki

Veins grafted into an arterial environment undergo a complex vascular remodeling process. Pathologic vascular remodeling often results in stenosed or occluded conduit grafts. Understanding this complex process is important for improving the outcome of patients with coronary and peripheral artery disease undergoing surgical revascularization. Using in vivo murine cell lineage-tracing models, we show that endothelial-derived cells contribute to neointimal formation through endothelial-to-mesenchymal transition (EndMT), which is dependent on early activation of the Smad2/3-Slug signaling pathway. Antagonism of transforming growth factor-β (TGF-β) signaling by TGF-β neutralizing antibody, short hairpin RNA-mediated Smad3 or Smad2 knockdown, Smad3 haploinsufficiency, or endothelial cell-specific Smad2 deletion resulted in decreased EndMT and less neointimal formation compared to controls. Histological examination of postmortem human vein graft tissue corroborated the changes observed in our mouse vein graft model, suggesting that EndMT is operative during human vein graft remodeling. These data establish that EndMT is an important mechanism underlying neointimal formation in interpositional vein grafts, and identifies the TGF-β-Smad2/3-Slug signaling pathway as a potential therapeutic target to prevent clinical vein graft stenosis.

Significance We describe a human disease linked to mutations in the linear deubiquitinase (DUB) OTULIN, which functions as a Met1-specific DUB to remove linear polyubiquitin chains that are assembled by the linear ubiquitin assembly complex (LUBAC). OTULIN has a role in regulating Wnt and innate immune signaling complexes. Hydrolysis of Met1-linked ubiquitin chains attenuates inflammatory signals in the NF-κB and ASC-mediated pathways. OTULIN-deficient patients have excessive linear ubiquitination of target proteins, such as NEMO, RIPK1, TNFR1, and ASC, leading to severe inflammation. Cytokine inhibitors have been efficient in suppressing constitutive inflammation in these patients. This study, together with the identification of haploinsufficiency of A20 (HA20), suggests a category of human inflammatory diseases, diseases of dysregulated ubiquitination.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

There has been considerable progress in engineering cardiac scaffolds for the treatment of myocardial infarction (MI). However, it is still challenging to replicate the structural specificity and variability of cardiac tissues using traditional bioengineering approaches. In this study, a four-dimensional (4D) cardiac patch with physiological adaptability has been printed by beam-scanning stereolithography. By combining a unique 4D self-morphing capacity with expandable microstructure, the specific design has been shown to improve both the biomechanical properties of the patches themselves and the dynamic integration of the patch with the beating heart. Our results demonstrate improved vascularization and cardiomyocyte maturation in vitro under physiologically relevant mechanical stimulation, as well as increased cell engraftment and vascular supply in a murine chronic MI model. This work not only potentially provides an effective treatment method for MI but also contributes a cutting-edge methodology to enhance the structural design of complex tissues for organ regeneration.

As an innovative additive manufacturing process, 4D printing can be utilized to generate predesigned, self-assembly structures which can actuate time-dependent, and dynamic shape-changes. Compared to other manufacturing techniques used for tissue engineering purposes, 4D printing has the advantage of being able to fabricate reprogrammable dynamic tissue constructs that can promote uniform cellular growth and distribution. For this study, a digital light processing (DLP)-based printing technique was developed to fabricate 4D near-infrared (NIR) light-sensitive cardiac constructs with highly aligned microstructure and adjustable curvature. As the curvature of the heart is varied across its surface, the 4D cardiac constructs can change their shape on-demand to mimic and recreate the curved topology of myocardial tissue for seamless integration. To mimic the aligned structure of the human myocardium and to achieve the 4D shape change, a NIR light-sensitive 4D ink material, consisting of a shape memory polymer and graphene, was created to fabricate microgroove arrays with different widths. The results of our study illustrate that our innovative NIR-responsive 4D constructs exhibit the capacity to actuate a dynamic and remotely controllable spatiotemporal transformation. Furthermore, the optimal microgroove width was discovered via culturing human induced pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells onto the constructs' surface and analyzing both their cellular morphology and alignment. The cell proliferation profiles and differentiation of tricultured human-induced pluripotent stem cell-derived cardiomyocytes, mesenchymal stem cells, and endothelial cells, on the printed constructs, were also studied using a Cell Counting Kit-8 and immunostaining. Our results demonstrate a uniform distribution of aligned cells and excellent myocardial maturation on our 4D curved cardiac constructs. This study not only provides an efficient method for manufacturing curved tissue architectures with uniform cell distributions, but also extends the potential applications of 4D printing for tissue regeneration.

Article1 July 2002free access A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression Manfred Boehm Manfred Boehm Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Takanobu Yoshimoto Takanobu Yoshimoto Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Martin F. Crook Martin F. Crook Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Shriram Nallamshetty Shriram Nallamshetty Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Andrea True Andrea True Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Gary J. Nabel Gary J. Nabel Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Elizabeth G. Nabel Corresponding Author Elizabeth G. Nabel Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Manfred Boehm Manfred Boehm Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Takanobu Yoshimoto Takanobu Yoshimoto Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Martin F. Crook Martin F. Crook Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Shriram Nallamshetty Shriram Nallamshetty Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Andrea True Andrea True Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Gary J. Nabel Gary J. Nabel Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Elizabeth G. Nabel Corresponding Author Elizabeth G. Nabel Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA Search for more papers by this author Author Information Manfred Boehm1, Takanobu Yoshimoto1, Martin F. Crook1, Shriram Nallamshetty1, Andrea True1, Gary J. Nabel2 and Elizabeth G. Nabel 1 1Cardiovascular Branch, NHLBI, Bethesda, MD, 20892 USA 2Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, MD, 20892 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3390-3401https://doi.org/10.1093/emboj/cdf343 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The cyclin-dependent kinase inhibitor, p27Kip1, which regulates cell cycle progression, is controlled by its subcellular localization and subsequent degradation. p27Kip1 is phosphorylated on serine 10 (S10) and threonine 187 (T187). Although the role of T187 and its phosphorylation by Cdks is well-known, the kinase that phosphorylates S10 and its effect on cell proliferation has not been defined. Here, we identify the kinase responsible for S10 phosphorylation as human kinase interacting stathmin (hKIS) and show that it regulates cell cycle progression. hKIS is a nuclear protein that binds the C-terminal domain of p27Kip1 and phosphorylates it on S10 in vitro and in vivo, promoting its nuclear export to the cytoplasm. hKIS is activated by mitogens during G0/G1, and expression of hKIS overcomes growth arrest induced by p27Kip1. Depletion of KIS using small interfering RNA (siRNA) inhibits S10 phosphorylation and enhances growth arrest. p27−/− cells treated with KIS siRNA grow and progress to S/G2similar to control treated cells, implicating p27Kip1 as the critical target for KIS. Through phosphorylation of p27Kip1 on S10, hKIS regulates cell cycle progression in response to mitogens. Introduction The protein p27Kip1 is an important regulator of the mammalian cell cycle (Sherr and Roberts, 1999). An increase in p27Kip1 causes proliferating cells to exit from the cell cycle, while a decrease in p27Kip1 is required for quiescent cells to resume cell division (Loda et al., 1997; Porter et al., 1997). Low levels of p27Kip1 are associated with excessive cell proliferation in pathological conditions such as inflammation and cancers (Fero et al., 1998; Ophascharoensuk et al., 1998). High levels of p27Kip1 are observed in conditions of diminished cell proliferation such as in the late stages of arterial wound repair in atherosclerosis (Tanner et al., 1998). p27Kip1 is regulated by transcriptional (Servant et al., 2000), translational (Agrawal et al., 1996; Hengst and Reed, 1996; Millard et al., 1997) and proteolytic mechanisms. A major mechanism in the regulation of p27Kip1 abundance is proteolysis by the ubiquitin–proteasome pathway (Pagano et al., 1995). Phosphorylation of p27Kip1 on threonine 187 (T187) by Cdk2 creates a binding site for a Skp2-containing E3 ubiquitin-protein ligase, SCF (Feldman et al., 1997; Skowyrs et al., 1997), and ubiquitylation of p27Kip1 by SCF results in degradation of p27Kip1 by the proteasome (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al, 1999). This pathway is operational in the S and G2 phases of the cell cycle, after Cdk2 is activated by cyclins E and A. A second proteolytic pathway for controlling p27Kip1 is activated by mitogens and degrades p27Kip1 during G0/G1 (Malek et al., 2001). Inactivation of p27Kip1 also occurs by sequestration into cyclin D–Cdk complexes (Sherr and Roberts, 1999). Serine 10 (S10) is another phosphorylation site on p27Kip1 (Ishida et al., 2000). Phosphorylation of S10 signals the nuclear export of p27Kip1 to the cytoplasm upon cell cycle re-entry (Rodier et al., 2001), and it is generally believed that the S10 phosphorylation pathway plays a role in p27Kip1 degradation. Despite these observations, the mechanisms regulating S10 phosphorylation are poorly understood. Specifically, the protein that phosphorylates p27Kip1 on S10 and its role in the regulation of cell cycle progression have not been defined. Here, we identify the serine-threonine kinase hKIS (human kinase interacting stathmin) as the major kinase that phosphorylates p27Kip1 on S10. We demonstrate that phosphorylation of p27Kip1 on S10 by hKIS is activated by mitogens in G0/G1 cells and that this modification of S10 facilitates nuclear export of p27Kip1 to the cytoplasm. In addition, we show that the physiological significance of S10 phosphorylation by hKIS is that it regulates cell cycle progression. Results Identification of a serine/threonine kinase, hKIS, which interacts with the C-terminal of p27Kip1 We hypothesized that the C-terminal of p27Kip1 would be important in p27Kip1 protein–protein interactions, and hence we employed a yeast two-hybrid screen using a human B-cell library (Durfee et al., 1993). The yeast two-hybrid screen yielded several cDNAs that interacted with the p27Kip1 C-terminal, as well as with full-length p27Kip1, but not the N-terminal region of p27Kip1, p57Kip2 or p21Cip1 (Figure 1A). One clone, KIS(C21), encoded a 49 kDa protein that is 98% homologous to a rat serine/threonine protein kinase, KIS (DDBJ/EMBL/GenBank accession No. X98374). We determined that this clone was the human homologue of rat KIS, whose function was unknown (Maucuer et al., 1995). hKIS has an N-terminal serine/threonine kinase consensus region and a C-terminal region with 42% sequence identity to hU2AF65, a 65 kDa subunit of the splicing factor U2AF (Valcarcel et al., 1993). hKIS binding was specific for C-terminal p27Kip1 because it interacted poorly in the two-hybrid assay with N-terminal p27Kip1, p57Kip2, p21Cip1 and several negative controls (Figure 1A and legend). Figure 1.Identification of hKIS and interaction with p27Kip1. (A) hKIS interacts with the QT domain of p27Kip1 in a yeast two-hybrid assay. hKIS was cotransfected into yeast with either p27Kip1, p27Kip1 [1–88 amino acids (aa)], p27Kip1 (144–198 aa), p57Kip2, p57Kip2 (1–89 aa), p57Kip2 (260–335 aa) or p21Cip1. β-galactosidase assay on selection plates was performed and the intensity of staining was categorized. CDK CS, cyclin-dependent kinase consensus sequence; NLS, nuclear localization signal; PCNA, proliferating cell nuclear antigen. +++, very strong; ++, strong; (+/−), weak; −, no staining. (B) Interaction between hKIS and CKIs. hKIS is coimmunoprecipated with p27Kip1, p16Ink4, p21Cip1 or p57Kip2. GST indicates pGEX-6P backbone control. Input is [35S]methionine-labelled hKIS control (10% of the total amount). Data are from an experiment that was repeated twice with similar results. Download figure Download PowerPoint Human KIS was mapped using radiation hybridization to human chromosome 1q23.1, closest to marker SHGCOOH-36663. Northern blot analysis of multiple adult human tissues revealed a single 9.4 kb band in all tissues using full-length hKIS cDNA as a probe. The highest levels of hKIS mRNA expression were observed in skeletal muscle, kidney, placenta and peripheral blood leukocytes (Supplementary figure 1, available at The EMBO Journal Online). hKIS interacts with p27Kip1 To determine whether hKIS interacts directly with p27Kip1 and other CKIs, glutathione S-transferase (GST) fusion proteins were incubated with labelled human hKIS generated by in vitro translation. hKIS directly bound GST–p27Kip1 at levels significantly higher than p21Cip1 or p57Kip2 fusion proteins (Figure 1B, lane 3 compared with lanes 5 and 6), and binding of hKIS to p16Ink4 was barely detectable (Figure 1B), suggesting specificity of hKIS binding to the Cip/Kip CKIs, predominantly p27Kip1. The kinase activity of hKIS was examined by incubation of in vitro translated and immunoprecipitated hKIS or a kinase-inactive hKIS mutant, K54A, with p27Kip1. hKIS readily phosphorylated p27Kip1, in contrast to the kinase inactive mutant K54A or a negative control (Figure 2A, lane 4 compared with lanes 2 and 3). hKIS and the K54A mutant were expressed at equivalent levels (data not shown). Under the same experimental conditions, hKIS did not phosphorylate p16Ink4, p21Cip1 or p57Kip2 (data not shown), documenting the specificity of p27Kip1 phosphorylation by hKIS. In addition, hKIS underwent autophosphorylation (Figure 2B, lanes 3, 5 and 7), as previously described for the rat homologue (Maucuer et al., 1997). Figure 2.Phosphorylation of CKIs by hKIS. (A) hKIS phosphorylates p27Kip1. In vitro-transcribed and -translated hKIS was immunoprecipitated with Anti-Xpress antibody and incubated with GST- purified p27Kip1. (Lane 1) [35S]methionine-labelled hKIS control; (lane 2) immunoprecipitated pcDNA 3.1 Anti-Xpress tag backbone incubated with p27Kip1 as a negative control; (lane 3) immunoprecipitated hKIS(K54A) mutant incubated with p27Kip1; (lane 4) immunoprecipitated hKIS incubated with p27Kip1. (B) hKIS phosphorylates p27Kip1 at the N-terminal domain. N-terminal (codons 1–88) or C-terminal (codons 144–198) fragments of p27Kip1 were purified as GST fusion proteins, and a kinase assay with hKIS was performed (upper panel). An equal amount of the GST fusion protein (40 pmol) was used in the kinase assay (upper panel), as stained by Coomassie Brilliant Blue (lower panel). (C) hKIS phosphorylates p27Kip1 on S10. Serine 10 was substituted with alanine in a GST fusion protein p27Kip1(S10A). The kinase assay was performed with hKIS (+, lanes 2 and 4) or a kinase-dead mutant, hKIS(K54A) (−, lanes 1 and 3). Download figure Download PowerPoint hKIS phosphorylates p27Kip1 on S10 To determine the hKIS phosphorylation site on p27Kip1, we generated additional GST fusion proteins and tested hKIS phosphorylation. We found that while hKIS bound C-terminal p27Kip1, hKIS phosphorylated N-terminal p27Kip1 and not C-terminal p27Kip1 (Figure 2B, lane 7 compared with lane 5). Since hKIS did not phosphorylate C-terminal p27Kip1, S178 and T187 were excluded as hKIS phosphorylation sites. To determine whether S10 was the putative phosphorylation site, mutational analyses of the N-terminal region were performed. Mutation of S10 to alanine [GST–p27(S10A)] abolished phosphorylation of GST–p27Kip1 (Figure 2C, lane 4 compared with lane 2), indicating that hKIS phosphorylated p27Kip1 on S10. Next, we determined the two-dimensional (2D) phosphopeptide map of expressed p27Kip1, p27(S10A) or p27(T187A) mutants following kinase assays with purified recombinant hKIS. Several radioactive spots were reproducibly detected, two of which (spots 1 and 2) appeared common to all maps. Two intensely labelled peptides (double arrows), however, were detected only in the maps of wild-type p27Kip1 and the T187A mutant, but not in the map of the S10A mutant (Figure 3A), suggesting that these phosphopeptides contain S10 and confirming that hKIS phosphorylated p27Kip1 on S10. The observation that the phosphopeptide containing S10 yielded two spots is probably attributable to treatment during sample preparation. Figure 3.hKIS phosphorylates p27Kip1 on S10. (A) Two-dimensional tryptic phosphopeptide mapping of wild-type and mutant p27Kip1. p27Kip1, p27(S10A) or p27(T187A) were expressed in HEK 293 cells and metabolically labelled with [32P]orthophosphate in the presence of 50 μM lactacystin. The recombinant proteins were immunoprecipitated with p27Kip1 C19 antibody and subjected to 2D tryptic phosphopeptide mapping. Major phosphopeptides are numbered 1 and 2. Phosphopeptides containing S10 are indicated by arrows. An asterisk indicates the origin of migration, and arrows show the directions of separation by thin layer chromatography (TLC) and electrophoresis. (B) Mouse polyclonal antibodies recognize phosphorylated p27Kip1 on S10. A western blot of recombinant p27Kip1 phosphorylated by hKIS was performed with a p27S10-p antibody absorbed with non- phosphorylated recombinant p27Kip1 protein (Control, lane 2) or S10-p p27Kip1 peptide (lane 1). Phosphorylated p27Kip1 was incubated with (+) or without (−) CIAP, and a western blot with p27S10-p antibody was performed (lanes 3 and 4). The same blot was stripped and reprobed with a monoclonal p27Kip1 K25020 antibody (lanes 5 and 6). Recombinant p27Kip1 was phosphorylated by hKIS in the presence of [γ-32P]ATP incubated with (+, lane 8) or without CIAP (−, lane 7). HEK 293 cell lysates were analysed on SDS–PAGE and a western blot was performed using the p27S10-p antibody (upper panel, lanes 9 and 10). The same blot was stripped and reprobed with a p27Kip1 C19 antibody (lower panel, lanes 9 and 10). (C) hKIS phosphorylates p27Kip1 on S10. Recombinant p27Kip1 protein was incubated with immunoprecipitated, in vitro-transcribed and -translated hKIS (+, lane 2), and a western blot with p27S10-p antibody was performed. The same blot was stripped and reprobed with a p27Kip1 K25020 antibody (lanes 1 and 2, lower panel). Download figure Download PowerPoint It is possible that mutation of S to A might induce a change in the structure of p27Kip1, which in turn may be responsible for the observed decrease in phosphorylation of the p27(S10A) mutant by hKIS. To determine whether hKIS phosphorylates p27Kip1 on S10 directly, we prepared an antibody to a p27 S10 phosphopeptide (p27S10-p). The specificity of this antibody was analysed by western blot analysis and absorption tests. p27S10-p antibody was absorbed with the S10-p peptide (Figure 3B, lane 1), but not by the control protein, producing the expected 27 kDa band (Figure 3B, lane 2). Recombinant phosphorylated GST–p27Kip1 protein was treated with calf intestinal alkaline phosphatase (CIAP) and probed with the p27S10-p antibody. Treatment with CIAP resulted in the disappearance of phosphorylated p27Kip1 (Figure 3B, lane 4 compared with lane 3), and dephosphorylation of 32P-labelled p27Kip1 by CIAP was observed (Figure 3B, lane 8 compared with lane 7). To ensure that equal amounts of protein were used, the western blot was stripped and reprobed with a monoclonal p27Kip1 antibody (Figure 3B, lanes 5 and 6). This specificity was confirmed further using 32P-labelled p27Kip1 (Figure 3B, lane 8 compared with lane 7). Specificity of the p27S10-p antibody was also confirmed in human embryonic kidney (HEK) 293 cells transfected with wild-type p27Kip1 or p27(S10A) (Figure 3B, lanes 9 and 10). hKIS phosphorylation of S10 was further confirmed with an in vitro kinase assay, incubating recombinant p27Kip1 with or without hKIS. The addition of hKIS to the kinase assay resulted in S10 phosphorylation, detected by p27S10-p antibodies, while phosphorylated S10 was not detected in the absence of hKIS (Figure 3C, lane 2 compared with lane 1, upper panel), despite equal amounts of p27Kip1 protein on the western blot (Figure 3C, lower panel). These findings provide additional evidence that hKIS phosphorylates p27Kip1 on S10. hKIS phosphorylates p27Kip1 in vivo We next examined whether hKIS phosphorylates p27Kip1 in vivo. HEK 293 cells transiently expressing haemagglutinin (HA)-tagged p27Kip1, p27(S10A), hKIS, or the hKIS kinase inactive mutant K54A were metabolically labelled with [32P]orthophosphate and lysed. Recombinant p27Kip1 protein was immunoprecipitated, subjected to SDS–PAGE and analysed by autoradiography. To ensure equivalent hKIS and p27Kip1 protein levels, a western blot analysis was performed with hKIS and HA-tagged p27Kip1 antibodies. hKIS phosphorylated p27Kip1 (Figure 4, lane 3), but the hKIS kinase inactive mutant K54 did not (lane 5). Mutation of S10 prevented phosphorylation of p27Kip1 by hKIS (Figure 4, lane 4). These data indicate that S10 is required for hKIS phosphorylation of p27Kip1 in vivo. Figure 4.hKIS phosphorylates p27Kip1 in vivo. HEK 293 cells transiently expressing HA-tagged wild-type p27Kip1, hKIS, an hKIS(K54A) mutant or a p27(S10A) mutant were metabolically labelled with [32P]orthophosphate. HA-tagged p27Kip1 and p27(S10A) proteins were immunoprecipitated with an HA antibody, subjected to SDS–PAGE and analysed by autoradiography. (Lane 1) a control immunoprecipitation using IgG; (lane 2) wild-type p27Kip1; (lane 3) expression of wild-type p27Kip1 and hKIS; (lane 4) expression of p27Kip1(S10A) and hKIS; (lane 5) expression of wild-type p27Kip1 and hKIS(K54A). A decrease in p27Kip1 protein might be expected following expression with hKIS (lane 3), but was not observed as the ratio of hKIS to p27Kip1 was 3:1. Download figure Download PowerPoint Expression of endogenous hKIS To investigate the expression and function of endogenous hKIS, polyclonal antibodies were raised in rabbit (hKIS 291 antibodies), and a monoclonal antibody (hKIS 3H2 antibody) was derived from mice. The specificity of both hKIS antibodies was confirmed by absorption using GST fusion proteins followed by western blot analysis of hKIS. The expected 49 kDa band was detected by western blotting with the hKIS 291 antibodies and 3H2 antibody in NIH 3T3 cells or primary smooth muscle cells (data not shown). Similar reactivity was observed in HEK 293 cells (Figure 5A, lanes 1 and 3, and B, lane 2). This band was not observed when the antibody was pre-absorbed with GST–hKIS (Figure 5A, lane 2, and B, lane 1). Figure 5.hKIS is a growth factor-dependent kinase and localizes to the nucleus. (A) Specificity of polyclonal hKIS 291 antibodies. The antibodies were absorbed with GST fusion proteins, and immunoblotting was performed using NIH 3T3 lysates. (Lane 1) GST alone; (lane 2) hKIS blocked with GST–hKIS protein; (lane 3) an irrelevant GST fusion protein, GST–C19. (B) A mouse monoclonal 3H2 antibody recognizes hKIS. An immunoblot was performed using NIH 3T3 lysates after incubation of the mouse hKIS antibody with GST fusion proteins. (Lane 1) hKIS blocked with GST–hKIS protein; (lane 2) GST alone. (C) hKIS localizes to the nucleus. NIH 3T3 cells were serum starved for 36 h (0.1% FBS, upper panel), and then cells were serum stimulated for 6 h in 10% FBS (lower panel). Immunofluorescence and confocal microscopy were performed using hKIS 291 antibodies (left panel), a p27Kip1 antibody K25020 (second panel from left) or both antibodies (third panel from left). A nuclear stain, DAPI, is shown on the far right panel. Download figure Download PowerPoint We determined the subcellular localization of endogenous hKIS by immunofluorescence and confocal microscopy. In asynchronously growing NIH 3T3 cells, endogenous hKIS was detected mainly in the nucleus (Figure 5C, left lower panel). During serum starvation, nuclear hKIS expression was reduced (Figure 5C, left upper panel). In contrast, endogenous p27Kip1 was expressed in the nucleus during serum starvation and shifted to the cytoplasm during serum stimulation (Figure 5C, p27, upper panel compared with lower panel). hKIS and p27Kip1 colocalize in the nucleus (Figure 5C, hKIS + p27). These findings demonstrate that endogenous hKIS is a nuclear protein and colocalizes with p27Kip1 in the nucleus during serum starvation. Furthermore, the data suggests that hKIS expression in vivo is regulated by serum growth factors. Endogenous hKIS interacts with p27Kip1 in vivo To investigate the interaction between endogenous p27Kip1 and hKIS, we examined mouse skin fibroblasts from p27Kip1 wild-type (p27+/+) and null (p27−/−) mice. hKIS coimmunoprecipitated with p27Kip1 in p27+/+ cells (Figure 6, lane 3) but not in p27−/− cells (Figure 6, lane 2). The absence of p27Kip1 in null cells was confirmed by western blot [Figure 6, lane 4 compared with lane 5, p27Kip1 (p27) arrow], and comparable levels of KIS protein were present in p27−/− and p27+/+ cells [Figure 6, lanes 4 and 5, hKIS (KIS) arrow]. In addition, p27Kip1 was detected in immunoprecipitates using hKIS 291 antibodies but not with control IgG (Figure 6, lane 7 compared with lane 6). These data demonstrate that endogenous hKIS interacts with endogenous p27Kip1 in vivo. Figure 6.hKIS kinase activity is growth factor-dependent and regulates p27Kip1 in vivo. hKIS interacts with endogenous p27Kip1. Fibroblasts from p27Kip1 wild-type (p27+/+) and null (p27−/−) mice were incubated with AMP-PNP, p27Kip1 was immunoprecipitated using p27Kip1 C19 antibody, and a western blot was performed (lanes 2 and 3). hKIS and p27Kip1 protein levels in p27+/+ and p27−/− cells were determined using a western blot (lanes 4 and 5). To demonstrate a reciprocal p27Kip1 and hKIS interaction, hKIS was immunoprecipitated with hKIS 291 antibodies, and a western blot was performed with p27Kip1 K25020 antibody (lanes 6 and 7). Download figure Download PowerPoint hKIS phosphorylation of p27Kip1 on S10 in vivo is growth factor-dependent To examine whether endogenous p27Kip1 is phosphorylated by hKIS following serum stimulation, NIH 3T3 cells were serum starved for 36 h, followed by serum stimulation for 0–8 h. In these cells, hKIS kinase activity was present at a low level in G0 cells, increased with serum stimulation, and was accompanied by an increase in hKIS protein levels (Figure 7A and B). In additional experiments, NIH 3T3 cells were serum starved for 36 h, followed by serum stimulation for 6 h in 10% fetal bovine serum (FBS). hKIS was expressed at ∼5-fold higher levels during serum stimulation compared with serum starvation (data not shown). Figure 7.hKIS kinase activity is associated with S10 phosphorylation of endogenous p27Kip1. (A) KIS kinase activity increases following serum induction. NIH 3T3 cells were serum starved for 36 h, followed by serum stimulation for 0–8 h. hKIS protein was immunoprecipitated with hKIS 291 antibodies, and a kinase assay was performed with recombinant p27Kip1 as a substrate (lanes 1–4, upper panel). hKIS protein levels were visualized by western blot analysis (lanes 5–7, lower panel). (B) Quantification of phosphorylated p27Kip1 in NIH 3T3 cells treated with lactacystin. The relative intensity of phosphorylated recombinant p27Kip1 by hKIS was determined at the indicated time points by densitometry (left). Cells were lysed at the indicated time points. Data are from an experiment that was repeated twice with similar results. (C) S10 is a major phosphorylation site following serum stimulation. Two-dimensional tryptic phosphopeptide mapping was performed on NIH 3T3 cells serum starved for 36 h, followed by serum stimulation for 0–8 h. Phosphopeptides containing S10 are indicated by arrows. An asterisk indicates the origin of migration, and arrows show the directions of separation by TLC and electrophoresis. Download figure Download PowerPoint Next, we performed 2D phosphopeptide mapping of endogenous p27Kip1 following serum stimulation for 0–8 h. Following serum starvation, no radioactive labelled peptides were detected (Figure 7C, t0). Following serum stimulation, two labelled peptides were weakly detected, and the intensity of these spots increased by 8 h (Figure 7C, arrows). The two intensely labelled peptides had a migration pattern that corresponded to the S10-containing phosphopeptides in Figure 3A. The findings suggested that S10 is a major phosphorylation site of endogenous p27Kip1 and that serum stimulation increases hKIS kinase activity and p27Kip1 S10 phosphorylation. Phosphorylation of S10 by hKIS stabilizes p27Kip1 in G1 We examined the effect of hKIS phosphorylation on S10 on p27Kip1 stability during G1 in a pulse–chase analysis. Samples were taken at 0–8 h, and p27Kip1 immunoprecipitation was analysed. The expression of hKIS in cells stabilized p27Kip1, compared with its absence (Figure 8A), with half-lives of 7.6 and 5.0 h, respectively (Figure 8B). The half-life of p27(S10A) was 4.2 h, in contrast to the phosphomimetic p27(S10D) mutant, which stabilized p27Kip1 and prolonged its half-life to >8 h (Figure 8B). Inhibition of S10 phosphorylation by overexpression of the kinase-inactive mutant hKIS(K54A) decreased slightly the stability of endogenous p27, similar to the S10A mutant (data not shown). Our data are consistent with previous reports (Ishida et al., 2000; Rodier et al., 2001), and demonstrate that KIS phosphorylation of S10 stabilizes p27Kip1 protein. Figure 8.hKIS stabilizes p27Kip1 in G1 cells. (A) NIH 3T3 cells were serum starved for 24 h and transfected with wild-type p27Kip1, p27(S10A), p27(S10D), or p27Kip1 and hKIS. The cells were pulse- labelled for 2 h with [35S]methionine, and chased for the indicated time points in media containing 20% FBS. Cell lysates were immunoprecipitated with p27Kip1 C19 antibodies, and the labelled p27Kip1 protein was analysed by autoradiography. (B) Densitometric analysis of p27Kip1 degradation rate. The intensity of the bands in (A) is expressed as a percentage of the time point t0. Data are from an experiment that was repeated twice with similar results. Download figure Download PowerPoint hKIS phosphorylation on S10 causes nuclear export of p27Kip1 We reasoned that hKIS regulates the transport of p27Kip1 and that the subcellular localization of p27Kip1 controls its stability (Tomoda et al., 1999; Rodier et al., 2001). We examined the subcellular distribution of endogenous p27Kip1 during cell cycle progression in NIH 3T3 cells expressing hKIS. In quiescent cells that expressed hKIS, p27Kip1 was located in both the nucleus and the cytoplasm (Figure 9A and B). Serum stimulation caused a time-dependent redistribution of p27Kip1 to the cytoplasm, with ∼80% of cells showing cytoplasmic staining after 8 h. Treatment of cells with an inhibitor of nuclear export, leptomycin B (Nishi et al., 1994), prevented the cytoplasmic redistribution of p27Kip1 after 8 h. Kinetic studies revealed that the cytoplasmic localization of p27Kip1 occurred prior to activation of Cdk2 and most p27Kip1 degradation (Figure 9C). These findings were confirmed in additional experiments by expressing p27-HA in NIH 3T3 cells and visualizing p27 localization with an HA antibody using immunofluorescence (see Supplementary figure 2) as shown previously (Rodier et al., 2001). Taken together, these data demonstrate that hKIS phosphorylation on S10 leads to nuclear export of p27Kip1 to the cytoplasm. Figure 9.hKIS phosphorylation on S10 causes nuclear export of p27Kip1. (A) Subcellular localization of endogenous p27Kip1 in NIH 3T3 cells transfected with hKIS. Cells were serum starved for 24 h, then serum stimulated for 8 h in the absence (−) or presence (+) of leptomycin B (LMB) 2 ng/ml, stained with p27Kip1 or hKIS antibodies, and examined by confocal microscopy. In serum-starved cells not expressing KIS, endogenous p27Kip1 is nuclear. Serum stimulation leads to redistribution to the cytoplasm. In contrast, in serum-starved cells expressing KIS, endogenous p27Kip1 is nuclear and cytoplasmic. After serum stimulation, endogenous 27Kip1 is cytoplasmic in these cells. The arrows indicate endogenous p27Kip1 in cells expressing KIS (upper panel) and transfected KIS (middle panel). A nuclear DAPI stain is shown in the lower panel. (B) Quantitative analysis of the cellular localization of endogenous p27Kip1 in cells transfected with hKIS. Results are expressed as the percentage of cells demonstrating both cytoplasmic and nuclear staining. Data are expressed as means ± SEM of three experiments. (C) Nuclear export precedes Cdk2 activation. Cells were serum starved for 24 h and stimulated with serum for the indicated times. p27Kip1 expression was examined by immunoblotting with p27Kip1 antibodies. Cdk2 activity was assayed using histone H1 as substrate. Download figure Download PowerPoint hKIS promotes cell cycle progression To determine whether hKIS phosphorylation of p27Kip1 abolishes growth arrest, we measured the cell cycle distribution of HEK 293 cells expressing hKIS, p27Kip1, the kinase inactive mutant hKIS(K54A), the S10A mutant p27Kip1(S10A), the

The cyclin-dependent kinase inhibitors (CKIs) have different patterns of expression in vascular diseases. The Kip/Cip CKIs, p27(Kip1) and p21(Cip1), are upregulated during arterial repair and negatively regulate the growth of vascular smooth muscle cells (VSMCs). In contrast, the Ink CKI, p16(Ink4), is not expressed in vascular lesions. We hypothesized that a variation in the inactivation of cdk2 and cdk4 during the G(1) phase of the cell cycle by p27(Kip1), p21(Cip1), and p16(Ink4) leads to different effects on VSMC growth in vitro and in vivo.The expression of p27(Kip1) and p21(Cip1) in serum-stimulated VSMCs inactivated cdk2 and cdk4, leading to G(1) growth arrest. p16(Ink4) inhibited cdk4, but not cdk2, kinase activity, producing partial inhibition of VSMC growth in vitro. In an in vivo model of vascular injury, overexpression of p27(Kip1) reduced intimal VSMC proliferation by 52% (P<0.01) and the intima/media area ratio by 51% (P<0.005) after vascular injury and gene transfer to pig arteries, when compared with control arteries. p16(Ink4) was a weak inhibitor of intimal VSMC proliferation in injured arteries (P=NS), and it did not significantly reduce intima/media area ratios (P=NS), which is consistent with its minor effects on VSMC growth in vitro.p27(Kip1) and p21(Cip1) are potent inhibitors of VSMC growth compared with p16(Ink4) because of their different molecular mechanisms of cyclin-dependent kinase inhibition in the G(1) phase of the cell cycle. These findings have important implications for our understanding of the pathophysiology of vascular proliferative diseases and for the development of molecular therapies.

Relatively little is known regarding the role of mitochondrial metabolism in stem cell biology. Here we demonstrate that mouse embryonic stem cells sorted for low and high resting mitochondrial membrane potential (DeltaPsi(m)L and DeltaPsi(m)H) are indistinguishable morphologically and by the expression of pluripotency markers, whereas markedly differing in metabolic rates. Interestingly, DeltaPsi(m)L cells are highly efficient at in vitro mesodermal differentiation yet fail to efficiently form teratomas in vivo, whereas DeltaPsi(m)H cells behave in the opposite fashion. We further demonstrate that DeltaPsi(m) reflects the degree of overall mammalian target of rapamycin (mTOR) activation and that the mTOR inhibitor rapamycin reduces metabolic rate, augments differentiation, and inhibits tumor formation of the mouse embryonic stem cells with a high metabolic rate. Taken together, our results suggest a coupling between intrinsic metabolic parameters and stem cell fate that might form a basis for novel enrichment strategies and therapeutic options.

Heme oxygenase (HO)-1 (encoded by Hmox1) catalyzes the oxidative degradation of heme to biliverdin and carbon monoxide. HO-1 is induced during inflammation and oxidative stress to protect tissues from oxidative damage. Because intravascular thrombosis forms at sites of tissue inflammation, we hypothesized that HO-1 protects against arterial thrombosis during oxidant stress. To investigate the direct function of HO-1 on thrombosis, we used photochemical-induced vascular injury in Hmox1-/- and Hmox1+/+ mice. Hmox1-/- mice developed accelerated, occlusive arterial thrombus compared with Hmox1+/+ mice, and we detected several mechanisms accounting for this antithrombotic effect. First, endothelial cells in Hmox1-/- arteries were more susceptible to apoptosis and denudation, leading to platelet-rich microthrombi in the subendothelium. Second, tissue factor, von Willebrand Factor, and reactive oxygen species were significantly elevated in Hmox1-/- mice, consistent with endothelial cell damage and loss. Third, following transplantation of Hmox1-/- donor bone marrow into Hmox1+/+ recipients and subsequent vascular injury, we observed rapid arterial thrombosis compared with Hmox1+/+ mice receiving Hmox1+/+ bone marrow. Fourth, inhaled carbon monoxide and biliverdin administration rescued the prothrombotic phenotype in Hmox1-/- mice. Fifth, using a transcriptional analysis of arterial tissue, we found that HO-1 determined a transcriptional response to injury, with specific effects on cell cycle regulation, coagulation, thrombosis, and redox homeostasis. These data provide direct genetic evidence for a protective role of HO-1 against thrombosis and reactive oxygen species during vascular damage. Induction of HO-1 may be beneficial in the prevention of thrombosis associated with vascular oxidant stress and inflammation.

Hypoxia-triggered neovascularization occurs in many types of disease. Endothelial cells must be able to cope with hypoxic stress, which in other cell types can induce a DNA repair response and inhibit replication. Matina Economopoulou et al. now show that hypoxia induces the generation of a hallmark of the DNA repair response, phosphorylated histone H2AX, in proliferating endothelial cells and that H2AX function is required for neovascularization under hypoxic or ischemic conditions in vivo pages 491–493 .. H2A histone family member X (H2AX, encoded by H2AFX) and its C-terminal phosphorylation (γ-H2AX) participates in the DNA damage response and mediates DNA repair1,2,3,4,5,6. Hypoxia is a physiological stress that induces a replication-associated DNA damage response7. Moreover, hypoxia is the major driving force for neovascularization8, as the hypoxia-mediated induction of vascular growth factors triggers endothelial cell proliferation8. Here we studied the role of the hypoxia-induced DNA damage response in endothelial cell function and in hypoxia-driven neovascularization in vivo. Hypoxia induced replication-associated generation of γ-H2AX in endothelial cells in vitro and in mice. Both in cultured cells and in mice, endothelial cell proliferation under hypoxic conditions was reduced by H2AX deficiency. Whereas developmental angiogenesis was not affected in H2afx−/− mice, hypoxia-induced neovascularization during pathologic proliferative retinopathy, in response to hind limb ischemia or during tumor angiogenesis was substantially lower in H2afx−/− mice. Moreover, endothelial-specific H2afx deletion resulted in reduced hypoxia-driven retina neovascularization and tumor neovascularization. Our findings establish that H2AX, and hence activation of the DNA repair response, is needed for endothelial cells to maintain their proliferation under hypoxic conditions and is crucial for hypoxia-driven neovascularization.

Type I interferon (IFN-α/β or IFN-I) signals through two receptor subunits, IFNAR1 and IFNAR2, to orchestrate sterile and infectious immunity. Cellular pathways that regulate IFNAR1 are often targeted by viruses to suppress the antiviral effects of IFN-I. Here we report that encephalitic flaviviruses, including tick-borne encephalitis virus and West Nile virus, antagonize IFN-I signaling by inhibiting IFNAR1 surface expression. Loss of IFNAR1 was associated with binding of the viral IFN-I antagonist, NS5, to prolidase (PEPD), a cellular dipeptidase implicated in primary immune deficiencies in humans. Prolidase was required for IFNAR1 maturation and accumulation, activation of IFNβ-stimulated gene induction, and IFN-I-dependent viral control. Human fibroblasts derived from patients with genetic prolidase deficiency exhibited decreased IFNAR1 surface expression and reduced IFNβ-stimulated signaling. Thus, by understanding flavivirus IFN-I antagonism, prolidase is revealed as a central regulator of IFN-I responses.

Mice genetically deficient in endothelial nitric oxide synthase (eNOS(-/-)) are hypertensive with lower circulating nitrite levels, indicating the importance of constitutively produced nitric oxide (NO•) to blood pressure regulation and vascular homeostasis. Although the current paradigm holds that this bioactivity derives specifically from the expression of eNOS in endothelium, circulating blood cells also express eNOS protein. A functional red cell eNOS that modulates vascular NO• signaling has been proposed.To test the hypothesis that blood cells contribute to mammalian blood pressure regulation via eNOS-dependent NO• generation, we cross-transplanted wild-type and eNOS(-/-) mice, producing chimeras competent or deficient for eNOS expression in circulating blood cells. Surprisingly, we observed a significant contribution of both endothelial and circulating blood cell eNOS to blood pressure and systemic nitrite levels, the latter being a major component of the circulating NO• reservoir. These effects were abolished by the NOS inhibitor L-NG-nitroarginine methyl ester and repristinated by the NOS substrate L-arginine and were independent of platelet or leukocyte depletion. Mouse erythrocytes were also found to carry an eNOS protein and convert (14)C-arginine into (14)C-citrulline in NOS-dependent fashion.These are the first studies to definitively establish a role for a blood-borne eNOS, using cross-transplant chimera models, that contributes to the regulation of blood pressure and nitrite homeostasis. This work provides evidence suggesting that erythrocyte eNOS may mediate this effect.

Arterial Calcification due to Deficiency of CD73 (ACDC) results from mutations in the NT5E gene encoding the 5′ exonucleotidase, CD73. We now describe the third familial case of ACDC, including radiological and histopathological details of the arterial calcifications. The medial lesions involve the entire circumference of the elastic lamina, in contrast to the intimal plaque-like disease of atherosclerosis. The demonstration of broken and fragmented elastic fibers leading to generalized vascular calcification suggests an analogy to pseudoxanthoma elasticum (PXE), which exhibits similar histopathology. Classical PXE is caused by deficiency of ABCC6, a C type ABC transporter whose ligand is unknown. Other C type ABC proteins transport nucleotides, so the newly described role of adenosine in inhibiting vascular calcification, along with the similarity of ACDC and PXE with respect to vascular pathology, suggests that adenosine may be the ligand for ABCC6.

Several studies have demonstrated the expression of odorant receptors (OR) in various human tissues and their involvement in different physiological and pathophysiological processes. However, the functional role of ORs in the human heart is still unclear. Here, we firstly report the functional characterization of an OR in the human heart. Initial next-generation sequencing analysis revealed the OR expression pattern in the adult and fetal human heart and identified the fatty acid-sensing OR51E1 as the most highly expressed OR in both cardiac development stages. An extensive characterization of the OR51E1 ligand profile by luciferase reporter gene activation assay identified 2-ethylhexanoic acid as a receptor antagonist and various structurally related fatty acids as novel OR51E1 ligands, some of which were detected at receptor-activating concentrations in plasma and epicardial adipose tissue. Functional investigation of the endogenous receptor was carried out by Ca2+ imaging of human stem cell-derived cardiomyocytes. Application of OR51E1 ligands induced negative chronotropic effects that depended on activation of the OR. OR51E1 activation also provoked a negative inotropic action in cardiac trabeculae and slice preparations of human explanted ventricles. These findings indicate that OR51E1 may play a role as metabolic regulator of cardiac function.

The deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessively inherited disease that has undergone extensive phenotypic expansion since being first described in patients with fevers, recurrent strokes, livedo racemosa, and polyarteritis nodosa in 2014. It is now recognized that patients may develop multisystem disease that spans multiple medical subspecialties. Here, we describe the findings from a large single center longitudinal cohort of 60 patients, the broad phenotypic presentation, as well as highlight the cohort's experience with hematopoietic cell transplantation and COVID-19. Disease manifestations could be separated into three major phenotypes: inflammatory/vascular, immune dysregulatory, and hematologic, however, most patients presented with significant overlap between these three phenotype groups. The cardinal features of the inflammatory/vascular group included cutaneous manifestations and stroke. Evidence of immune dysregulation was commonly observed, including hypogammaglobulinemia, absent to low class-switched memory B cells, and inadequate response to vaccination. Despite these findings, infectious complications were exceedingly rare in this cohort. Hematologic findings including pure red cell aplasia (PRCA), immune-mediated neutropenia, and pancytopenia were observed in half of patients. We significantly extended our experience using anti-TNF agents, with no strokes observed in 2026 patient months on TNF inhibitors. Meanwhile, hematologic and immune features had a more varied response to anti-TNF therapy. Six patients received a total of 10 allogeneic hematopoietic cell transplant (HCT) procedures, with secondary graft failure necessitating repeat HCTs in three patients, as well as unplanned donor cell infusions to avoid graft rejection. All transplanted patients had been on anti-TNF agents prior to HCT and received varying degrees of reduced-intensity or non-myeloablative conditioning. All transplanted patients are still alive and have discontinued anti-TNF therapy. The long-term follow up afforded by this large single-center study underscores the clinical heterogeneity of DADA2 and the potential for phenotypes to evolve in any individual patient.

It is important to evaluate whether a new treatment for heart failure with reduced ejection fraction (HFrEF) provides additive benefit to background foundational treatments. As such, we aimed to evaluate the efficacy and safety of empagliflozin in patients with HFrEF in addition to baseline treatment with specific doses and combinations of disease-modifying therapies.We performed a post-hoc analysis of the EMPEROR-Reduced randomised, double-blind, parallel-group trial, which took place in 520 centres (hospitals and medical clinics) in 20 countries in Asia, Australia, Europe, North America, and South America. Patients with New York Heart Association (NYHA) classification II-IV with an ejection fraction of 40% or less were randomly assigned (1:1) to receive the addition of either oral empagliflozin 10 mg per day or placebo to background therapy. The primary composite outcome was cardiovascular death and heart failure hospitalisation; the secondary outcome was total heart failure hospital admissions. An extended composite outcome consisted of inpatient and outpatient HFrEF events was also evaluated. Outcomes were analysed according to background use of angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs) or angiotensin receptor neprilysin inhibitors (ARNIs), as well as β blockers and mineralocorticoid receptor antagonists (MRAs) at less than 50% or 50% or more of target doses and in various combinations. This study is registered with ClinicalTrials.gov, NCT03057977.In this post-hoc analysis of 3730 patients (mean age 66·8 years [SD 11·0], 893 [23·9%] women; 1863 [49·9%] in the empagliflozin group, 1867 [50·1%] in the placebo group) assessed between March 6, 2017, and May 28, 2020, empagliflozin reduced the risk of the primary outcome (361 in 1863 participants in the empagliflozin group and 462 of 1867 in the placebo group; HR 0·75 [95% CI 0·65-0·86]) regardless of background therapy or its target doses for ACE inhibitors or ARBs at doses of less than 50% of the target dose (HR 0·85 [0·69-1·06]) and for doses of 50% or more of the target dose (HR 0·67 [0·52-0·88]; pinteraction=0·18). A similar result was seen for β blockers at doses of less than 50% of the target dose (HR 0·66 [0·54-0·80]) and for doses of 50% or more of the target dose (HR 0·81 [0·66-1·00]; pinteraction=0·15). Empagliflozin also reduced the risk of the primary outcome irrespective of background use of triple therapy with an ACE inhibitor, ARB, or ARNI plus β blocker plus MRA (given combination HR 0·73 [0·61-0·88]; not given combination HR 0·76 [0·62-0·94]; pinteraction=0·77). Similar patterns of benefit were observed for the secondary and extended composite outcomes. Empagliflozin was well tolerated and rates of hypotension, symptomatic hypotension, and hyperkalaemia were similar across all subgroups.Empagliflozin reduced serious heart failure outcomes across doses and combinations of disease-modifying therapies for HFrEF. Clinically, these data suggest that empagliflozin might be considered as a foundational therapy in patients with HFrEF regardless of their existing background therapy.Boehringer Ingelheim and Eli Lilly and Company.

It is known that some people age faster than others, some people live into old age disease-free, while others develop age-related chronic diseases. With a rapidly aging population and an emerging chronic diseases epidemic, finding mechanisms and implementing preventive measures that could slow down the aging process has become a new challenge for biomedical research and public health. In mice, lifelong water restriction shortens the lifespan and promotes degenerative changes. Here, we test the hypothesis that optimal hydration may slow down the aging process in humans.We performed a cohort analysis of data from the Atherosclerosis Risk in Communities study with middle-age enrollment (45-66 years, n = 15,752) and 25 years follow-up. We used serum sodium, as a proxy for hydration habits. To estimate the relative speed of aging, we calculated the biological age (BA) from age-dependent biomarkers and assessed risks of chronic diseases and premature mortality.The analysis showed that middle age serum sodium >142 mmol/l is associated with a 39% increased risk to develop chronic diseases (hazard ratio [HR] = 1.39, 95% confidence interval [CI]:1.18-1.63) and >144 mmol/l with 21% elevated risk of premature mortality (HR = 1.21, 95% CI:1.02-1.45). People with serum sodium >142 mmol/l had up to 50% higher odds to be older than their chronological age (OR = 1.50, 95% CI:1.14-1.96). A higher BA was associated with an increased risk of chronic diseases (HR = 1.70, 95% CI:1.50-1.93) and premature mortality (HR = 1.59, 95% CI 1.39-1.83).People whose middle-age serum sodium exceeds 142 mmol/l have increased risk to be biologically older, develop chronic diseases and die at younger age. Intervention studies are needed to confirm the link between hydration and aging.This work was funded by Intramural Research program of the National Heart, Lung, and Blood Institute (NHLBI). The ARIC study has been funded in whole or in part with federal funds from the NHLBI; the National Institutes of Health (NIH); and the Department of Health and Human Services.

Vascular aging impacts multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular read-outs, remain vastly incomplete. Here we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and uncovered that Notch3 inactivation alters regulation of calcium, contractile function, and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of CADASIL patients – a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3 null mice has unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of β-catenin. In this study we demonstrate directly that growth factors stimulate β-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether β-catenin/TCF signaling regulates VSMC proliferation via modulation of the β-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited β-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of β-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by β-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting β-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and β-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 β-catenin/TCF responsive genes.

The renin–angiotensin system is a master regulator of human physiology. It controls blood pressure and fluid and electrolyte balance through coordinated effects on the heart, blood vessels, and kidneys. In the classic pathway of the renin–angiotensin system, renin is secreted from the juxtaglomerular apparatus of the kidney and acts on the circulating precursor angiotensinogen to generate angiotensin I (Figure 1). Angiotensin I has little effect on blood pressure and is converted in the lungs by angiotensin-converting enzyme (ACE) to angiotensin II. A potent vasopressor, angiotensin II acts on the heart and the kidneys by binding to the G protein–coupled receptors . . .

Inflammation is a key component of arterial injury, with VSMC proliferation and neointimal formation serving as the final outcomes of this process. However, the acute events transpiring immediately after arterial injury that establish the blueprint for this inflammatory program are largely unknown. We therefore studied these events in mice and found that immediately following arterial injury, medial VSMCs upregulated Rantes in an acute manner dependent on Stat3 and NF-kappaB (p65 subunit). This led to early T cell and macrophage recruitment, processes also under the regulation of the cyclin-dependent kinase inhibitor p21Cip1. Unique to VSMCs, Rantes production was initiated by Tnf-alpha, but not by Il-6/gp130. This Rantes production was dependent on the binding of a p65/Stat3 complex to NF-kappaB-binding sites within the Rantes promoter, with shRNA knockdown of either Stat3 or p65 markedly attenuating Rantes production. In vivo, acute NF-kappaB and Stat3 activation in medial VSMCs was identified, with acute Rantes production after injury substantially reduced in Tnfa-/- mice compared with controls. Finally, we generated mice with SMC-specific conditional Stat3 deficiency and confirmed the Stat3 dependence of acute Rantes production by VSMCs. Together, these observations unify inflammatory events after vascular injury, demonstrating that VSMCs orchestrate the arterial inflammatory response program via acute Rantes production and subsequent inflammatory cell recruitment.

Patient-derived induced pluripotent stem cells reveal treatment strategies for a rare genetic form of arterial calcification.

The musculocontractural type of Ehlers-Danlos syndrome (MC-EDS) has been recently recognized as a clinical entity. MC-EDS represents a differential diagnosis within the congenital neuromuscular and connective tissue disorders spectrum. Thirty-one and three patients have been reported with MC-EDS so far with bi-allelic mutations identified in CHST14 and DSE, respectively, encoding two enzymes necessary for dermatan sulfate (DS) biosynthesis. We report seven additional patients with MC-EDS from four unrelated families, including the follow-up of a sib-pair originally reported with the kyphoscoliotic type of EDS in 1975. Brachycephaly, a characteristic facial appearance, an asthenic build, hyperextensible and bruisable skin, tapering fingers, instability of large joints, and recurrent formation of large subcutaneous hematomas are always present. Three of seven patients had mildly elevated serum creatine kinase. The oldest patient was blind due to retinal detachment at 45 years and died at 59 years from intracranial bleeding; her affected brother died at 28 years from fulminant endocarditis. All patients in this series harbored homozygous, predicted loss-of-function CHST14 mutations. Indeed, DS was not detectable in fibroblasts from two unrelated patients with homozygous mutations. Patient fibroblasts produced higher amounts of chondroitin sulfate, showed intracellular retention of collagen types I and III, and lacked decorin and thrombospondin fibrils compared with control. A great proportion of collagen fibrils were not integrated into fibers, and fiber bundles were dispersed into the ground substance in one patient, all of which is likely to contribute to the clinical phenotype. This report should increase awareness for MC-EDS.

During IgE-mediated immediate hypersensitivity reactions, vascular endothelial cells permeabilize in response to mast cell mediators. We have demonstrated previously that patients and mice with signal transducer and activator of transcription 3 (STAT3) mutations (autosomal dominant hyper-IgE syndrome [AD-HIES]) are partially protected from anaphylaxis.We sought to study the mechanism by which STAT3 contributes to anaphylaxis and determine whether small-molecule inhibition of STAT3 can prevent anaphylaxis.Using unaffected and STAT3-inhibited or genetic loss-of-function samples, we performed histamine skin prick tests, investigated the contribution of STAT3 to animal models of anaphylaxis, and measured endothelial cell permeability, gene and protein expression, and histamine receptor-mediated signaling.Although mouse mast cell degranulation was minimally affected by STAT3 blockade, mast cell mediator-induced anaphylaxis was blunted in Stat3 mutant mice with AD-HIES and in wild-type mice subjected to small-molecule STAT3 inhibition. Histamine skin prick test responses were diminished in patients with AD-HIES. Human umbilical vein endothelial cells derived from patients with AD-HIES or treated with a STAT3 inhibitor did not signal properly through Src or cause appropriate dissolution of the adherens junctions made up of the proteins vascular endothelial-cadherin and β-catenin. Furthermore, we found that diminished STAT3 target microRNA17-92 expression in human umbilical vein endothelial cells from patients with AD-HIES is associated with increased phosphatase and tensin homolog (PTEN) expression, which inhibits Src, and increased E2F transcription factor 1 expression, which regulates β-catenin cellular dynamics.These data demonstrate that STAT3-dependent transcriptional activity regulates critical components for the architecture and functional dynamics of endothelial junctions, thus permitting vascular permeability.

Endothelial-mesenchymal transition (EndMT) is associated with various cardiovascular diseases and in particular with atherosclerosis and plaque instability. However, the molecular pathways that govern EndMT are poorly defined. Specifically, the role of epigenetic factors and histone deacetylases (HDACs) in controlling EndMT and the atherosclerotic plaque phenotype remains unclear. Here, we identified histone deacetylation, specifically that mediated by HDAC9 (a class IIa HDAC), as playing an important role in both EndMT and atherosclerosis. Using in vitro models, we found class IIa HDAC inhibition sustained the expression of endothelial proteins and mitigated the increase in mesenchymal proteins, effectively blocking EndMT. Similarly, ex vivo genetic knockout of Hdac9 in endothelial cells prevented EndMT and preserved a more endothelial-like phenotype. In vivo, atherosclerosis-prone mice with endothelial-specific Hdac9 knockout showed reduced EndMT and significantly reduced plaque area. Furthermore, these mice displayed a more favorable plaque phenotype, with reduced plaque lipid content and increased fibrous cap thickness. Together, these findings indicate that HDAC9 contributes to vascular pathology by promoting EndMT. Our study provides evidence for a pathological link among EndMT, HDAC9, and atherosclerosis and suggests that targeting of HDAC9 may be beneficial for plaque stabilization or slowing the progression of atherosclerotic disease.

Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disorder caused by a loss of functional ADA2 protein. TNF inhibition (TNFi) has proven to be highly effective in treating inflammatory manifestations.We sought to explore the pathophysiology and the underlying mechanisms of TNF-inhibitor response in these patients.We performed Sanger sequencing of the ADA2 gene. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry, and cell differentiation experiments to define an inflammatory signature in patients with DADA2 and studied their response to TNF-inhibitor treatment.We demonstrated increased inflammatory signals and overproduction of cytokines mediated by IFN and nuclear factor kappa B pathways in patients' primary cells. Treatment with TNFi led to reduction in inflammation, rescued the skewed differentiation toward the proinflammatory M1 macrophage subset, and restored integrity of endothelial cells in blood vessels. We also report 8 novel disease-associated variants in 7 patients with DADA2.Our data explore the cellular mechanism underlying effective treatment with TNFi therapies in DADA2. DADA2 vasculitis is strongly related to the presence of activated myeloid cells, and the endothelial cell damage is rescued with anti-TNF treatment.

Abstract Aims With increasing prevalence of heart failure (HF) owing to the ageing population, identification of modifiable risk factors is important. In a mouse model, chronic hypohydration induced by lifelong water restriction promotes cardiac fibrosis. Hypohydration elevates serum sodium. Here, we evaluate the association of serum sodium at middle age as a measure of hydration habits with risk to develop HF. Methods and results We analysed data from Atherosclerosis Risk in Communities study with middle age enrolment (45–66 years) and 25 years of follow-up. Participants without water balance dysregulation were selected: serum sodium within normal range (135–146 mmol/L), not diabetic, not obese and free of HF at baseline (N = 11 814). In time-to-event analysis, HF risk was increased by 39% if middle age serum sodium exceeded 143 mmol/L corresponding to 1% body weight water deficit [hazard ratio 1.39, 95% confidence interval (CI) 1.14–1.70]. In a retrospective case-control analysis performed on 70- to 90-year-old attendees of Visit 5 (N = 4961), serum sodium of 142.5–143 mmol/L was associated with 62% increase in odds of left ventricular hypertrophy (LVH) diagnosis [odds ratio (OR) 1.62, 95% CI 1.03–2.55]. Serum sodium above 143 mmol/L was associated with 107% increase in odds of LVH (OR 2.07, 95% CI 1.30–3.28) and 54% increase in odds of HF (OR 1.54, 95% CI 1.06–2.23). As a result, prevalence of HF and LVH was increased among 70- to 90-year-old participants with higher middle age serum sodium. Conclusion Middle age serum sodium above 142 mmol is a risk factor for LVH and HF. Maintaining good hydration throughout life may slow down decline in cardiac function and decrease prevalence of HF.

Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch's-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.

Abstract Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN , p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of β2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

AP-4 is a member of the family of heterotetrameric adaptor protein (AP) complexes that mediate the sorting of integral membrane proteins in post-Golgi compartments. This complex consists of four subunits (epsilon, beta4, mu4 and sigma4) and localizes to the cytoplasmic face of the trans-Golgi network (TGN). Here, we show that the recruitment of endogenous AP-4 to the TGN in vivo is regulated by the small GTP-binding protein ARF1. In addition, we demonstrate a direct interaction of the epsilon and mu4 subunits of AP-4 with ARF1. epsilon binds only to ARF1-GTP and requires residues in the switch I and switch II regions of ARF1. In contrast, mu4 binds equally well to the GTP- and GDP-bound forms of ARF1 and is less dependent on switch I and switch II residues. These observations establish AP-4 as an ARF1 effector and suggest a novel mode of interaction between ARF1 and an AP complex involving both constitutive and regulated interactions.

The protein arginine methyltransferases (PRMTs) include a family of proteins with related putative methyltransferase domains that modify chromatin and regulate cellular transcription. Although some family members, PRMT1 and PRMT4, have been implicated in transcriptional modulation or intracellular signaling, the roles of other PRMTs in diverse cellular processes have not been fully established. Here, we report that PRMT2 inhibits NF-kappaB-dependent transcription and promotes apoptosis. PRMT2 exerted this effect by blocking nuclear export of IkappaB-alpha through a leptomycin-sensitive pathway, increasing nuclear IkappaB-alpha and decreasing NF-kappaB DNA binding. The highly conserved S-adenosylmethionine-binding domain of PRMT2 mediated this effect. PRMT2 also rendered cells susceptible to apoptosis by cytokines or cytotoxic drugs, likely due to its effects on NF-kappaB. Mouse embryo fibroblasts from PRMT2 genetic knockouts showed elevated NF-kappaB activity and decreased susceptibility to apoptosis compared to wild-type or complemented cells. Taken together, these data suggest that PRMT2 inhibits cell activation and promotes programmed cell death through this NF-kappaB-dependent mechanism.

When the term 'angioblast' was initially used, almost a century ago, the cells bearing this name were thought to give rise to plasma, red blood cells and endothelium, with new blood vessels arising from 'within the (cell) bodies of these angioblasts'. More recently, putative circulating 'progenitor endothelial cells', often also referred to as angioblasts, were first described in the adult human a mere decade ago. Therefore, while our understanding of progenitor cell biology and other relevant vascular developmental programs has progressed enormously, key terms such as 'angioblast' and 'angiogenesis' have remained constant. With the recent intense interest in angioblasts and endothelial progenitor cells, and their potential with respect to cardiovascular regenerative medicine, our knowledge of how these and other nonprogenitor cells might contribute to new vessel formation has taken a further leap in understanding. In this review, we focus on the controversial use and definition of these terms based upon our current understanding of vascular biology and progenitor cells.

The retinoblastoma gene product (RB) is an important regulator of E2F activity. RB recruits a number of proteins, including HDACs, SWI/SNF complex, lysine methyl transferase (SUV39H1) and DNA methyltransferase (DNMT1), all of which negatively regulate E2F activity with RB. Here, we show that RB interacts with PRMT2, a member of the protein arginine methyltransferase family, to regulate E2F activity. PRMT2 directly bound and interacted with RB through its AdoMet binding domain, in contrast to other PRMT proteins, including PRMT1, PRMT3 and PRMT4. In reporter assays, PRMT2 repressed E2F1 transcriptional activity in an RB-dependent manner. PRMT2 formed a ternary complex with E2F1 in the presence of RB. To further explore the role of endogenous PRMT2 in the regulation of E2F activity, the PRMT2 gene was ablated in mice by gene targeting. Compared with PRMT2+/+ mouse embryonic fibroblasts (MEFs), PRMT2−/− MEFs demonstrated increased E2F activity and early S phase entry following release of serum starvation. Vascular injury to PRMT2−/− arteries results in a hyperplastic response, consistent with increased G1–S phase progression. Taken together, these findings demonstrate a novel mechanism for the regulation of E2F activity by a member of the protein arginine methyltransferase family.

Throughout development and adult life the vasculature exhibits a remarkably dynamic capacity for growth and repair. The vasculature also plays a pivotal role in the execution of other diverse biologic processes, such as the provisioning of early hematopoietic stem cells during embryonic development or the regulation of vascular tone and blood pressure. Adding to this importance, from an anatomical perspective, the vasculature is clearly an omnipresent organ, with few areas of the body that it does not penetrate. Given these impressive characteristics, it is perhaps to be expected that the vasculature should require, or at least be associated with, a ready supply of stem and progenitor cells. However, somewhat surprisingly, it is only now just beginning to be broadly appreciated that the vasculature plays host to a range of vessel-resident stem and progenitor cells. The possibility that these vessel-resident cells are implicated in processes as diverse as tumor vascularization and adaptive vascular remodeling appears likely, and several exciting avenues for clinical translation are already under investigation. This review explores the various stem and progenitor cell populations that are resident in the microvasculature, endothelium, and vessel walls and vessel-resident cells capable of phenotypic transformation.

Spinal and bulbar muscular atrophy (SBMA, Kennedy's disease) is a motor neuron disease caused by polyglutamine repeat expansion in the androgen receptor. Although degeneration occurs in the spinal cord and muscle, the exact mechanism is not clear. Induced pluripotent stem cells from spinal and bulbar muscular atrophy patients provide a useful model for understanding the disease mechanism and designing effective therapy. Stem cells were generated from six patients and compared to control lines from three healthy individuals. Motor neurons from four patients were differentiated from stem cells and characterized to understand disease-relevant phenotypes. Stem cells created from patient fibroblasts express less androgen receptor than control cells, but show androgen-dependent stabilization and nuclear translocation. The expanded repeat in several stem cell clones was unstable, with either expansion or contraction. Patient stem cell clones produced a similar number of motor neurons compared to controls, with or without androgen treatment. The stem cell-derived motor neurons had immunoreactivity for HB9, Isl1, ChAT, and SMI-32, and those with the largest repeat expansions were found to have increased acetylated α-tubulin and reduced HDAC6. Reduced HDAC6 was also found in motor neuron cultures from two other patients with shorter repeats. Evaluation of stably transfected mouse cells and SBMA spinal cord showed similar changes in acetylated α-tubulin and HDAC6. Perinuclear lysosomal enrichment, an HDAC6 dependent process, was disrupted in motor neurons from two patients with the longest repeats. SBMA stem cells present new insights into the disease, and the observations of reduced androgen receptor levels, repeat instability, and reduced HDAC6 provide avenues for further investigation of the disease mechanism and development of effective therapy.

Objectives To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1 , a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. Methods We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients’ primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). Results We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1β were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients’ fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. Conclusions Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

Abstract Human induced pluripotent stem cells (iPSCs) have great potential as source cells for therapeutic uses. However, reports indicate that iPSCs carry genetic abnormalities, which may impede their medical use. Little is known about mechanisms contributing to intrinsic DNA damage in iPSCs that could lead to genomic instability. In this report, we investigated the level of DNA damage in human iPSC lines compared with their founder fibroblast line and derived mesenchymal stromal cell (MSC) lines using the phosphorylated histone variant, γH2AX, as a marker of DNA damage. We show that human iPSCs have elevated basal levels of γH2AX, which correlate with markers of DNA replication: 5-ethynyl-2′-deoxyuridine and the single-stranded binding protein, replication protein A. γH2AX foci in iPSCs also colocalize to BRCA1 and RAD51, proteins in the homologous repair pathway, implying γH2AX in iPSCs marks sites of double strand breaks. Our study demonstrates an association between increased basal levels of γH2AX and the rapid replication of iPSCs.

Recent advances in genetic evaluation improved the identification of several variants in the NOTCH3 gene causing Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). Despite improved diagnosis, the disease mechanism remains an elusive target and an increasing number of scientific/clinical groups are investigating CADASIL to better understand it. The purpose of this review is to summarize the current knowledge in CADASIL.CADASIL is a genotypically and phenotypically diverse condition involving multiple molecular systems affecting small blood vessels. Cerebral white matter changes observed by MRI are a key CADASIL characteristic in young adult patients often before severe symptoms and trigger NOTCH3 genetic testing. NOTCH3 mutation locations are highly variable, correlate to disease severity and consistently affect the cysteine balance within extracellular Notch3. Granular osmiophilic material deposits around blood vessels are also a unique CADASIL feature and appear to have a role in sequestering proteins that are essential for blood vessel homeostasis. As potential biomarkers and therapeutic targets are being actively investigated, neurofilament light chain can be detected in patient serum and may be a promising circulating biomarker.CADASIL is a complex, devastating disease with unknown mechanism and no treatment options. As we increase our understanding of CADASIL, translational research bridging basic science and clinical findings needs to drive biomarker and therapeutic target discovery.

The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries.These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.

Spinal muscular atrophy (SMA) is one of the most common severe hereditary diseases of infancy and early childhood in North America, Europe, and Asia. SMA is usually caused by deletions of the survival motor neuron 1 (SMN1) gene. A closely related gene, SMN2, modifies the disease severity. SMA carriers have only 1 copy of SMN1 and are relatively common (1 in 30-50) in populations of European and Asian descent. SMN copy numbers and SMA carrier frequencies have not been reliably estimated in Malians and other sub-Saharan Africans.We used a quantitative polymerase chain reaction assay to determine SMN1 and SMN2 copy numbers in 628 Malians, 120 Nigerians, and 120 Kenyans. We also explored possible mechanisms for SMN1 and SMN2 copy number differences in Malians, and investigated their effects on SMN mRNA and protein levels.The SMA carrier frequency in Malians is 1 in 209, lower than in Eurasians. Malians and other sub-Saharan Africans are more likely to have ≥3 copies of SMN1 than Eurasians, and more likely to lack SMN2 than Europeans. There was no evidence of gene conversion, gene locus duplication, or natural selection from malaria resistance to account for the higher SMN1 copy numbers in Malians. High SMN1 copy numbers were not associated with increased SMN mRNA or protein levels in human cell lines.SMA carrier frequencies are much lower in sub-Saharan Africans than in Eurasians. This finding is important to consider in SMA genetic counseling in individuals with black African ancestry.

Introduction: Since the initial discoveries of human embryonic and induced pluripotent stem cells, many strategies have been developed to utilize the potential of these cells for translational research and disease modeling. The success of these aims and the development of future applications in this area will depend on the ability to generate high-quality and large numbers of differentiated cell types that genetically, epigenetically, and functionally mimic the cells found in the body.Areas covered: In this review, we highlight the current strategies used to maintain stem cell pluripotency (a measure of stem cell quality), as well as provide an overview of the various differentiation strategies being used to generate cells from all three germ lineages. We also discuss the particular considerations that must be addressed when utilizing these cells for translational therapy, and provide an example of a cell type currently used in clinical trials.Expert opinion: The major challenge in regenerative medicine and disease modeling will be in generating functional cells of sufficient quality that are physiologically and epigenetically similar to the diverse cells that they are modeled after. By meeting these criteria, these differentiated products can be successfully used in disease modeling, drug/toxicology screens, and cellular replacement therapy.

With increased life expectancy worldwide, there is an urgent need for improving preventive measures that delay the development of age-related degenerative diseases. Here, we report evidence from mouse and human studies that this goal can be achieved by maintaining optimal hydration throughout life. We demonstrate that restricting the amount of drinking water shortens mouse lifespan with no major warning signs up to 14 months of life, followed by sharp deterioration. Mechanistically, water restriction yields stable metabolism remodeling toward metabolic water production with greater food intake and energy expenditure, an elevation of markers of inflammation and coagulation, accelerated decline of neuromuscular coordination, renal glomerular injury, and the development of cardiac fibrosis. In humans, analysis of data from the Atherosclerosis Risk in Communities (ARIC) study revealed that hydration level, assessed at middle age by serum sodium concentration, is associated with markers of coagulation and inflammation and predicts the development of many age-related degenerative diseases 24 years later. The analysis estimates that improving hydration throughout life may greatly decrease the prevalence of degenerative diseases, with the most profound effect on dementia, heart failure (HF), and chronic lung disease (CLD), translating to the development of these diseases in 3 million fewer people in the United States alone.

ormation of a healed arterial wound is a landmark event during the repair of vascular lesions.Adult coronary, peripheral, and cerebral arteries are exposed to multiple stresses, including mechanical trauma, endothelial denudation, damage to intimal and medial smooth muscle, abnormal shear stresses, and oxidation of LDL by macrophages, and they must undergo repair.This wound repair process, like in many other organs, requires that a complex network of molecular signals be regulated within the cytoplasm and nucleus of an endothelial or smooth muscle cell.The cell cycle is a key regulator of these signals and has direct effects on multiple cell processes, including cell proliferation, DNA repair, apoptosis, and cell migration.

Cell cycle withdrawal associated with terminal differentiation is responsible for the incapability of many organs to regenerate after injury. Here, we employed a cell-free system to analyze the molecular mechanisms underlying cell cycle arrest in cardiomyocytes. In this assay, incubation of S phase nuclei mixed with cytoplasmic extract of S phase cells and adult primary cardiomyocytes results in a dramatic reduction of proliferating cell nuclear antigen (PCNA) protein levels. This effect was blocked by the proteasome inhibitors MG132 and lactacystin, whereas actinomycin D and cycloheximide had no effect. Immunodepletion and addback experiments revealed that the effect of cardiomyocyte extract on PCNA protein levels is maintained by p21 but not p27. In serum-stimulated cardiomyocytes PCNA expression was reconstituted, whereas the protein level of p21 but not that of p27 was reduced. Cytoplasmic extract of serum-stimulated cardiomyocytes did not influence the PCNA protein level in S phase nuclei. Moreover, the hypertrophic effect of serum stimulation was blocked by ectopic expression of p21 and the PCNA protein level was found to be upregulated in adult cardiomyocytes derived from p21 knockout mice. Our data provide evidence that p21 regulates the PCNA protein level in adult cardiomyocytes, which has implications for cardiomyocyte growth control.

The cyclin-dependent kinase inhibitors are key regulators of cell cycle progression.Although implicated in carcinogenesis, they inhibit the proliferation of a variety of normal cell types, and their role in diverse human diseases is not fully understood.Here, we report that p27 Kip1 plays a major role in cardiovascular disease through its effects on the proliferation of bone marrow-derived (BM-derived) immune cells that migrate into vascular lesions.Lesion formation after mechanical arterial injury was markedly increased in mice with homozygous deletion of p27 Kip1 , characterized by prominent vascular infiltration by immune and inflammatory cells.Vascular occlusion was substantially increased when BM-derived cells from p27 -/-mice repopulated vascular lesions induced by mechanical injury in p27 +/+ recipients, in contrast to p27 +/+ BM donors.To determine the contribution of immune cells to vascular injury, transplantation was performed with BM derived from RAG -/-and RAG +/+ mice.RAG +/+ BM markedly exacerbated vascular proliferative lesions compared with what was found in RAG -/-donors.Taken together, these findings suggest that vascular repair and regeneration is regulated by the proliferation of BM-derived hematopoietic and nonhematopoietic cells through a p27 Kip1dependent mechanism and that immune cells largely mediate these effects.

Recruitment and retention of circulating progenitor cells at the site of injured or ischemic tissues facilitates adult neo-vascularization. We hypothesized that cell therapy could modulate local neo-vascularization through the vascular endothelial growth factor (VEGF)/stromal cell-derived factor-1 (SDF-1) axis and by paracrine effects on local endothelial cells. We isolated from rat bone marrow a subset of multipotent adult progenitor cell-derived progenitor cells (MDPC). In vitro, MDPCs secreted multiple cytokines related to inflammation and angiogenesis, including monocyte chemotactic protein-1, SDF-1, basic fibroblast growth factor, and VEGF, and expressed the chemokine receptors CXCR4 and VEGFR1. To investigate in vivo properties, we transplanted MDPCs into the ischemic hind limbs of rats. Elevated levels of the chemokine SDF-1 and colocalization of CD11b+ cells marked the initial phase of tissue remodeling after cell transplantation. Prolonged engraftment was observed in the adventitial–medial border region of arterioles of ischemic muscles. However, engrafted cells did not differentiate into endothelial or smooth muscle cells. Limb perfusion normalized 4 weeks after cell injection. Inhibition of SDF-1 reduced the engraftment of transplanted cells and decreased endothelial cell proliferation. These findings suggest a two-stage model whereby transplanted MDPCs modulate wound repair through recruitment of inflammatory cells to ischemic tissue. This is an important potential mechanism for cell transplantation, in addition to the direct modulation of local vascular cells through paracrine mechanisms.

Cyclin-dependent kinase inhibitors, including p21Cip1, are implicated in cell turnover and are active players in cardiovascular wound repair. Here, we show that p21Cip1 orchestrates the complex interactions between local vascular and circulating immune cells during vascular wound repair. In response to femoral artery mechanical injury, mice with homozygous deletion of p21Cip1 displayed accelerated proliferation of VSMCs and increased immune cell infiltration. BM transplantation experiments indicated that local p21Cip1 plays a pivotal role in restraining excessive proliferation during vascular wound repair. Increased local vascular stromal cell-derived factor-1 (SDF-1) levels were observed after femoral artery injury in p21+/+ and p21-/- mice, although this was significantly greater in p21-/- animals. In addition, disruption of SDF-1/CXCR4 signaling inhibited the proliferative response during vascular remodeling in both p21+/+ and p21-/- mice. We provide evidence that the JAK/STAT signaling pathway is an important regulator of vascular SDF-1 levels and that p21Cip1 inhibits STAT3 binding to the STAT-binding site within the murine SDF-1 promoter. Collectively, these results suggest that p21Cip1 activity is essential for the regulation of cell proliferation and inflammation after arterial injury in local vascular cells and that the SDF-1/CXCR4 signaling system is a key mediator of vascular proliferation in response to injury.

In murine embryonic stem cells, the onset of vascular endothelial growth factor receptor 2 (VEGFR-2) expression identifies endothelial precursors. Undifferentiated human embryonic stem cells express VEGFR-2, and VEGFR-2 expression persists on differentiation. The objective of our study was to identify a single population of endothelial precursors with common identifying features from both human and murine embryonic stem cells.We report that expression of the VEGF coreceptor neuropilin-1 (NRP-1) coincides with expression of Brachyury and VEGFR-2 and identifies endothelial precursors in murine and human embryonic stem cells before CD31 or CD34 expression. When sorted and differentiated, VEGFR-2(+)NRP-1(+) cells form endothelial-like colonies that express CD31 and CD34 7-fold more efficiently than NRP-1 cells. Finally, antagonism of both the VEGF and Semaphorin binding functions of NRP-1 impairs the differentiation of vascular precursors to endothelial cells.The onset of NRP-1 expression identifies endothelial precursors in murine and human stem cells. The findings define the origin of a single population of endothelial precursors from human and murine stem cells to endothelial cells. Additionally, the function of both the VEGF and Semaphorin binding activities of NRP-1 has important roles in the differentiation of stem cells to endothelial cells, providing novel insights into the role of NRP-1 in a model of vasculogenesis.

Pulmonary hypertension is a vascular proliferative disease characterized by pulmonary artery remodeling because of dysregulated endothelial and smooth muscle cell proliferation.Although the role of inflammation in the development of the disease is not welldefined, plexogenic lesions in human disease are characterized by perivascular inflammation composed, in part, of T cells.We explored the role of T-cell infiltration on pulmonary vascular remodeling after endothelial cell damage.We induced endothelial cell damage using monocrotaline and isolated the role of T cells by using Rag1 tm1Mom mice and performing adoptive T-cell transfer.We found that monocrotaline causes pulmonary vascular endothelial cell injury followed by a perivascular inflammatory response.The infiltration of inflammatory cells primarily involves CD4 1 T cells and leads to the progressive muscularization of small (,30 mm) arterioles.Pulmonary vascular proliferative changes were accompanied by progressive and persistent elevations in right ventricular pressure and right ventricular hypertrophy.Supporting the central role of CD4 1 T cells in the inflammatory response, Rag1 tm1Mom (Rag1 2/2 ) mice, which are devoid of T and B cells, were protected from the development of vascular injury when exposed to monocrotaline.The introduction of T cells from control mice into Rag1 2/2 mice reproduced the vascular injury phenotype.These data indicate that after endothelial cell damage, CD4 1 T-cell infiltration participates in pulmonary vascular remodeling.This finding suggests that a CD4 1 T-cell immune response may contribute to the pathogenesis of inflammatory vascular lesions seen in some forms of pulmonary hypertension.

Summary The majority of the biomedical research workforce and funds are focused on studying common diseases and the development of drugs to treat them. However, some of the most remarkable discoveries in physiology and medicine are uncovered by studying rare conditions, because the importance of certain molecular mechanisms is revealed only when their dysfunction results in disease. In 2008, the National Institutes of Health (NIH) launched the NIH Undiagnosed Diseases Program (UDP), which recruits and selects patients who suffer from diseases of unknown etiology, and studies their causes at the clinical, genetic and cellular levels. In this Editorial, we discuss how the UDP has enabled the discovery of several new diseases and disease mechanisms through collaborations between clinical and basic science teams, using the power of both clinical medicine and biological models. Establishing programs with similar infrastructure at other centers around the world could help to benefit patients, their families and the entire medical community, by enhancing research productivity for rare and novel diseases.

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies.

One of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff™, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation. Both hematopoietic and non-hematopoietic cells were functionally characterized throughout differentiation. In the hematopoietic fraction, an early transient population of primitive CD235a+ erythroid progenitor cells first emerged, followed by hematopoietic progenitors with multilineage differentiation activity in vitro but no long-term engraftment potential in vivo. In later stages of differentiation, a nearly exclusive production of definitive erythroid progenitors was observed. In the non-hematopoietic fraction, we identified a prevalent population of mesenchymal stromal cells and limited arterial vascular endothelium (VE), suggesting that the cellular constitution of the monolayer may be inadequate to support the generation of HSCs with durable repopulating potential. Quantitative modulation of WNT/β-catenin and activin/nodal/TGFβ signaling pathways with CHIR/SB molecules during differentiation enhanced formation of arterial VE, definitive multilineage and erythroid progenitors, but was insufficient to orchestrate the generation of engrafting HSCs. Overall, STEMdiff™ provides a clinically-relevant and readily adaptable platform for the generation of erythroid and multilineage hematopoietic progenitors from human pluripotent stem cells.

The increasing incidence of cardiovascular disease (CVD) has led to a significant ongoing need to address this surgically through coronary artery bypass grafting (CABG) and percutaneous coronary interventions (PCI). From this, there continues to be a substantial burden of mortality and morbidity due to complications arising from endothelial damage, resulting in restenosis. Whilst mast cells (MC) have been shown to have a causative role in atherosclerosis and other vascular diseases, including restenosis due to vein engraftment; here, we demonstrate their rapid response to arterial wire injury, recapitulating the endothelial damage seen in PCI procedures. Using wild-type mice, we demonstrate accumulation of MC in the femoral artery post-acute wire injury, with rapid activation and degranulation, resulting in neointimal hyperplasia, which was not observed in MC-deficient KitW-sh/W-sh mice. Furthermore, neutrophils, macrophages, and T cells were abundant in the wild-type mice area of injury but reduced in the KitW-sh/W-sh mice. Following bone-marrow-derived MC (BMMC) transplantation into KitW-sh/W-sh mice, not only was the neointimal hyperplasia induced, but the neutrophil, macrophage, and T-cell populations were also present in these transplanted mice. To demonstrate the utility of MC as a target for therapy, we administered the MC stabilizing drug, disodium cromoglycate (DSCG) immediately following arterial injury and were able to show a reduction in neointimal hyperplasia in wild-type mice. These studies suggest a critical role for MC in inducing the conditions and coordinating the detrimental inflammatory response seen post-endothelial injury in arteries undergoing revascularization procedures, and by targeting the rapid MC degranulation immediately post-surgery with DSCG, this restenosis may become a preventable clinical complication.

Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS-/- mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS-/- VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.

The potential impact of stem cell technology on medical and dental practice is vast. Stem cell research will not only provide the foundation for future therapies, but also reveal unique insights into basic disease mechanisms. Therefore, an understanding of stem cell technology will be necessary for clinicians in the future. Herein, we give a basic overview of stem cell biology and therapeutics for the practicing clinician.

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy, but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with >85% purity in 10 days, using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes, we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies, including in vivo tissue regeneration models and drug screening.

We have previously shown that an F9 teratocarcinoma retinoic acid receptor beta(2) (RARbeta(2)) knockout cell line exhibits no growth arrest in response to all-trans-retinoic acid (RA), whereas F9 wild type (Wt), F9 RARalpha(-/-), and F9 RARgamma(-/-) cell lines do growth arrest in response to RA. To examine the role of RARbeta(2) in growth inhibition, we analyzed the cell cycle regulatory proteins affected by RA in F9 Wt and F9 RARbeta(2)(-/-) cells. Flow microfluorimetry analyses revealed that RA treatment of F9 Wt cells greatly increased the percentage of cells in the G1/G0 phase of the cell cycle. In contrast, RA did not alter the cell cycle distribution profile of RARbeta(2)(-/-) cells. In F9 Wt cells, cyclin D1, D3, and cyclin E protein levels decreased, while cyclin D2 and p27 levels increased after RA treatment. Compared to the F9 Wt cells, the F9 RARbeta(2)(-/-) cells exhibited lower levels of cyclins D1, D2, D3, and E in the absence of RA, but did not exhibit further changes in the levels of these cell cycle regulators after RA addition. Since RA significantly increased the level of p27 protein (approximately 24-fold) in F9 Wt as compared to the F9 RARbeta(2)(-/-) cells, we chose to study p27 in greater detail. The p27 mRNA level and the rate of p27 protein synthesis were increased in RA-treated F9 Wt cells, but not in F9 RARbeta(2)(-/-) cells. Moreover, RA increased the half-life of p27 protein in F9 Wt cells. Reduced expression of RARbeta(2) is associated with the process of carcinogenesis and RARbeta(2) can mediate the growth arrest induced by RA in a variety of cancer cells. Using both genetic and molecular approaches, we have identified some of the molecular mechanisms, such as the large elevation of p27, through which RARbeta(2) mediates these growth inhibitory effects of RA in F9 cells.

Rationale: Cryptogenic strokes, those of unknown cause, have been estimated as high as 30% to 40% of strokes. Inflammation has been suggested as a critical etiologic factor. However, there is lack of experimental evidence. Objective: In this study, we investigated inflammation-associated stroke using a mouse model that developed spontaneous stroke because of myeloid deficiency of TGF-β (transforming growth factor-β) signaling. Methods and Results: We report that mice with deletion of Tgfbr2 in myeloid cells ( Tgfbr2 Myeko ) developed cerebrovascular inflammation in the absence of significant pathology in other tissues, culminating in stroke and severe neurological deficits with 100% penetrance. The stroke phenotype can be transferred to syngeneic wild-type mice via Tgfbr2 Myeko bone marrow transplant and can be rescued in Tgfbr2 Myeko mice with wild-type bone marrow. The underlying mechanisms involved an increased type 1 inflammation and cerebral endotheliopathy, characterized by elevated NF-κB (nuclear factor-κB) activation and TNF (tumor necrosis factor) production by myeloid cells. A high-fat diet accelerated stroke incidence. Anti-TNF treatment, as well as metformin and methotrexate, which are associated with decreased stroke risk in population studies, delayed stroke occurrence. Conclusions: Our studies show that TGF-β signaling in myeloid cells is required for maintenance of vascular health and provide insight into inflammation-mediated cerebrovascular disease and stroke.

Arterial calcification due to deficiency of CD73 (ACDC) is a hereditary autosomal recessive ectopic mineralization syndrome caused by loss-of-function mutations in the ecto-5'-nucleotidase gene. Periarticular calcification has been reported but the clinical characterization of arthritis as well as the microstructure and chemical composition of periarticular calcifications and SF crystals has not been systematically investigated.Eight ACDC patients underwent extensive rheumatological and radiological evaluation over a period of 11 years. Periarticular and synovial biopsies were obtained from four patients. Characterization of crystal composition was evaluated by compensated polarized light microscopy, Alizarin Red staining for synovial fluid along with X-ray diffraction and X-ray micro tomosynthesis scanner for periarticular calcification.Arthritis in ACDC patients has a clinical presentation of mixed erosive-degenerative joint changes with a median onset of articular symptoms at 17 years of age and progresses over time to the development of fixed deformities and functional limitations of small peripheral joints with, eventually, larger joint and distinct axial involvement later in life. We have identified calcium pyrophosphate and calcium hydroxyapatite (CHA) crystals in SF specimens and determined that CHA crystals are the principal component of periarticular calcifications.This is the largest study in ACDC patients to describe erosive peripheral arthropathy and axial enthesopathic calcifications over a period of 11 years and the first to identify the composition of periarticular calcifications and SF crystals. ACDC should be considered among the genetic causes of early-onset OA, as musculoskeletal disease signs may often precede vascular symptoms.

Prolidase deficiency is a rare inborn error of metabolism causing ulcers and other skin disorders, splenomegaly, developmental delay, and recurrent infections. Most of the literature is constituted of isolated case reports. We aim to provide a quantitative description of the natural history of the condition by describing 19 affected individuals and reviewing the literature.Nineteen patients were phenotyped per local institutional procedures. A systematic review following PRISMA criteria identified 132 articles describing 161 patients. Main outcome analyses were performed for manifestation frequency, diagnostic delay, overall survival, symptom-free survival, and ulcer-free survival.Our cohort presented a wide variability of severity. Autoimmune disorders were found in 6/19, including Crohn disease, systemic lupus erythematosus, and arthritis. Another immune finding was hemophagocytic lymphohistiocytosis (HLH). Half of published patients were symptomatic by age 4 and had a delayed diagnosis (mean delay 11.6 years). Ulcers were present initially in only 30% of cases, with a median age of onset at 12 years old.Prolidase deficiency has a broad range of manifestations. Symptoms at onset may be nonspecific, likely contributing to the diagnostic delay. Testing for this disorder should be considered in any child with unexplained autoimmunity, lower extremity ulcers, splenomegaly, or HLH.

Arterial calcification due to deficiency of CD73 (ACDC; OMIM 211800) is a rare genetic disease resulting in calcium deposits in arteries and small joints causing claudication, resting pain, severe joint pain, and deformities. Currently, there are no standard treatments for ACDC. Our previous work identified etidronate as a potential targeted ACDC treatment, using in vitro and in vivo disease models with patient-derived cells. In this study, we test the safety and effectiveness of etidronate in attenuating the progression of lower-extremity arterial calcification and vascular blood flow based on the computed tomography (CT) calcium score and ankle-brachial index (ABI).Seven adult patients with a confirmed genetic diagnosis of ACDC were enrolled in an open-label, nonrandomized, single-arm pilot study for etidronate treatment. They took etidronate daily for 14 days every 3 months and were examined at the NIH Clinical Center bi-annually for 3 years. They received a baseline evaluation as well as yearly follow up after treatment. Study visits included imaging studies, exercise tolerance tests with ABIs, clinical blood and urine testing, and full dental exams.Etidronate treatment appeared to have slowed the progression of further vascular calcification in lower extremities as measured by CT but did not have an effect in reversing vascular and/or periarticular joint calcifications in our small ACDC cohort.Etidronate was found to be safe and well tolerated by our patients and, despite the small sample size, appeared to show an effect in slowing the progression of calcification in our ACDC patient cohort.(ClinicalTrials.gov Identifier NCT01585402).

Donor-recipient cell interactions are essential for functional engraftment after nonautologous cell transplantation. During this process, transplant engraftment is characterized and defined by interactions between transplanted cells with local and recruited inflammatory cells. The outcome of these interactions determines donor cell fate. Here, we provide evidence that lineage-committed embryonic stem cell (ESC)-derived vascular progenitor cells are the target of major histocompatibility complex (MHC) class I-dependent, natural killer (NK) cell-mediated elimination in vitro and in vivo. Treatment with interferon γ was found to significantly upregulate MHC class I expression on ESC-derived vascular progenitor cells, rendering them less susceptible to syngeneic NK cell-mediated killing in vitro and enhancing their survival and differentiation potential in vivo. Furthermore, in vivo ablation of NK cells led to enhanced progenitor cell survival after transplantation into a syngeneic murine ischemic hindlimb model, providing additional evidence that NK cells mediate ESC-derived progenitor cell transplant rejection. These data highlight the importance of recipient immune-donor cell interactions, and indicate a functional role for MHC-I antigen expression during successful ESC-derived syngeneic transplant engraftment.

To clarify how smoking leads to heart attack and stroke, we developed an endothelial cell model (iECs) generated from human induced Pluripotent Stem Cells (iPSC) and evaluated its responses to tobacco smoke. These iECs exhibited a uniform endothelial morphology, and expressed markers PECAM1/CD31, VWF/ von Willebrand Factor, and CDH5/VE-Cadherin. The iECs also exhibited tube formation and acetyl-LDL uptake comparable to primary endothelial cells (EC). RNA sequencing (RNA-Seq) revealed a robust correlation coefficient between iECs and EC (R = 0.76), whereas gene responses to smoke were qualitatively nearly identical between iECs and primary ECs (R = 0.86). Further analysis of transcriptional responses implicated 18 transcription factors in regulating responses to smoke treatment, and identified gene sets regulated by each transcription factor, including pathways for oxidative stress, DNA damage/repair, ER stress, apoptosis, and cell cycle arrest. Assays for 42 cytokines in HUVEC cells and iECs identified 23 cytokines that responded dynamically to cigarette smoke. These cytokines and cellular stress response pathways describe endothelial responses for lymphocyte attachment, activation of coagulation and complement, lymphocyte growth factors, and inflammation and fibrosis; EC-initiated events that collectively lead to atherosclerosis. Thus, these studies validate the iEC model and identify transcriptional response networks by which ECs respond to tobacco smoke. Our results systematically trace how ECs use these response networks to regulate genes and pathways, and finally cytokine signals to other cells, to initiate the diverse processes that lead to atherosclerosis and cardiovascular disease.

There are more than 7000 described rare diseases, most lacking specific treatment. Autosomal-dominant hyper-IgE syndrome (AD-HIES, also known as Job’s syndrome) is caused by mutations in STAT3. These patients present with immunodeficiency accompanied by severe nonimmunological features, including skeletal, connective tissue, and vascular abnormalities, poor postinfection lung healing, and subsequent pulmonary failure. No specific therapies are available for these abnormalities. Here, we investigated underlying mechanisms in order to identify therapeutic targets. Histological analysis of skin wounds demonstrated delayed granulation tissue formation and vascularization during skin-wound healing in AD-HIES patients. Global gene expression analysis in AD-HIES patient skin fibroblasts identified deficiencies in a STAT3-controlled transcriptional network regulating extracellular matrix (ECM) remodeling and angiogenesis, with hypoxia-inducible factor 1α (HIF-1α) being a major contributor. Consistent with this, histological analysis of skin wounds and coronary arteries from AD-HIES patients showed decreased HIF-1α expression and revealed abnormal organization of the ECM and altered formation of the coronary vasa vasorum. Disease modeling using cell culture and mouse models of angiogenesis and wound healing confirmed these predicted deficiencies and demonstrated therapeutic benefit of HIF-1α–stabilizing drugs. The study provides mechanistic insights into AD-HIES pathophysiology and suggests potential treatment options for this rare disease.

The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin-dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor-dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin-dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.

The cyclin-dependent kinase inhibitor p27(Kip1) arrests cell cycle progression through G1/S phases and is regulated by phosphorylation of serine/threonine residues. Recently, we identified the serine/threonine kinase, KIS, which phosphorylates p27(Kip1) on serine 10 leading to nuclear export of p27(Kip1) and protein degradation. However, the molecular mechanisms of transcriptional activation of the human KIS gene and its biological activity are not known. We mapped the transcription initiation site approximately 116 bp 5' to the translation start site, and sequences extending to -141 were sufficient for maximal promoter activity. Mutation in either of two Ets-binding sites in this region resulted in an approximately 75-80% decrease in promoter activity. These sites form at least 3 specific complexes, which contained GA-binding protein (GABP). Knocking down GABPalpha by siRNA in vascular smooth muscle cells (VSMCs) diminished KIS gene expression and reduced cell migration. Correspondingly, in serum stimulated GABPalpha-deficient mouse embryonic fibroblasts (MEFs), KIS gene expression was also significantly reduced, which was associated with an increase in p27(Kip1) protein levels and a decreased percentage of cells in S-phase. Consistent with these findings, following vascular injury in vivo, GABPalpha-heterozygous mice demonstrated reduced KIS gene expression within arterial lesions and these lesions were significantly smaller compared to GABP+/+ mice. In summary, serum-responsive GABP binding to Ets-binding sites activates the KIS promoter, leading to KIS gene expression, cell migration, and cell cycle progression.

Cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), are upregulated during vascular cell proliferation and negatively regulate growth of vascular cells. We hypothesized that absence of either p21(Cip1) or p27(Kip1) in apolipoprotein E (apoE)-deficiency may increase atherosclerotic plaque formation. Compared to apoE(-/-) aortae, both apoE(-/-)/p21(-/-) and apoE(-/-)/p27(-/-) aortae exhibited significantly more atherosclerotic plaque following a high-cholesterol regimen. This increase was particularly observed in the abdominal aortic regions. Deficiency of p27(Kip1) accelerated plaque formation significantly more than p21(-/-) in apoE(-/-) mice. This increased plaque formation was in parallel with increased intima/media area ratios. Deficiency of p21(Cip1) and p27(Kip1) accelerates atherogenesis in apoE(-/-) mice. These findings have significant implications for our understanding of the molecular basis of atherosclerosis associated with excessive proliferation of vascular cells.

The history of revascularization for cardiac ischemia dates back to the early 1960’s when the first coronary artery bypass graft procedures were performed in humans. With this 50 year history of providing a new vasculature to ischemic and hibernating myocardium, a profound depth of experience has been amassed in clinical cardiovascular medicine as to what does, and does not work in the context of cardiac revascularization, alleviating ischemia and adequacy of myocardial perfusion. These issues are of central relevance to contemporary cell-based cardiac regenerative approaches. While the cardiovascular cell therapy field is surging forward on many exciting fronts, several well accepted clinical axioms related to the cardiac arterial supply appear to be almost overlooked by some of our current basic conceptual and experimental cell therapy paradigms. We present here information drawn from five decades of the clinical revascularization experience, review relevant new data on vascular formation via cell therapy, and put forward the case that for optimal cell-based cardiac regeneration due attention must be paid to providing an adequate vascular supply.

Nature Communications 8: Article number: 11853 (2016); Published: 24 June 2016; Updated: 16 February 2017 In this Article, the catalogue number for the anti-Fap-Alexa Fluor 647 antibody is listed incorrectly and should have read bs-5758R-A647 instead of bs-5760R-A647.

As the field of medicine is striving forward heralded by a new era of next-generation sequencing (NGS) and integrated technologies such as bioprinting and biological material development, the utility of rare monogenetic vascular disease modeling in this landscape is starting to emerge. With their genetic simplicity and broader applicability, these patient-specific models are at the forefront of modern personalized medicine. As a collective, rare diseases are a significant burden on global healthcare systems, and rare vascular diseases make up a significant proportion of this. High costs are due to a lengthy diagnostic process, affecting all ages from infants to adults, as well as the severity and chronic nature of the disease. Their complex nature requires sophisticated disease models and integrated approaches involving multidisciplinary teams. Here, we review these emerging vascular disease models, how they contribute to our understanding of the pathomechanisms in rare vascular diseases and provide useful platforms for therapeutic discovery.

ABSTRACT The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL1 and SHARPIN, and is essential for proper immune responses. Patients with HOIP and HOIL1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage. In mice, the loss of Sharpin leads to severe dermatitis due to excessive cell death in keratinocytes. Here we report the first patient with SHARPIN deficiency, manifesting fever, arthritis, colitis, chronic otitis media and hepatic glycogenosis but unexpectedly, not associated with dermatologic manifestations. Mechanistically, fibroblasts and B cells from patients with all three LUBAC deficiencies showed attenuated canonical NF-B response and propensity to apoptosis mediated by TNF superfamily members. Furthermore, the SHARPIN deficient patient showed substantial reduction of adenoidal germinal center B cell development. Treatment of the SHARPIN deficient patient with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical role of LUBAC as a gatekeeper for apoptosis-mediated immune dysregulation in humans.

This work was supported in full by funding from the National Institutes of Health, Division of Intramural Research, National Institute of Dental and Craniofacial Research, and National Heart, Lung, and Blood Institute. Disclosure Summary: The authors have nothing to declare. For article see page E189 Abbreviations: ALP, Alkaline phosphatase; Ank, ankylosis protein; CCAL2, chondrocalcinosis-2; CMD, craniometaphyseal dysplasia; DMP1, dentin matrix protein 1; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; FGF23, fibroblast growth factor 23; HA, hydroxyapatite; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; TNAP, “tissue nonspecific” ALP.

Key teaching points •Malignant atrophic papulosis is a rare vasculopathy that may cause isolated cutaneous disease or severe, widespread disease involving the skin, gastrointestinal tract, central nervous system, and cardiovascular system. •Early skin lesions of malignant atrophic papulosis appear as multiple rose-colored papules that evolve over several weeks to form a scar with an atrophic, porcelain-white center surrounded by an inflamed, telangiectatic border. •Traditional therapeutic approaches appear to be ineffective, but new drugs, including eculizumab and treprostinil, have shown benefit in a small number of patients. •Malignant atrophic papulosis is a rare vasculopathy that may cause isolated cutaneous disease or severe, widespread disease involving the skin, gastrointestinal tract, central nervous system, and cardiovascular system. •Early skin lesions of malignant atrophic papulosis appear as multiple rose-colored papules that evolve over several weeks to form a scar with an atrophic, porcelain-white center surrounded by an inflamed, telangiectatic border. •Traditional therapeutic approaches appear to be ineffective, but new drugs, including eculizumab and treprostinil, have shown benefit in a small number of patients.

Abstract Techniques that enable longitudinal tracking of cell fate after myocardial delivery are imperative for optimizing the efficacy of cell-based cardiac therapies. However, these approaches have been underutilized in preclinical models and clinical trials, and there is considerable demand for site-specific strategies achieving long-term expression of reporter genes compatible with safe noninvasive imaging. In this study, the rhesus sodium/iodide symporter (NIS) gene was incorporated into rhesus macaque induced pluripotent stem cells (RhiPSCs) via CRISPR/Cas9. Cardiomyocytes derived from NIS-RhiPSCs (NIS-RhiPSC-CMs) exhibited overall similar morphological and electrophysiological characteristics compared to parental control RhiPSC-CMs at baseline and with exposure to physiological levels of sodium iodide. Mice were injected intramyocardially with 2 million NIS-RhiPSC-CMs immediately following myocardial infarction, and serial positron emission tomography/computed tomography was performed with 18F-tetrafluoroborate to monitor transplanted cells in vivo. NIS-RhiPSC-CMs could be detected until study conclusion at 8 to 10 weeks postinjection. This NIS-based molecular imaging platform, with optimal safety and sensitivity characteristics, is primed for translation into large-animal preclinical models and clinical trials.

Clinical investigations have established that vascular-associated medical conditions are significant risk factors for various kinds of dementia. And yet, we are unable to associate certain types of vascular deficiencies with specific cognitive impairments. The reasons for this are many, not the least of which are that most vascular disorders are multi-factorial and the development of vascular dementia in humans is often a multi-year or multi-decade progression. To better study vascular disease and its underlying causes, the National Heart, Lung, and Blood Institute of the National Institutes of Health has invested considerable resources in the development of animal models that recapitulate various aspects of human vascular disease. Many of these models, mainly in the mouse, are based on genetic mutations, frequently using single-gene mutations to examine the role of specific proteins in vascular function. These models could serve as useful tools for understanding the association of specific vascular signaling pathways with specific neurological and cognitive impairments related to dementia. To advance the state of the vascular dementia field and improve the information sharing between the vascular biology and neurobehavioral research communities, National Heart, Lung, and Blood Institute convened a workshop to bring in scientists from these knowledge domains to discuss the potential utility of establishing a comprehensive phenotypic cognitive assessment of a selected set of existing mouse models, representative of the spectrum of vascular disorders, with particular attention focused on age, sex, and rigor and reproducibility. The workshop highlighted the potential of associating well-characterized vascular disease models, with validated cognitive outcomes, that can be used to link specific vascular signaling pathways with specific cognitive and neurobehavioral deficits.

Cardiovascular diseases are the leading cause of morbidity and mortality in industrialized countries. Most cardiovascular diseases result from complications of atherosclerosis, which is a chronic and progression inflammatory condition characterized by excessive cellular proliferation of vascular smooth muscle cells, endothelial cells and inflammatory cells leading to occlusive vascular disease, myocardial infarction and stroke. Recent studies have revealed the important role of the cyclins, the cyclin-dependent kinases (CDKs), and the cyclin-dependent kinase inhibitors (CKIs) in vascular and cardiac tissue injury, inflammation and wound repair. Tissue remodeling in the cardiovascular system is a regulated balance between pro- and anti-proliferative molecules, and this balance becomes derailed in cardiovascular pathology. Understanding the circuitry of the cyclin-CDK-CKI interactions in normal physiology and disease pathology allows a better understanding of the molecular mechanisms of cardiovascular diseases and permits the rationale design of new classes of therapeutic agents for these diseases.

The cyclin-dependent kinase inhibitors are key regulators of cell cycle progression. Although implicated in carcinogenesis, they inhibit the proliferation of a variety of normal cell types, and their role in diverse human diseases is not fully understood. Here, we report that p27(Kip1) plays a major role in cardiovascular disease through its effects on the proliferation of bone marrow-derived (BM-derived) immune cells that migrate into vascular lesions. Lesion formation after mechanical arterial injury was markedly increased in mice with homozygous deletion of p27(Kip1), characterized by prominent vascular infiltration by immune and inflammatory cells. Vascular occlusion was substantially increased when BM-derived cells from p27(-/-) mice repopulated vascular lesions induced by mechanical injury in p27(+/+) recipients, in contrast to p27(+/+) BM donors. To determine the contribution of immune cells to vascular injury, transplantation was performed with BM derived from RAG(-/-) and RAG(+/+) mice. RAG(+/+) BM markedly exacerbated vascular proliferative lesions compared with what was found in RAG(-/-) donors. Taken together, these findings suggest that vascular repair and regeneration is regulated by the proliferation of BM-derived hematopoietic and nonhematopoietic cells through a p27(Kip1)-dependent mechanism and that immune cells largely mediate these effects.

Chemokine receptor 5 (CCR5) is the primary coreceptor for HIV entry into macrophages. Individuals with a homozygous deletion of 32 bp in the CCR5 gene (CCR5Δ32) are highly resistant to HIV infection (Samson et al., 1996). Allogeneic stem cell transplantation from a healthy donor with the homozygous CCR5Δ32 variant to an HIV positive individual has demonstrated efficient long-term control of HIV. We identified three individuals with this homozygous CCR5Δ32 variant, and successfully generated induced pluripotent stem cell (iPSC) lines from their dermal fibroblasts. The iPSCs lines carrying homozygous CCR5Δ32 variant displayed phenotypically normal and the potential to differentiation toward the three germ layers.

Successful recovery from vascular diseases has typically relied on the surgical repair of damaged blood vessels (BVs), with the majority of current approaches involving the implantation of autologous BVs, which is plagued by donor site tissue damage. Researchers have attempted to develop artificial vessels as an alternative solution to traditional approaches to BV repair. However, the manufacturing of small-diameter (< 6 mm) BVs is still considered one of the biggest challenges due to its difficulty in the precise fabrication and the replication of biomimetic architectures. In this study, we successfully developed 3D printed flexible small-diameter BVs that consist of smooth muscle cells and a vascularized endothelium. In the developed artificial BV, a rubber-like elastomer was printed as the outermost layer of the vessel, which demonstrated enhanced mechanical properties, while and human induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iSMCs) and endothelial cells (iECs) embedded fibrinogen solutions were coaxially extruded with thrombin solution to form cell-laden fibrin gel inner layers. Our results showed that the 3D BVs possessed proper mechanical properties, and the cells in the fibrin layers substantially proliferated over time to form a stable BV construct. Our study demonstrated that the 3D printed flexible small-diameter BV using iPSCs could be a promising platform for the treatment of vascular diseases.

A 54-year old female patient with the genetic disease of arterial calcification due to deficiency of CD73 was studied under the Undiagnosed Disease Program of the National Institutes of Health. She presented with symptoms of claudication in her 40s and later developed arthritic symptoms, ectopic calcification in her left hand and severe arterial calcifications of the lower extremities. Since little was known about the composition of the calcifications in arterial calcification due to deficiency of CD73, we investigated their chemical identity and microscopic morphology in this patient with imaging and x-ray diffraction analysis. We found that, microscopically, the bulk calcifications consisted of fragments of either solid or porous internal structure. Both periarticular and arterial calcifications were primarily hydroxyapatite crystals of the same crystalline anisotropy, but different crystalline grain sizes. This was consistent with the presence of hydroxyapatite crystals along with birefringent calcium pyrophosphate dihydrate crystals in the synovial fluid of the patients by polarized light microscopy. The result suggests that tissue calcification in both locations follow a similar biochemical mechanism caused by an increase in extracellular tissue-nonspecific alkaline phosphatase activity.

Abstract Histopathology protocols often require sectioning and processing of numerous microscopy slides to survey a sample. Trade-offs between workload and sampling density means that small features can be missed. Aiming to reduce the workload of routine histology protocols and the concern over missed pathology in skipped sections, we developed a prototype x-ray tomographic scanner dedicated to rapid scouting and identification of regions of interest in pathology specimens, thereby allowing targeted histopathology analysis to replace blanket searches. In coronary artery samples of a deceased HIV patient, the scanner, called Tomopath, obtained depth-resolved cross-sectional images at 15 µm resolution in a 15-minute scan, which guided the subsequent histological sectioning and microscopy. When compared to a commercial tabletop micro-CT scanner, the prototype provided several-fold contrast-to-noise ratio in 1/11 th the scan time. Correlated tomographic and histological images revealed two types of micro calcifications: scattered loose calcifications typically found in atherosclerotic lesions; isolated focal calcifications in one or several cells in the internal elastic lamina and occasionally in the tunica media, which we speculate were the initiation of medial calcification linked to kidney disease, but rarely detected at this early stage due to their similarity to particle contaminants introduced during histological processing, if not for the evidence from the tomography scan prior to sectioning. Thus, in addition to its utility as a scouting tool, in this study it provided complementary information to histological microscopy. Overall, the prototype scanner represents a step toward a dedicated scouting and complementary imaging tool for routine use in pathology labs.

Autosomal dominant Hyper IgE syndrome (AD-HIES), a rare immune deficiency affecting fewer than one per million people, is caused by heterozygous deleterious mutations in STAT3. STAT3 signaling plays crucial roles in basic cellular functions affecting broad aspects of cellular homeostasis. Accordingly, in addition to immunological deficits, patients experience severe multisystem non-immunological features. Human induced pluripotent stem cells (hiPSC) are well established as in vivo disease models for various human pathologies. We describe the generation of iPSC from three AD-HIES patients. These iPSCs express pluripotency markers, differentiate into three germ layers, have normal karyotype and similar genome identity to parental cells.

We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts of a patient with a homozygous p.Leu272Pro mutation in the gene encoding the linear deubiquitinase OTULIN. Biallelic loss of function mutations in this gene are responsible for the OTULIN deficiency termed Otulipenia or OTULIN-related autoinflammatory syndrome (ORAS). The iPSC carrying homozygous L272P OTULIN gene mutations are phenotypically normal and they have capacity to differentiate toward the three germ layers. These iPSC have great potential to study the role of linear ubiquitination in the regulation of immune responses and other cellular pathways.

Abstract Living with an undiagnosed medical condition places a tremendous burden on patients, their families, and their healthcare providers. The Undiagnosed Diseases Program (UDP) was established at the National Institutes of Health (NIH) in 2008 with the primary goals of providing a diagnosis for patients with mysterious conditions and advancing medical knowledge about rare and common diseases. The program reviews applications from referring clinicians for cases that are considered undiagnosed despite a thorough evaluation. Those that are accepted receive clinical evaluations involving deep phenotyping and genetic testing that includes exome and genomic sequencing. Selected candidate gene variants are evaluated by collaborators using functional assays. Since its inception, the UDP has received more than 4500 applications and has completed evaluations on nearly 1300 individuals. Here we present six cases that exemplify the discovery of novel disease mechanisms, the importance of deep phenotyping for rare diseases, and how genetic diagnoses have led to appropriate treatment. The creation of the Undiagnosed Diseases Network (UDN) in 2014 has substantially increased the number of patients evaluated and allowed for greater opportunities for data sharing. Expansion to the Undiagnosed Diseases Network International (UDNI) has the possibility to extend this reach even farther. Together, networks of undiagnosed diseases programs are powerful tools to advance our knowledge of pathophysiology, accelerate accurate diagnoses, and improve patient care for patients with rare conditions.

We have successfully created induced pluripotent stem cells (iPSC) from patients carrying a heterozygous mutation in the gene encoding STING. The gain-of-function mutation leads to constitutive activation of STING which leads to the development of the disease STING-associated vasculopathy with onset in infancy (SAVI). The iPSC lines derived from the SAVI patitents are shown to be morphologically and phenotypically normal and have the potential to self renew and differentiate into the three germ layers. These iPSC provide a powerful tools to investigate the role of STING in the regulation of immune responses and vascular renegeration.

Abstract This study presents computerized automatic image analysis for quantitatively evaluating dynamic contrast‐enhanced MRI in an ischemic rat hindlimb model. MRI at 7 T was performed on animals in a blinded placebo‐controlled experiment comparing multipotent adult progenitor cell‐derived progenitor cell (MDPC)‐treated, phosphate buffered saline (PBS)‐injected, and sham‐operated rats. Ischemic and non‐ischemic limb regions of interest were automatically segmented from time‐series images for detecting changes in perfusion and late enhancement. In correlation analysis of the time–signal intensity histograms, the MDPC‐treated limbs correlated well with their corresponding non‐ischemic limbs. However, the correlation coefficient of the PBS control group was significantly lower than that of the MDPC‐treated and sham‐operated groups. In semi‐quantitative parametric maps of contrast enhancement, there was no significant difference in hypo‐enhanced area between the MDPC and PBS groups at early perfusion‐dependent time frames. However, the late‐enhancement area was significantly larger in the PBS than the MDPC group. The results of this exploratory study show that MDPC‐treated rats could be objectively distinguished from PBS controls. The differences were primarily determined by late contrast enhancement of PBS‐treated limbs. These computerized methods appear promising for assessing perfusion and late enhancement in dynamic contrast‐enhanced MRI. Published in 2007 by John Wiley &amp; Sons, Ltd.

Human induced pluripotent stem cell (iPSC) technology has opened exciting opportunities for stem-cell-based therapy. However, its wide adoption is precluded by several challenges including low reprogramming efficiency and potential for malignant transformation. Better understanding of the molecular mechanisms of the changes that cells undergo during reprograming is needed to improve iPSCs generation efficiency and to increase confidence for their clinical use safety. Here, we find that dominant negative mutations in STAT3 in patients with autosomal-dominant hyper IgE (Job's) syndrome (AD-HIES) result in greatly reduced reprograming efficiency of primary skin fibroblasts derived from skin biopsies. Analysis of normal skin fibroblasts revealed upregulation and phosphorylation of endogenous signal transducer and activator of transcription 3 (STAT3) and its binding to the NANOG promoter following transduction with OKSM factors. This coincided with upregulation of NANOG and appearance of cells expressing pluripotency markers. Upregulation of NANOG and number of pluripotent cells were greatly reduced throughout the reprograming process of AD-HIES fibroblasts that was restored by over-expression of functional STAT3. NANOGP8, the human-specific NANOG retrogene that is often expressed in human cancers, was also induced during reprogramming, to very low but detectable levels, in a STAT3-dependent manner. Our study revealed the critical role of endogenous STAT3 in facilitating reprogramming of human somatic cells.