Michael E. Boulton

Age-related macular degeneration (AMD) is the leading cause of blind registration in the developed world, and yet its pathogenesis remains poorly understood. Oxidative stress, which refers to cellular damage caused by reactive oxygen intermediates (ROI), has been implicated in many disease processes, especially age-related disorders. ROIs include free radicals, hydrogen peroxide, and singlet oxygen, and they are often the byproducts of oxygen metabolism. The retina is particularly susceptible to oxidative stress because of its high consumption of oxygen, its high proportion of polyunsaturated fatty acids, and its exposure to visible light. In vitro studies have consistently shown that photochemical retinal injury is attributable to oxidative stress and that the antioxidant vitamins A, C, and E protect against this type of injury. Furthermore, there is strong evidence suggesting that lipofuscin is derived, at least in part, from oxidatively damaged photoreceptor outer segments and that it is itself a photoreactive substance. However, the relationships between dietary and serum levels of the antioxidant vitamins and age-related macular disease are less clear, although a protective effect of high plasma concentrations of alpha-tocopherol has been convincingly demonstrated. Macular pigment is also believed to limit retinal oxidative damage by absorbing incoming blue light and/or quenching ROIs. Many putative risk-factors for AMD have been linked to a lack of macular pigment, including female gender, lens density, tobacco use, light iris color, and reduced visual sensitivity. Moreover, the Eye Disease Case-Control Study found that high plasma levels of lutein and zeaxanthin were associated with reduced risk of neovascular AMD. The concept that AMD can be attributed to cumulative oxidative stress is enticing, but remains unproven. With a view to reducing oxidative damage, the effect of nutritional antioxidant supplements on the onset and natural course of age-related macular disease is currently being evaluated.

The retina resides in an environment that is primed for the generation of reactive oxygen species (ROS) and resultant oxidative damage. The retina is one of the highest oxygen-consuming tissues in the human body. The highest oxygen levels are found in the choroid, but this falls dramatically across the outermost retina, creating a large gradient of oxygen towards the retina and inner segments of the photoreceptors which contain high levels of polyunsaturated fatty acids. This micro-environment together with abundant photosensitizers, visible light exposure and a high energy demand supports a highly oxidative milieu. However, oxidative damage is normally minimized by the presence of a range of antioxidant and efficient repair systems. Unfortunately, as we age oxidative damage increases, antioxidant capacity decreases and the efficiency of reparative systems become impaired. The result is retinal dysfunction and cell loss leading to visual impairment. It appears that these age-related oxidative changes are a hallmark of early age-related macular degeneration (AMD) which, in combination with hereditary susceptibility and other retinal modifiers, can progress to the pathology and visual morbidity associated with advanced AMD. This review reassesses the consequences of oxidative stress in AMD and strategies for preventing or reversing oxidative damage in retinal tissues.

OPA1 is a ubiquitously expressed, nuclear dynamin-related GTPase, targeted to the inner mitochondrial membrane, which plays a role in mitochondrial fusion. Mutations in the OPA1 gene on chromosome 3q28-qter are associated with autosomal dominant optic atrophy (ADOA), the most common inherited optic neuropathy, in which retinal ganglion cells (RGCs) are lost and visual acuity is impaired from an early age. We have generated a novel ENU-induced mutant mouse carrying a protein-truncating nonsense mutation in opa1 in order to explore the pathophysiology of ADOA. The heterozygous mutation, B6; C3- Opa1Q285STOP , located in exon 8 immediately before the central dynamin-GTPase, leads to ∼50% reduction in opa1 protein in retina and all tissues on western analysis. The homozygous mutation is embryonic lethal by 13.5 days post coitum, demonstrating the importance of Opa1 during early development. Fibroblasts taken from adult heterozygous mutant mice show an apparent alteration in morphology, with an increase in mitochondrial fission and fragmentation. Heterozygous mutants show a slow onset of degeneration in the optic nerve electron microscopy. Furthermore, they demonstrate a functional reduction in visual function on testing with the optokinetic drum and the circadian running wheel. These findings indicate that the opa1 GTPase contains crucial information required for the survival of RGCs and that Opa1 is essential for early embryonic survival. The Opa1 +/− mice described here provide a means to directly investigate the cellular pathophysiology of OPA1 ADOA.

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.

Age-related macular degeneration (AMD) is a complex, degenerative and progressive eye disease that usually does not lead to complete blindness, but can result in severe loss of central vision. Risk factors for AMD include age, genetics, diet, smoking, oxidative stress and many cardiovascular-associated risk factors. Autophagy is a cellular housekeeping process that removes damaged organelles and protein aggregates, whereas heterophagy, in the case of the retinal pigment epithelium (RPE), is the phagocytosis of exogenous photoreceptor outer segments. Numerous studies have demonstrated that both autophagy and heterophagy are highly active in the RPE. To date, there is increasing evidence that constant oxidative stress impairs autophagy and heterophagy, as well as increases protein aggregation and causes inflammasome activation leading to the pathological phenotype of AMD. This review ties together these crucial pathological topics and reflects upon autophagy as a potential therapeutic target in AMD.

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.

Age-related macular degeneration (AMD) is the leading cause of visual impairment in the aging population with limited understanding of its pathogenesis and a lack of effective treatment. The progression of AMD is initially characterized by atrophic alterations in the retinal pigment epithelium, as well as the formation of lysosomal lipofuscin and extracellular drusen deposits. Damage caused by chronic oxidative stress, protein aggregation and inflammatory processes may lead to geographic atrophy and/or choroidal neovascularization and fibrosis. The role of macroautophagy/autophagy in AMD pathology is steadily emerging. This review describes selective and secretory autophagy and their role in drusen biogenesis, senescence-associated secretory phenotype, inflammation and epithelial-mesenchymal transition in the pathogenesis of AMD.

Identifying the mechanisms determining species-specific life spans is a central challenge in understanding the biology of aging. Cellular stresses produce damage, that may accumulate and cause aging. Evolution theory predicts that long-lived species secure their longevity through investment in a more durable soma, including enhanced cellular resistance to stress. To investigate whether cells from long-lived species have better mechanisms to cope with oxidative and non-oxidative stress, we compared cellular resistance of primary skin fibroblasts from eight mammalian species with a range of life spans. Cell survival was measured by the thymidine incorporation assay following stresses induced by paraquat, hydrogen peroxide, tert-butyl hydroperoxide, sodium arsenite and alkaline pH (sodium hydroxide). Significant positive correlations between cell LD90 and maximum life span were found for all these stresses. Similar results were obtained when cell survival was measured by the MTT assay, and when lymphocytes from different species were compared. Cellular resistance to a variety of oxidative and non-oxidative stresses was positively correlated with mammalian longevity. Our results support the concept that the gene network regulating the cellular response to stress is functionally important in aging and longevity.

Age and advanced disease in the fellow eye are the two most important risk factors for age-related macular degeneration (AMD). In this study, the authors investigated the relationship between these variables and the optical density of macular pigment (MP) in a group of subjects from a northern European population.The optical density of MP was measured psychophysically in 46 subjects ranging in age from 21 to 81 years with healthy maculae and in 9 healthy eyes known to be at high-risk of AMD because of advanced disease in the fellow eye. Each eye in the latter group was matched with a control eye on the basis of variables believed to be associated with the optical density of MP (iris color, gender, smoking habits, age, and lens density).There was an age-related decline in the optical density of macular pigment among volunteers with no ocular disease (right eye: r(2) = 0.29, P = 0.0006; left eye: r(2) = 0.29, P < 0.0001). Healthy eyes predisposed to AMD had significantly less MP than healthy eyes at no such risk (Wilcoxon's signed rank test: P = 0.015).The two most important risk factors for AMD are associated with a relative absence of MP. These findings are consistent with the hypothesis that supplemental lutein and zeaxanthin may delay, avert, or modify the course of this disease.

Mitochondria are central to retinal cell function and survival. There is increasing evidence to support an association between mitochondrial dysfunction and a number of retinal pathologies including age-related macular degeneration (AMD), diabetic retinopathy and glaucoma. The past decade has highlighted mitochondrial genomic instability as an important factor in mitochondrial impairment culminating in age-related changes and age-related pathology. This represents a combination of the susceptibility of mitochondrial DNA (mtDNA) to oxidative damage and a limited base excision repair pathway. This random cumulative mtDNA damage leads to cellular heteroplasmy and, if the damage affects a sufficient proportion of mitochondria within a given cell, results in loss of cell function and greater susceptibility to stress. mtDNA damage is increased in the neural retina and RPE with ageing and appears to be greatest in AMD. It thus appears that the mitochondrial genome is a weak link in the antioxidant defenses of retinal cells and that deficits in mitochondrial DNA (mtDNA) repair pathways are important contributors to the pathogenesis of retinal degeneration. Specifically targeting mitochondria with pharmacological agents able to protect against oxidative stress or promote repair of mtDNA damage may offer potential alternatives for the treatment of retinal degenerations such as AMD.

Retinal neovascularization characterizes proliferative diabetic retinopathy (PDR). Pigment epithelium-derived factor (PEDF) has been shown to be a major antiangiogenic growth factor in the mammalian eye. PEDF expression is suppressed by hypoxia, and changes in PEDF have been correlated to the development of retinal neovascularization in animal models of hypoxic eye disease. However, whether this concept of a reduced angiogenesis inhibitor holds true in humans is as yet unclear. In this study, we analyzed the in vivo regulation of PEDF in patients with and without hypoxic eye disease. We used immunoblots to measure PEDF in ocular fluids obtained from 64 nondiabetic and diabetic patients. In addition, immunohistochemistry of PEDF was carried out in specimens of normal human retinas and retinas with various degrees of diabetic retinopathy. The PEDF concentrations in patients with PDR (P &amp;lt; 0.001) or extensive nondiabetic retinal neovascularization caused by retinal-vein occlusion (P &amp;lt; 0.001) were lower than in control patients. Levels of PEDF were replenished in PDR patients with previous retinal scatter photocoagulation compared with PDR patients without previous photocoagulation (P = 0.01). Immunohistochemistry revealed an interstitial staining pattern as expected for a secreted protein, with an intense staining in retinas of patients without proliferative eye disease. However, in patients with PDR, little or no staining was detectable. Our data strongly support the concept that retinal angiogenesis is induced by loss of the major angiogenesis inhibitor in the eye, PEDF, in combination with an increased expression of angiogenic growth factors such as vascular endothelial growth factor. Our findings suggest that substitution of angiogenesis inhibitors may be an effective approach in the treatment of PDR.

The present epidemic of diabetes is resulting in a worldwide increase in cardiovascular and microvascular complications including retinopathy. Current thinking has focused on local influences in the retina as being responsible for development of this diabetic complication. However, the contribution of circulating cells in maintenance, repair, and dysfunction of the vasculature is now becoming appreciated. Diabetic individuals have fewer endothelial progenitor cells (EPCs) in their circulation and these cells have diminished migratory potential, which contributes to their decreased reparative capacity. Using a rat model of type 2 diabetes, we show that the decrease in EPC release from diabetic bone marrow is caused by bone marrow neuropathy and that these changes precede the development of diabetic retinopathy. In rats that had diabetes for 4 mo, we observed a dramatic reduction in the number of nerve terminal endings in the bone marrow. Denervation was accompanied by increased numbers of EPCs within the bone marrow but decreased numbers in circulation. Furthermore, denervation was accompanied by a loss of circadian release of EPCs and a marked reduction in clock gene expression in the retina and in EPCs themselves. This reduction in the circadian peak of EPC release led to diminished reparative capacity, resulting in the development of the hallmark feature of diabetic retinopathy, acellular retinal capillaries. Thus, for the first time, diabetic retinopathy is related to neuropathy of the bone marrow. This novel finding shows that bone marrow denervation represents a new therapeutic target for treatment of diabetic vascular complications.

Aim-To elucidate the diurnal variation in human corneal thickness over a 48 hour period.Method-Changes in central corneal thickness were monitored in eight healthy subjects (four male, four female) aged between 10 and 63 years using an ultrasonic pachymeter.Measurements were made over a 48 hour period-immediately before sleep, immediately upon waking and at 15, 30, 45 minutes, 1, 1.5, 2, 2.5, 3 hours, and at 2 hour intervals thereafter throughout the remainder of each day.Results-The mean corneal thickness for the group (SD) was 546 ( 14) ptm, with a

Lipofuscin accumulates with age in a variety of highly metabolically active cells, including the retinal pigment epithelium (RPE) of the eye, where its photoreactivity has the potential for cellular damage. The aim of this study was to assess the phototoxic potential of lipofuscin in the retina. RPE cell cultures were fed isolated lipofuscin granules and maintained in basal medium for 7 d. Control cells lacking granules were cultured in an identical manner. Cultures were either maintained in the dark or exposed to visible light (2.8 mWcm2) at 37 degrees C for up to 48 h. Cells were subsequently assessed for alterations in cell morphology, cell viability, lysosomal stability, lipid peroxidation, and protein oxidation. Exposure of lipofuscin-fed cells to short wavelength visible light (390-550 nm) caused lipid peroxidation (increased levels of malondialdehyde and 4-hydroxy-nonenal), protein oxidation (protein carbonyl formation), loss of lysosomal integrity, cytoplasmic vacuolation, and membrane blebbing culminating in cell death. This effect was wavelength-dependent because light exposure at 550 to 800 nm had no adverse effect on lipofuscin-loaded cells. These results confirm the photoxicity of lipofuscin in a cellular system and implicate it in cell dysfunction such as occurs in ageing and retinal diseases.

<h3>AIM</h3> To determine the staining pattern of vascular endothelial growth factor (VEGF) at different stages of diabetic retinopathy (including post-laser photocoagulation) and to compare staining in excised fibrovascular and fibrocellular (non-diabetic) preretinal membranes. <h3>METHODS</h3> Immunohistochemical localisation of VEGF, using antibodies raised against VEGF₁₆₅ and VEGF_121,165,189, was carried out on specimens of normal human retina (n=15), diabetic retinas ((a) with no overt retinopathy (n=19), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n=6), (c) with active proliferative retinopathy (n=6), (d) with no residual proliferative retinopathy after photocoagulation therapy (n=15)), excised diabetic fibrovascular membranes (n=19), and non-diabetic fibrocellular membranes (n=7). The degree and pattern of immunostaining was recorded. <h3>RESULTS</h3> In general, VEGF was absent from the majority of normal retinas. VEGF staining was apparent in most diabetic tissues but the staining pattern was dependent on both the specificity of the antibody used and the category of tissue. Staining with the VEGF₁₆₅ antibody was generally confined to endothelial cells and perivascular regions while the VEGF_121,165,189antibody was also associated with extravascular components of the inner retina. Intensity of immunostaining of diabetic eyes was dependent on the severity of retinopathy being least in diabetics with no overt retinopathy and greatest in retinas with proliferative retinopathy. Interestingly, the intensity of immunostaining in diabetic retinas which had undergone laser surgery for proliferative retinopathy was reduced to basal levels. Moderate to intense immunostaining was observed in all fibrovascular and fibrocellular membranes examined. <h3>CONCLUSIONS</h3> This study supports a circumstantial role for VEGF in the pathogenesis of both the preclinical and proliferative stages of diabetic retinopathy.

PURPOSE. A fluorescent component of lipofuscin, A2-E (N-retinylidene-N-retinylethanol-amine) has been shown to impair lysosomal function and to increase the intralysosomal pH of human retinal pigment epithelial (RPE) cells. In addition to its lysosomotropic properties A2-E is known to be photoreactive. The purpose of this study was to determine the phototoxic potential of A2-E on RPE cells. METHODS. A2-E (synthesized by coupling all-trans-retinaldehyde to ethanolamine) was complexed to low-density lipoprotein (LDL) to allow for specific loading of the lysosomal compartment. Human RPE cell cultures were loaded with the A2-E-LDL complex four times within 2 weeks. A2-E accumulation was confirmed by fluorescence microscopy and flow cytometry analysis. Acridine orange staining allowed assessment of lysosomal integrity and intralysosomal pH. The phototoxic properties of A2-E were determined by exposing A2-E-free and A2-E-fed RPE cell cultures to short wavelength visible light (400-500 nm) and assessing cell viability and lysosomal integrity. RESULTS. Fluorescence microscopy and flow cytometry analysis demonstrated that the intralysosomal accumulation of A2-E in cultured RPE cells increased with the number of feedings. Acridine orange staining confirmed that the A2-E was located in the lysosomal compartment and induced an elevation of intralysosomal pH. Exposure of A2-E-fed cells to light resulted in a significant loss of cell viability by 72 hours, which was not observed in either RPE cells maintained in the dark or A2-E-free cultures exposed to light. Toxicity was associated with a loss of lysosomal integrity. CONCLUSIONS. A2-E is detrimental to RPE cell function by a variety of mechanisms: inhibition of lysosomal degradative capacity, loss of membrane integrity, and phototoxicity. Such mechanisms could contribute to retinal aging as well as retinal diseases associated with excessive lipofuscin accumulation-for example, age-related macular degeneration and Stargardt's disease.

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein (∼2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-<i>trans</i>-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E₂ adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

The angiopoietin (Ang) system plays an important role in vascular stabilization and pathologic neovascularization. The hypothesis for the study was that, in addition to modulating endothelial cell behavior, the angiopoietin/Tie-2 system also regulates the pericyte apoptosis and/or the vessel maturation associated with diabetic retinopathy.Tie-2 expression in cultured retinal pericytes was analyzed by using ELISA, Western Blot analysis, and flow cytometry. CD13 (aminopeptidase N) expression in pericytes was determined by Western blot analysis and Ang effects verified with Tie-2 antisense treatment. Cell proliferation was monitored by crystal violet uptake, and pericyte migration was assessed in a scrape wound. Annexin V-FITC flow cytometry was used to quantify pericyte apoptosis.Pericytes expressed a functionally active Tie-2 receptor upregulated by both Ang-1 and -2 (P < 0.05). In pericytes undergoing apoptosis induced by either TNF-alpha or high glucose Ang-1 increased survival (P < 0.05 for TNF-alpha; P < 0.01 for high glucose), whereas Ang-2 increased apoptosis. Ang-1 enhanced CD13 expression in a dose-dependent manner (P < 0.05) and stimulated pericyte migration in a synthetic matrix wound-healing assay that was associated with a change in F-actin organization. Addition of Tie-2 antisense confirmed that angiopoietins act through Tie-2.These findings demonstrate that Tie-2 is functional in pericytes and may play an important role in the progression of diabetic retinopathy, by regulating pericyte loss and influencing the activation state and recruitment of pericytes.

Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of beta-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of low-density lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts, were also elevated in the DR models. The high glucose-induced activation of beta-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target.

Mitochondrial DNA (mtDNA) damage may be associated with age-related diseases, such as age-related macular degeneration (AMD). The present study was designed to test whether the frequency of mtDNA damage, heteroplasmic mtDNA mutations, and repair capacity correlate with progression of AMD.Macular and peripheral RPE cells were isolated and cultured from human donor eyes with and without AMD. The stages of AMD were graded according to the Minnesota Grading System. Confluent primary RPE cells were used to test the frequency of endogenous mtDNA damage by quantitative PCR. Mutation detection kits were used to detect heteroplasmic mtDNA mutation. To test the mtDNA repair capacity, cultured RPE cells were allowed to recover for 3 and 6 hours after exposure to H(2)O(2), and repair was assessed by quantitative PCR. The levels of human OGG1 protein, which is associated with mtDNA repair, were analyzed by Western blot.This study showed that mtDNA damage increased with aging and that more lesions occurred in RPE cells from the macular region than the periphery. Furthermore, mtDNA repair capacity decreased with aging, with less mtDNA repair capacity in the macular region compared with the periphery in samples from aged subjects. Most interestingly, the mtDNA damage was positively correlated with the grading level of AMD, whereas repair capacity was negatively correlated. In addition, more mitochondrial heteroplasmic mutations were detected in eyes with AMD.These data show macula-specific increases in mtDNA damage, heteroplasmic mutations, and diminished repair that are associated with aging and AMD severity.

β-Secretase (BACE1) is a major drug target for combating Alzheimer's disease (AD). Here we show that BACE1(-/-) mice develop significant retinal pathology including retinal thinning, apoptosis, reduced retinal vascular density and an increase in the age pigment, lipofuscin. BACE1 expression is highest in the neural retina while BACE2 was greatest in the retinal pigment epithelium (RPE)/choroid. Pigment epithelial-derived factor, a known regulator of γ-secretase, inhibits vascular endothelial growth factor (VEGF)-induced in vitro and in vivo angiogenesis and this is abolished by BACE1 inhibition. Moreover, intravitreal administration of BACE1 inhibitor or BACE1 small interfering RNA (siRNA) increases choroidal neovascularization in mice. BACE1 induces ectodomain shedding of vascular endothelial growth factor receptor 1 (VEGFR1) which is a prerequisite for γ-secretase release of a 100 kDa intracellular domain. The increase in lipofuscin following BACE1 inhibition and RNAI knockdown is associated with lysosomal perturbations. Taken together, our data show that BACE1 plays a critical role in retinal homeostasis and that the use of BACE inhibitors for AD should be viewed with extreme caution as they could lead to retinal pathology and exacerbate conditions such as age-related macular degeneration.

Age-related macular degeneration (AMD) is associated with multiple genetic and cellular defects which lead to a common endpoint, retinal degeneration. Aging and oxidative stress, significant features in the pathogenesis of AMD, are associated with an increase in damaged intracellular organelles and defective autophagy flux in a range of age-related and neurodegenerative diseases. Autophagy is a key process in the maintenance of cellular homeostasis that serves to remove dysfunctional organelles and proteins. Autophagy proteins are strongly expressed in the retina and there is now strong evidence that mitochondrial damage and defective autophagy are a feature of the aging retina and that this is further exacerbated in AMD. It is apparent that autophagy makes a significant contribution to lipofuscin accumulation in the RPE. Pharmacological manipulation of autophagy may offer an alternative therapeutic target in AMD.

Two independent clinical studies have reported that fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has robust therapeutic effects on microvascular complications of diabetes, including diabetic retinopathy (DR) in type 2 diabetic patients. However, the expression and function of PPARα in the retina are unclear. Here, we demonstrated that PPARα is expressed in multiple cell types in the retina. In both type 1 and type 2 diabetes models, expression of PPARα, but not PPARβ/δ or PPARγ, was significantly down-regulated in the retina. Furthermore, high-glucose medium was sufficient to down-regulate PPARα expression in cultured retinal cells. To further investigate the role of PPARα in DR, diabetes was induced in PPARα knockout (KO) mice and wild-type (WT) mice. Diabetic PPARα KO mice developed more severe DR, as shown by retinal vascular leakage, leukostasis, pericyte loss, capillary degeneration, and over-expression of inflammatory factors, compared with diabetic WT mice. In addition, overexpression of PPARα in the retina of diabetic rats significantly alleviated diabetes-induced retinal vascular leakage and retinal inflammation. Furthermore, PPARα overexpression inhibited endothelial cell migration and proliferation. These findings revealed that diabetes-induced down-regulation of PPARα plays an important role in DR. Up-regulation or activation of PPARα may represent a novel therapeutic strategy for DR.

Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1(flox/flox)). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

Individuals with diabetes suffering from coronavirus disease 2019 (COVID-19) exhibit increased morbidity and mortality compared with individuals without diabetes. In this Perspective, we critically evaluate and argue that this is due to a dysregulated renin-angiotensin system (RAS). Previously, we have shown that loss of angiotensin-I converting enzyme 2 (ACE2) promotes the ACE/angiotensin-II (Ang-II)/angiotensin type 1 receptor (AT1R) axis, a deleterious arm of RAS, unleashing its detrimental effects in diabetes. As suggested by the recent reports regarding the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), upon entry into the host, this virus binds to the extracellular domain of ACE2 in nasal, lung, and gut epithelial cells through its spike glycoprotein subunit S1. We put forth the hypothesis that during this process, reduced ACE2 could result in clinical deterioration in COVID-19 patients with diabetes via aggravating Ang-II–dependent pathways and partly driving not only lung but also bone marrow and gastrointestinal pathology. In addition to systemic RAS, the pathophysiological response of the local RAS within the intestinal epithelium involves mechanisms distinct from that of RAS in the lung; however, both lung and gut are impacted by diabetes-induced bone marrow dysfunction. Careful targeting of the systemic and tissue RAS may optimize clinical outcomes in subjects with diabetes infected with SARS-CoV-2.

The retinal vasculature of postmortem normal human and diabetic eyes was studied using an immunohistochemical technique in conjunction with confocal laser scanning microscopy. The technique, which stained for von Willebrand factor, allowed both large areas of the retinal vasculature to be visualised and abnormalities to be studied in detail without disturbing the tissue architecture. Only one microaneurysm, defined as any focal capillary dilation, was observed in 10 normal eyes but numerous microaneurysms were seen in 4 out of 5 diabetic retinas; counts varied between 0 and 26 per 0.41 mm 2 sample area. Microaneurysms were classified into 3 categories according to morphology: saccular, fusiform and focal bulges. Most were saccular, these having no preferred orientation. The majority of microaneurysms were associated with just 2 vessels suggesting they were unlikely to develop at vascular junctions. The majority were observed to originate from the inner nuclear layer and were therefore in the deeper part of the inner retinal capillary plexus. Variation in the staining of microaneurysms may correlate with endothelial dysfunction seen clinically as dye leakage during fluorescein angiography.

Mitochondria are critical for ocular function as they represent the major source of a cell’s supply of energy and play an important role in cell differentiation and survival. Mitochondrial dysfunction can occur as a result of inherited mitochondrial mutations (e.g. Leber’s hereditary optic neuropathy and chronic progressive external ophthalmoplegia) or stochastic oxidative damage which leads to cumulative mitochondrial damage and is an important factor in age-related disorders (e.g. age-related macular degeneration, cataract and diabetic retinopathy). Mitochondrial DNA (mtDNA) instability is an important factor in mitochondrial impairment culminating in age-related changes and pathology, and in all regions of the eye mtDNA damage is increased as a consequence of aging and age-related disease. It is now apparent that the mitochondrial genome is a weak link in the defenses of ocular cells since it is susceptible to oxidative damage and it lacks some of the systems that protect the nuclear genome, such as nucleotide excision repair. Accumulation of mitochondrial mutations leads to cellular dysfunction and increased susceptibility to adverse events which contribute to the pathogenesis of numerous sporadic and chronic disorders in the eye.

The pathogenesis of diabetic retinopathy (DR) is not fully understood. Clinical studies suggest that dyslipidemia is associated with the initiation and progression of DR. However, no direct evidence supports this theory.Immunostaining of apolipoprotein B100 (ApoB100, a marker of low-density lipoprotein [LDL]), macrophages, and oxidized LDL was performed in retinal sections from four different groups of subjects: nondiabetic, type 2 diabetic without clinical retinopathy, diabetic with moderate nonproliferative diabetic retinopathy (NPDR), and diabetic with proliferative diabetic retinopathy (PDR). Apoptosis was characterized using the TUNEL assay. In addition, in cell culture studies using in vitro-modified LDL, the induction of apoptosis by heavily oxidized-glycated LDL (HOG-LDL) in human retinal capillary pericytes (HRCPs) was assessed.Intraretinal immunofluorescence of ApoB100 increased with the severity of DR. Macrophages were prominent only in sections from diabetic patients with PDR. Merged images revealed that ApoB100 partially colocalized with macrophages. Intraretinal oxidized LDL was absent in nondiabetic subjects but present in all three diabetic groups, increasing with the severity of DR. TUNEL-positive cells were present in retinas from diabetic subjects but absent in those from nondiabetic subjects. In cell culture, HOG-LDL induced the activation of caspase, mitochondrial dysfunction, and apoptosis in HRCPs.These findings suggest a potentially important role for extravasated, modified LDL in promoting DR by promoting apoptotic pericyte loss.

Advanced glycation end products (AGEs) accumulate during aging and have been observed in postmortem eyes within the retinal pigment epithelium (RPE), Bruch's membrane, and subcellular deposits (drusen). AGEs have been associated with age-related dysfunction of the RPE-in particular with development and progression to age-related macular degeneration (AMD). In the present study the impact of AGEs at the RPE-Bruch's membrane interface was evaluated, to establish how these modifications may contribute to age-related disease.AGEs on Bruch's membrane were evaluated using immunohistochemistry. A clinically relevant in vitro model of substrate AGE accumulation was established to mimic Bruch's membrane ageing. Responses of ARPE-19 growing on AGE-modified basement membrane (AGE-BM) for 1 month were investigated by using a microarray approach and validated by quantitative (q)RT-PCR. In addition to identified AGE-related mRNA alterations, lysosomal enzyme activity and lipofuscin accumulation were also studied in ARPE-19 grown on AGE-BM.Autofluorescent and glycolaldehyde-derived AGEs were observed in clinical specimens on Bruch's membrane and choroidal extracellular matrix. In vitro analysis identified a range of dysregulated mRNAs in ARPE-19 exposed to AGE-BM. Altered ARPE-19 degradative enzyme mRNA expression was observed on exposure to AGE-BM. AGE-BM caused a significant reduction in cathepsin-D activity in ARPE-19 (P < 0.05) and an increase in lipofuscin accumulation (P < 0.01).AGEs influence ARPE-19 mRNA expression profiles and may contribute to reduced lysosomal enzyme degradative capacity and enhanced accumulation of lipofuscin. Formation of AGEs on Bruch's membrane may have important consequences for age-related dysfunction of the RPE, perhaps leading to age-related outer retinal disease.

The aryl hydrocarbon receptor (AhR) is a nuclear receptor that regulates xenobiotic metabolism and detoxification. Herein, we report a previously undescribed role for the AhR signaling pathway as an essential defense mechanism in the pathogenesis of early dry age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. We found that AhR activity and protein levels in human retinal pigment epithelial (RPE) cells, cells vulnerable in AMD, decrease with age. This finding is significant given that age is the most established risk factor for development of AMD. Moreover, AhR(-/-) mice exhibit decreased visual function and develop dry AMD-like pathology, including disrupted RPE cell tight junctions, accumulation of RPE cell lipofuscin, basal laminar and linear-like deposit material, Bruch's membrane thickening, and progressive RPE and choroidal atrophy. High-serum low-density lipoprotein levels were also observed in AhR(-/-) mice. In its oxidized form, this lipoprotein can stimulate increased secretion of extracellular matrix molecules commonly found in deposits from RPE cells, in an AhR-dependent manner. This study demonstrates the importance of cellular clearance via the AhR signaling pathway in dry AMD pathogenesis, implicating AhR as a potential target, and the mouse model as a useful platform for validating future therapies.

The retinal pigment epithelium contains three major types of pigment granules; melanosomes, lipofuscin and melanolipofuscin. Melanosomes in the retinal pigment epithelium (RPE) are formed during embryogenesis and mature during early postnatal life while lipofuscin and melanolipofuscin granules accumulate as a function of age. The difficulty in studying the formation and consequences of melanosomes and lipofuscin granules in RPE cell culture is compounded by the fact that these pigment granules do not normally occur in established RPE cell lines and pigment granules are rapidly lost in adult human primary culture. This review will consider options available for overcoming these limitations and permitting the study of melanosomes and lipofuscin in cell culture and will briefly evaluate the advantages and disadvantages of the different protocols.

All explosive volcanic eruptions generate volcanic ash, fragments of rock that are produced when magma or vent material is explosively disintegrated. Volcanic ash is then convected upwards within the eruption column and carried downwind, falling out of suspension and potentially affecting communities across hundreds, or even thousands, of square kilometres. Ash is the most frequent, and often widespread, volcanic hazard and is produced by all explosive volcanic eruptions. Although ash falls rarely endanger human life directly, threats to public health and disruption to critical infrastructure services, aviation and primary production can lead to potentially substantial societal impacts and costs, even at thicknesses of only a few millimetres. Communities exposed to any magnitude of ash fall commonly report anxiety about the health impacts of inhaling or ingesting ash (as well as impacts to animals and property damage), which may lead to temporary socio-economic disruption (e.g. evacuation, school and business closures, cancellations). The impacts of any ash fall can therefore be experienced across large areas and can also be long-lived, both because eruptions can last weeks, months or even years and because ash may be remobilised and re-deposited by wind, traffic or human activities.

Abstract Ocular neovascularisation underlies blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. These diseases cause irreversible vision loss, and provide a significant health and economic burden. Biologics targeting vascular endothelial growth factor (VEGF) are the major approach for treatment. However, up to 30% of patients are non-responsive to these drugs and they are associated with ocular and systemic side effects. Therefore, there is a need for small molecule ocular angiogenesis inhibitors to complement existing therapies. We examined the safety and therapeutic potential of SH-11037, a synthetic derivative of the antiangiogenic homoisoflavonoid cremastranone, in models of ocular neovascularisation. SH-11037 dose-dependently suppressed angiogenesis in the choroidal sprouting assay ex vivo and inhibited ocular developmental angiogenesis in zebrafish larvae. Additionally, intravitreal SH-11037 (1 μM) significantly reduced choroidal neovascularisation (CNV) lesion volume in the laser-induced CNV mouse model, comparable to an anti-VEGF antibody. Moreover, SH-11037 synergised with anti-VEGF treatments in vitro and in vivo . Up to 100 μM SH-11037 was not associated with signs of ocular toxicity and did not interfere with retinal function or pre-existing retinal vasculature. SH-11037 is thus a safe and effective treatment for murine ocular neovascularisation, worthy of further mechanistic and pharmacokinetic evaluation.

Chronic inflammation and dysregulation of circadian rhythmicity are involved in the pathogenesis of diabetic retinopathy. MicroRNAs (miRNAs) can regulate inflammation and circadian clock machinery. We tested the hypothesis that altered daily rhythm of miR-146a expression in diabetes contributes to retinal inflammation.Nondiabetic and STZ-induced diabetic rats kept in 12/12 light/dark cycle were killed every 2 hours over a 72-hour period. Human retinal endothelial cells (HRECs) were synchronized with dexamethasone. Expression of miR-146a, IL-1 receptor-associated kinase 1 (IRAK1), IL-1β, VEGF and ICAM-1, as well as clock genes was examined by real-time PCR and Western blot. To modulate expression levels of miR-146a, mimics and inhibitors were used.Diabetes inhibited amplitude of negative arm (per1) and enhanced amplitude of the positive arm (bmal1) of clock machinery in retina. In addition to clock genes, miR-146a and its target gene IRAK1 also exhibited daily oscillations in antiphase; however, these patterns were lost in diabetic retina. This loss of rhythmic pattern was associated with an increase in ICAM-1, IL-β, and VEGF expression. Human retinal endothelial cells had robust miR-146a expression that followed circadian oscillation pattern; however, HRECs isolated from diabetic donors had reduced miR-146a amplitude but increased amplitude of IRAK1 and ICAM-1. In HRECs, miR-146a mimic or inhibitor caused 1.6- and 1.7-fold decrease or 1.5- and 1.6-fold increase, respectively, in mRNA and protein expression levels of ICAM-1 after 48 hours.Diabetes-induced dysregulation of daily rhythms of miR-146a and inflammatory pathways under miR-146a control have potential implications for the development of diabetic retinopathy.

Background: We examined components of systemic and intestinal renin-angiotensin system on gut barrier permeability, glucose homeostasis, systemic inflammation, and progression of diabetic retinopathy (DR) in human subjects and mice with type 1 diabetes (T1D). Methods: T1D individual with (n=18) and without (n=20) DR and controls (n=34) were examined for changes in gut-regulated components of the immune system, gut leakage markers (FABP2 [fatty acid binding protein 2] and peptidoglycan), and Ang II (angiotensin II); Akita mice were orally administered a Lactobacillus paracasei (LP) probiotic expressing humanized ACE2 (angiotensin-converting enzyme 2) protein (LP-ACE2) as either a prevention or an intervention. Akita mice with genetic overexpression of humanAce2 by small intestine epithelial cells ( Vil-Cre.hAce2KI-Akita ) were similarly examined. After 9 months of T1D, circulatory, enteral, and ocular end points were assessed. Results: T1D subjects exhibit elevations in gut-derived circulating immune cells (ILC1 cells) and higher gut leakage markers, which were positively correlated with plasma Ang II and DR severity. The LP-ACE2 prevention cohort and genetic overexpression of intestinal ACE2 preserved barrier integrity, reduced inflammatory response, improved hyperglycemia, and delayed development of DR. Improvements in glucose homeostasis were due to intestinal MasR activation, resulting in a GSK-3β (glycogen synthase kinase-3 beta)/c-Myc (cellular myelocytomatosis oncogene)-mediated decrease in intestinal glucose transporter expression. In the LP-ACE2 intervention cohort, gut barrier integrity was improved and DR reversed, but no improvement in hyperglycemia was observed. These data support that the beneficial effects of LP-ACE2 on DR are due to the action of ACE2, not improved glucose homeostasis. Conclusions: Dysregulated systemic and intestinal renin-angiotensin system was associated with worsening gut barrier permeability, gut-derived immune cell activation, systemic inflammation, and progression of DR in human subjects. In Akita mice, maintaining intestinal ACE2 expression prevented and reversed DR, emphasizing the multifaceted role of the intestinal renin-angiotensin system in diabetes and DR.

Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury.To examine the mechanism of IGFBP-3-mediated repair following vascular injury.We used 2 complementary vascular injury models: laser occlusion of retinal vessels in adult green fluorescent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3-expressing plasmid into lasered GFP chimeric mice stimulated homing of EPCs, whereas reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous NO generation. Thus, we asked whether the migratory effects of IGFBP-3 were attributable to stimulation of NO generation. IGFBP-3 increased endothelial NO synthase expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator-stimulated phosphoprotein, an NO regulated protein critical for cell migration. IGFBP-3-mediated NO generation required high-density lipoprotein receptor activation and stimulation of phosphatidylinositol 3-kinase/Akt pathway.These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.

Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and β-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and β-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.

In this study, we assessed whether Per2 clock gene–mutant mice exhibit a vascular phenotype similar to diabetes. Per2 (B6.129-Per2tm1Drw/J) or wild-type control mice 4 and 12 months of age were used. To evaluate diabetes-like phenotype in Per2 mutant mice, retina was quantified for mRNA expression, and degree of diabetic retinopathy was evaluated. Bone marrow neuropathy was studied by staining femurs for tyrosine hydroxylase (TH) and neurofilament 200 (NF-200). The rate of proliferation and quantification of bone marrow progenitor cells (BMPCs) was performed, and a threefold decrease in proliferation and 50% reduction in nitric oxide levels were observed in Per2 mutant mice. TH-positive nerve processes and NF-200 staining were reduced in Per2 mutant mice. Both retinal protein and mRNA expression of endothelial nitric oxide synthase were decreased by twofold. Other endothelial function genes (VEGFR2, VEGFR1) were downregulated (1.5–2-fold) in Per2 mutant retinas, whereas there was an upregulation of profibrotic pathway mediated by transforming growth factor-β1. Our studies suggest that Per2 mutant mice recapitulate key aspects of diabetes without the metabolic abnormalities, including retinal vascular damage, neuronal loss in the bone marrow, and diminished BMPC function.

Effective inhibition of angiogenesis targeting the tumor endothelial cells requires identification of key cellular and molecular mechanisms associated with survival of vasculatures within the tumor microenvironment. Intracellular autocrine (intracrine) VEGF production by endothelial cells plays a critical role on the vasculature homeostasis. In vitro breast cancer cell-stimulated activation of the unfolded protein response (UPR) of the endothelial cells contributes to maintenance of the intracrine VEGF levels in the endothelial cells through the upregulation of a previous undescribed downstream effector- αB-crystallin (CRYAB). siRNA-mediated knockdown of two major UPR proteins-inositol requiring kinase 1 and ATF6, led to attenuated CRYAB expression of the endothelial cells. Finally, inhibition of CRYAB blocked the breast cancer cell-stimulated increase in the endogenous VEGF levels of the endothelial cells. A VEGF limited proteolysis assay further revealed that CRYAB protected VEGF for proteolytic degradation. Here, we report that the molecular chaperone-CRYAB was significantly increased and colocalized with tumor vessels in a breast cancer xenograft. Specifically, neutralization of VEGF induced higher levels of CRYAB expression in the endothelial cells cocultured with MDA-MB-231 or the breast cancer xenograft with a significant survival benefit. However, knockdown of CRYAB had a greater inhibitory effect on endothelial survival. These findings underscore the importance of defining a role for intracrine VEGF signaling in sustaining aberrant tumor angiogenesis and strongly implicate UPR/CRYAB as dichotomous parts of a crucial regulation pathway for maintaining intracrine VEGF signaling.

By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow–neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow–derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45⁺/CCR2⁺/GR-1⁺/Iba-1⁺ bone marrow–derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.

We hypothesized that endothelial progenitor cells derived from individuals with diabetes would exhibit functional defects including inability to respond to hypoxia and altered paracrine/autocrine function that would impair the angiogenic potential of these cells. Circulating mononuclear cells isolated from diabetic (n = 69) and nondiabetic (n = 46) individuals were used to grow endothelial colony forming cells (ECFC), early endothelial progenitor cells (eEPCs) and isolate CD34+ cells. ECFCs and eEPCs were established from only 15% of the diabetic individuals tested thus directing our main effort toward examination of CD34+ cells. CD34+ cells were plated in basal medium to obtain cell-free conditioned medium (CM). In CM derived from CD34+ cells of diabetic individuals (diabetic-CM), the levels of stem cell factor, hepatocyte growth factor, and thrombopoietin were lower, and IL-1β and tumor necrosis factor (TNFα) levels were higher than CM derived from nondiabetic individuals (nondiabetic-CM). Hypoxia did not upregulate HIF1α in CD34+ cells of diabetic origin. Migration and proliferation of nondiabetic CD34+ cells toward diabetic-CM were lower compared to nondiabetic-CM. Attenuation of pressure-induced constriction, potentiation of bradykinin relaxation, and generation of cGMP and cAMP in arterioles were observed with nondiabetic-CM, but not with diabetic-CM. Diabetic-CM failed to induce endothelial tube formation from vascular tissue. These results suggest that diabetic subjects with microvascular complications exhibit severely limited capacity to generate ex-vivo expanded endothelial progenitor populations and that the vasoreparative dysfunction observed in diabetic CD34+ cells is due to impaired autocrine/paracrine function and reduced sensitivity to hypoxia.

Although anti-vascular endothelial growth factor (VEGF) treatments reduce pathological neovascularization in the eye and in tumors, the regression is often not sustainable or is incomplete. We investigated whether vascular endothelial cells circumvent anti-VEGF therapies by activating the unfolded protein response (UPR) to override the classic extracellular VEGF pathway. Exposure of endothelial cells to VEGF, high glucose, or H2O2 up-regulated the X-box binding protein-1/inositol-requiring protein-1 (IRE1) α and activating transcription factor 6 (ATF6) arms of the UPR compared with untreated cells. This was associated with increased expression in α-basic crystallin (CRYAB), which has previously bound VEGF. siRNA knockdown or pharmacological blockade of IRE1α, ATF6, or CRYAB increased intracellular VEGF degradation and decreased full-length intracellular VEGF. Inhibition of IRE1α, ATF6, or CRYAB resulted in an approximately 40% reduction of in vitro angiogenesis, which was further reduced in combination with a neutralizing antibody against extracellular VEGF. Blockade of IRE1α or ATF6 in the oxygen-induced retinopathy or choroidal neovascularization mouse models caused an approximately 35% reduction in angiogenesis. However, combination therapy of VEGF neutralizing antibody with UPR inhibitors or siRNAs reduced retinal/choroidal neovascularization by a further 25% to 40%, and this inhibition was significantly greater than either treatment alone. In conclusion, activation of the UPR sustains angiogenesis by preventing degradation of intracellular VEGF. The IRE1α/ATF6 arms of the UPR offer a potential therapeutic target in the treatment of pathological angiogenesis. Although anti-vascular endothelial growth factor (VEGF) treatments reduce pathological neovascularization in the eye and in tumors, the regression is often not sustainable or is incomplete. We investigated whether vascular endothelial cells circumvent anti-VEGF therapies by activating the unfolded protein response (UPR) to override the classic extracellular VEGF pathway. Exposure of endothelial cells to VEGF, high glucose, or H2O2 up-regulated the X-box binding protein-1/inositol-requiring protein-1 (IRE1) α and activating transcription factor 6 (ATF6) arms of the UPR compared with untreated cells. This was associated with increased expression in α-basic crystallin (CRYAB), which has previously bound VEGF. siRNA knockdown or pharmacological blockade of IRE1α, ATF6, or CRYAB increased intracellular VEGF degradation and decreased full-length intracellular VEGF. Inhibition of IRE1α, ATF6, or CRYAB resulted in an approximately 40% reduction of in vitro angiogenesis, which was further reduced in combination with a neutralizing antibody against extracellular VEGF. Blockade of IRE1α or ATF6 in the oxygen-induced retinopathy or choroidal neovascularization mouse models caused an approximately 35% reduction in angiogenesis. However, combination therapy of VEGF neutralizing antibody with UPR inhibitors or siRNAs reduced retinal/choroidal neovascularization by a further 25% to 40%, and this inhibition was significantly greater than either treatment alone. In conclusion, activation of the UPR sustains angiogenesis by preventing degradation of intracellular VEGF. The IRE1α/ATF6 arms of the UPR offer a potential therapeutic target in the treatment of pathological angiogenesis. It is evident that there exists a plethora of pro- and anti-angiogenic factors that regulate the ocular vasculature and influence the development and progression of aberrant neovascularization, such as in diabetic retinopathy and age-related macular degeneration (AMD). Furthermore, the spatiotemporal balance of these pro- and anti-angiogenic factors is critical in determining whether vascular homeostasis or a pathological condition predominates. The collective evidence suggests that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogenesis,1Penn J.S. Madan A. Caldwell R.B. Bartoli M. Caldwell R.W. Hartnett M.E. Vascular endothelial growth factor in eye disease.Prog Retin Eye Res. 2008; 27: 331-371Crossref PubMed Scopus (533) Google Scholar and treatment of patients with AMD who have choroidal neovascularization (CNV) using inhibitors of extracellular VEGF, such as ranibizumab (Lucentis) or bevacizumab (Avastin), significantly reduces CNV.2Abouammoh M. Sharma S. Ranibizumab versus bevacizumab for the treatment of neovascular age-related macular degeneration.Curr Opin Ophthalmol. 2011; 22: 152-158Crossref PubMed Scopus (37) Google Scholar, 3Chiang A. Regillo C.D. Preferred therapies for neovascular age-related macular degeneration.Curr Opin Ophthalmol. 2011; 22: 199-204Crossref PubMed Scopus (25) Google Scholar However, as shown by the ANCHOR, MARINA, and VISION clinical trials, regression is often not sustainable or is incomplete and only approximately 50% of patients benefit significantly from this therapeutic strategy.4Brown D.M. Michels M. Kaiser P.K. Heier J.S. Sy J.P. Ianchulev T. ANCHOR Study GroupRanibizumab versus verteporfin photodynamic therapy for neovascular age-related macular degeneration: two-year results of the ANCHOR study.Ophthalmology. 2009; 116: 57-65.e5Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar, 5Chang T.S. Bressler N.M. Fine J.T. Dolan C.M. Ward J. Klesert T.R. Improved vision-related function after ranibizumab treatment of neovascular age-related macular degeneration: results of a randomized clinical trial.Arch Ophthalmol. 2007; 125: 1460-1469Crossref PubMed Scopus (200) Google Scholar Furthermore, similar limitations are associated with the use of these anti-VEGF agents in treatment of proliferative diabetic retinopathy and diabetic macular edema.6Waisbourd M. Goldstein M. Loewenstein A. Treatment of diabetic retinopathy with anti-VEGF drugs.Acta Ophthalmol. 2011; 89: 203-207Crossref PubMed Scopus (48) Google Scholar The challenge, therefore, is to find an adjunct to the current therapy that will obviate the repeated injections, lower the dose of exogenous VEGF blocker, and act synergistically to elicit complete regression of the vascular lesion. There is increasing evidence from the cancer field that endothelial cells protect themselves and sustain tumor angiogenesis by intracellular accumulation of VEGF and self-regulation through an intracellular pathway.7Lee S. Chen T.T. Barber C.L. Jordan M.C. Murdock J. Desai S. Ferrara N. Nagy A. Roos K.P. Iruela-Arispe M.L. Autocrine VEGF signaling is required for vascular homeostasis.Cell. 2007; 130: 691-703Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar, 8Gerber H.P. Ferrara N. The role of VEGF in normal and neoplastic hematopoiesis.J Mol Med. 2003; 81: 20-31Crossref PubMed Scopus (185) Google Scholar Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR)9Glembotski C.C. The role of the unfolded protein response in the heart.J Mol Cell Cardiol. 2008; 44: 453-459Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 10Malhotra J.D. Kaufman R.J. The endoplasmic reticulum and the unfolded protein response.Semin Cell Dev Biol. 2007; 18: 716-731Crossref PubMed Scopus (795) Google Scholar play a critical role in transcriptional regulation of VEGF-A11Ghosh R. Lipson K.L. Sargent K.E. Mercurio A.M. Hunt J.S. Ron D. Urano F. Transcriptional regulation of VEGF-A by the unfolded protein response pathway.PLoS One. 2010; 5: e9575Crossref PubMed Scopus (201) Google Scholar and protect VEGF from intracellular degradation.12Ruan Q. Han S. Jiang W.G. Boulton M.E. Chen Z.J. Law B.K. Cai J. alphaB-crystallin, an effector of unfolded protein response, confers anti-VEGF resistance to breast cancer via maintenance of intracrine VEGF in endothelial cells.Mol Cancer Res. 2011; 9: 1632-1643Crossref PubMed Scopus (53) Google Scholar ER stress can activate one or more of the three ER stress sensors [protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring protein-1 (IRE1), and activating transcription factor 6 (ATF6)], leading to activation of the UPR pathway and production of chaperone proteins.9Glembotski C.C. The role of the unfolded protein response in the heart.J Mol Cell Cardiol. 2008; 44: 453-459Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 10Malhotra J.D. Kaufman R.J. The endoplasmic reticulum and the unfolded protein response.Semin Cell Dev Biol. 2007; 18: 716-731Crossref PubMed Scopus (795) Google Scholar α-Basic crystallin (CRYAB) is a classic small heat shock protein that is up-regulated by ER stress and has interactive sequences for VEGF.13Ghosh J.G. Shenoy Jr., A.K. Clark J.I. Interactions between important regulatory proteins and human alphaB crystallin.Biochemistry. 2007; 46: 6308-6317Crossref PubMed Scopus (88) Google Scholar A strong association between CRYAB expression and angiogenesis has been shown. By using CRYAB−/− mice, Kase et al14Kase S. He S. Sonoda S. Kitamura M. Spee C. Wawrousek E. Ryan S.J. Kannan R. Hinton D.R. alphaB-crystallin regulation of angiogenesis by modulation of VEGF.Blood. 2010; 115: 3398-3406Crossref PubMed Scopus (140) Google Scholar reported an attenuation of CNV. Dimberg et al15Dimberg A. Rylova S. Dieterich L.C. Olsson A.K. Schiller P. Wikner C. Bohman S. Botling J. Lukinius A. Wawrousek E.F. Claesson-Welsh L. alphaB-crystallin promotes tumor angiogenesis by increasing vascular survival during tube morphogenesis.Blood. 2008; 111: 2015-2023Crossref PubMed Scopus (86) Google Scholar showed that tumors in CRYAB−/− mice are significantly less vascularized than in wild-type mice. Based on these observations, we assessed whether activation of the UPR and downstream CRYAB up-regulation may sustain the VEGF signaling pathway and if targeting both extracellular VEGF and the UPR will represent a more effective strategy than current extracellular VEGF therapies alone. Bovine retinal microvascular endothelial cells were isolated and cultured, as previously described.16Cai J. Jiang W.G. Grant M.B. Boulton M. Pigment epithelium-derived factor inhibits angiogenesis via regulated intracellular proteolysis of vascular endothelial growth factor receptor 1.J Biol Chem. 2006; 281: 3604-3613Crossref PubMed Scopus (221) Google Scholar Briefly, freshly isolated bovine retinas were homogenized, and after trapping on an 83-μm nylon mesh, they were transferred into an enzyme mixture [500 μg/mL collagenase, 200 μg/mL protease (Pronase), and DNase] at 37°C for 20 minutes. The resultant vessel fragments were trapped on a 53-μm mesh, washed, and pelleted, and cells were plated in endothelial cell basal medium with growth supplement (Invitrogen, Carlsbad, CA) at 37°C, 5% CO2, for 3 days. The cells were used within three passages. Retinal microvascular endothelial cells were treated with 100 ng/mL VEGF, 25 mmol/L glucose, or 200 μmol/L H2O2 for 24 hours. The 5.2 mmol/L medium alone acted as the baseline control because 5.2 mmol/L glucose is considered the normoglycemic control for cultured endothelial cells and hyperglycemia is usually in the range from 15 to 30 mmol/L glucose. We chose to use 25 mmol/L glucose based on our previous studies and 25 mmol/L mannitol as an osmotic control.17Zhao B. Cai J. Boulton M. Expression of placenta growth factor is regulated by both VEGF and hyperglycaemia via VEGFR-2.Microvasc Res. 2004; 68: 239-246Crossref PubMed Scopus (46) Google Scholar The expression levels of X-box binding protein (XBP)-1, ATF6, IRE1, PERK, CRYAB, and α-tubulin were determined by using Western blot analysis. Retinal endothelial cells were cultured in 6-well plates. After 24-hour treatment with 100 ng/mL VEGF, 25 mmol/L glucose, or 200 μmol/L H2O2, the cells were rinsed in PBS, and fresh microvascular endothelial basal medium with 5% fetal calf serum was added. After a further 24 hours, the culture medium was removed and centrifuged at 2000 × g for 10 minutes to remove cellular debris. The cell numbers of each well were counted before preparing total cell lysates using radioimmunoprecipitation assay (RIPA) buffer. VEGF concentrations were measured in duplicate in each sample from at least three separate experiments with a VEGF DuoSet enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The results are expressed as ng/106 cells per day VEGF. We selected a list of compounds reported to be capable of inhibiting different UPR pathways (Table 1) and then measured their cytotoxicity using cultured retinal microvascular endothelial cells. In brief, cells were exposed to a serial dilution of test compound for 24 hours, and cytotoxicity was determined using the Cytotoxicity Detection Kit (Roche Applied Science, Indianapolis, IN), according to the manufacturer’s instructions. This kit can detect lactate dehydrogenase activity released from damaged cells, and results are presented as the LD50. Vehicle was used as a baseline control.Table 1LD50 of Compounds That Inhibit UPR Proteins in Bovine Microvascular Endothelial CellsCompoundsID50 (μmol/L)UPR proteinsMG13275IRE1α/XBP-117-AAG2IRE1α1-NM-PP150IRE1αAEBSF1500ATF6Lactacystin35IRE1α/XBP-1AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride. Open table in a new tab AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride. Two siRNAs were synthesized by Life Technologies Corporation (Carlsbad, CA) against each of bovine CRYAB, IRE-1, ATF6, and PERK. The siRNA sequences are shown in Table 2. Scrambled siRNA acted as a negative control. Bovine microvascular endothelial cells were seeded in 6-well tissue culture dishes for 24 hours before transfection with respective siRNA (30 nmol/L) using siPORT transfection agent (Life Technologies Corporation, Carlsbad, CA) per the manufacturer instructions. Knockdown was confirmed by Western blot analysis.Table 2Corresponding Silencer siRNA Construction Kit Template SequencessiRNASense primerAntisense primerIRE15′-AAGTACCAAATGTCCTGCTTTCCTGTCTC-3′5′-AAAAAGCAGGACATTTGGTACCCTGTCTC-3′IRE15′-AAATGGCGATCATCTTCTCTACCTGTCTC-3′5′-AATAGAGAAGATGATCGCCATCCTGTCTC-3′ATF65′-AAAGTCACGGAAAGTTTTCCACCTGTCTC-3′5′-AATGGAAAACTTTCCGTGACTCCTGTCTC-3′ATF65′-AATCACTCGACCTCCCAACTTCCTGTCTC-3′5′-AAAAGTTGGGAGGTCGAGTGACCTGTCTC-3′PERK5′-AATGCCTTCGATGTTGTTGTACCTGTCTC-3′5′-AATACAACAACATCGAAGGCACCTGTCTC-3′PERK5′-AACATTGAAGACACCCCTCTTCCTGTCTC-3′5′-AAAAGAGGGGTGTCTTCAATGCCTGTCTC-3′CRYAB5′-AAATCCTGGCGCTCTTCATGTCCTGTCTC-3′5′-AAACATGAAGAGCGCCAGGATCCTGTCTC-3′CRYAB5′-AAGAATCCGTCAGCTTCAGCACCTGTCTC-3′5′-AATGCTGAAGCTGACGGATTCCCTGTCTC-3′Silencer siRNA construction kit (part No. AM1620; Life Technologies Corporation, Carlsbad, CA). Open table in a new tab Silencer siRNA construction kit (part No. AM1620; Life Technologies Corporation, Carlsbad, CA). Western blot analysis and immunoprecipitation were performed as previously described.18Cai J. Chen Z. Ruan Q. Han S. Liu L. Qi X. Boye S.L. Hauswirth W.W. Grant M.B. Boulton M.E. γ-Secretase and presenilin mediate cleavage and phosphorylation of vascular endothelial growth factor receptor-1.J Biol Chem. 2011; 286: 42514-42523Crossref PubMed Scopus (36) Google Scholar In brief, cells were washed with PBS and lysed in RIPA buffer at 4°C containing a cocktail of protease (Sigma-Aldrich, St. Louis, MO) and phosphatase (Roche Applied Science) inhibitors for 30 minutes. Lysates were centrifuged, supernatants were collected, and total protein concentration was determined by the MicroBCA reagent assay (Pierce, Rockford, IL). Western blot analysis was performed after SDS-PAGE (10% gel; equal aliquot of total proteins per lane) and transfer onto polyvinylidene difluoride membranes. The IRE1α, ATF6, CRYAB, and VEGF proteins were detected using an affinity-purified rabbit or mouse anti-IRE1α (Sigma-Aldrich), anti-ATF6 (Sigma-Aldrich), anti-CRYAB (Novus Biologicals, LLC, Littleton, CO), and anti-VEGF (Sigma-Aldrich), followed by incubation with polyclonal anti-rabbit in goat or mouse in rabbit horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich) at 1:2000 dilution. The proteins were detected and visualized with the ECL Plus Western Blotting Detection System (GE Healthcare Biosciences, Pittsburgh, PA), and resultant bands were normalized against α-tubulin (Sigma-Aldrich). The intensity of each immunoreactive band was quantified using ImageJ software version 1.46 (NIH, Bethesda, MD).18Cai J. Chen Z. Ruan Q. Han S. Liu L. Qi X. Boye S.L. Hauswirth W.W. Grant M.B. Boulton M.E. γ-Secretase and presenilin mediate cleavage and phosphorylation of vascular endothelial growth factor receptor-1.J Biol Chem. 2011; 286: 42514-42523Crossref PubMed Scopus (36) Google Scholar For immunoprecipitation, total proteins were immunoprecipitated with 10 μg of relevant antibodies for 2.5 hours at 4°C, followed by the addition of 20 μL of protein A/G-agarose overnight at 4°C. The resultant pellet was dissolved in 2× Laemmli buffer (Bio-Rad Laboratories, Hercules, CA) and subjected to Western blot analysis. In vitro tube formation assays were performed as previously described.16Cai J. Jiang W.G. Grant M.B. Boulton M. Pigment epithelium-derived factor inhibits angiogenesis via regulated intracellular proteolysis of vascular endothelial growth factor receptor 1.J Biol Chem. 2006; 281: 3604-3613Crossref PubMed Scopus (221) Google Scholar Briefly, near-confluent microvascular endothelial cells were pretreated with 100 ng/mL VEGF for 2 hours and then either treated for 24 hours with test compounds at serial concentrations, as indicated, or VEGF165 neutralizing antibody or washed and maintained in basal medium for 24 hours to establish the intracrine VEGF signaling pathway, before treatment with test compounds or VEGF165 neutralizing antibody (anti-human VEGF165, AB-293-NA; R&D Systems), which we have previously shown to bind strongly to bovine VEGF. Cells without VEGF treatment or with VEGF only were used as control. In addition, siRNA-treated cells, with or without VEGF, were also assessed. The cells were then detached and plated at the concentration of 2.5 × 104 per well on 24-well plates coated with 12.5% (v/v) growth factor–poor Matrigel (BD, Franklin Lakes, NJ) and left overnight. The medium was then aspirated, and 250 μL per well of 12.5% Matrigel was overlaid on the cells for 2 hours to allow the polymerization of Matrigel, followed by the addition of 500 μL per well of basal medium, MCD131, with 10% fetal calf serum for 24 hours. The following day, the culture plates were observed under a phase-contrast microscope and imaged using five random fields (original magnification, ×10). The tubule length (mm/mm2) per microscope field was quantified. VEGF degradation was assessed as previously described.12Ruan Q. Han S. Jiang W.G. Boulton M.E. Chen Z.J. Law B.K. Cai J. alphaB-crystallin, an effector of unfolded protein response, confers anti-VEGF resistance to breast cancer via maintenance of intracrine VEGF in endothelial cells.Mol Cancer Res. 2011; 9: 1632-1643Crossref PubMed Scopus (53) Google Scholar Briefly, the cells were trypsinized and pelleted at 2000 × g for 5 minutes. The resultant pellets were dissolved in 500 μL of denaturing buffer (20 mmol/L Tris-HCl, pH 8.0, 6 mol/L guanidine-HCl, 5 mmol/L EDTA, and 4 mmol/L dithiothreitol) at room temperature for 2 hours. Total protein concentration was estimated by measuring the UV absorbance at 280 nm and diluted with denaturing buffer to a final concentration of 1 mg/mL. After centrifugation at 46,000 × g at 4°C for 30 minutes, the supernatant was concentrated to 100 μL using Millipore Ultrafree centrifugal devices (molecular weight cutoff, 3.5 kDa) (Millipore, Bedford, MA). The samples were mixed with 1:2 standard sample buffer and analyzed by using Western blot analysis using an antibody against the C-terminal of human VEGF (Abcam, Cambridge, MA). In accordance with previous studies, the degradation of VEGF resulted in multiple lower molecular weight bands, of which a fragment of approximately 38 kDa is prominent.19Lauer G. Sollberg S. Cole M. Krieg T. Eming S.A. Generation of a novel proteolysis resistant vascular endothelial growth factor165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar All mice were housed under specific pathogen-free conditions and handled in accordance with the Association for Research in Vision and Ophthalmology statement for Use of Animals in Ophthalmic and Vision Research and the guidelines of the Institutional Animal Care and Use Committee at the University of Florida (Gainesville, FL). Two animal models were used; the oxygen-induced retinopathy (OIR) model, which is representative of retinal neovascularization, as occurs in retinopathy of prematurity and diabetic retinopathy, and the laser-induced CNV model, which is representative of aberrant vascular growth in AMD. For the OIR model, C57BL/6 mouse pups were exposed to 75% oxygen from postnatal day 7 (P7) to P12, along with nursing mothers.20Caballero S. Sengupta N. Afzal A. Chang K.H. Li Calzi S. Guberski D.L. Kern T.S. Grant M.B. Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells.Diabetes. 2007; 56: 960-967Crossref PubMed Scopus (221) Google Scholar On day P12, the pups were removed from high oxygen conditions, and on P17, mice were sacrificed. Eyes were fixed in paraformaldehyde, paraffin embedding was performed, and serial sections (5 μm thick) were obtained. Representative sections (every 30th section) across the retina were stained with H&E to visualize retinal structure and note any overt toxicity. A masked investigator counted all endothelial cell nuclei above the internal limiting membrane in all representative sections through each eye. Vascular cell nuclei are considered to be associated with neovascularization if they are on the vitreous side of the internal limiting membrane and the region of the optic nerve is not considered. The data presented represent random sampling from a minimum of 12 pups per treatment group. Efficacy of treatment was calculated as the percentage average nuclei per section in the eyes treated with test agent compared with vehicle control. The laser procedure was undertaken as previously described.21Qi X. Cai J. Ruan Q. Liu L. Boye S.L. Chen Z. Hauswirth W.W. Ryals R.C. Shaw L. Caballero S. Grant M.B. Boulton M.E. Presenilin-1 Inhibition of Murine Choroidal neovascularization is Associated with Reduction of Superoxide and Proinflammatory Cytokines.Invest Ophthalmol Vis Sci. 2012; 53: 574-585Crossref PubMed Scopus (12) Google Scholar Briefly, 8-week-old female C57BL/6 mice were anesthetized with a mixture of 80 mg/kg ketamine and 10 mg/kg xylazine, and their pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine. Under the fundus microscope, an argon green ophthalmic laser, coupled to a slit lamp set to deliver a 50-millisecond pulse at 200 mW, with a 50-μm spot size, was used to rupture the Bruch’s membrane in three quadrants of the right eye, located approximately 50 mm from the optic disk at relative positions of 9 AM, 12 PM, and 3 PM. The left eye served as an untreated control. Mice were sacrificed 14 days after laser injury, and the eyes were enucleated. For measuring lesion volume, we used a vascular-specific dye, Ricinus Communis Agglutinin I conjugated to rhodamine, to label whole flat mounts of retinal pigment epithelium (RPE)/choroid/sclera. The laser lesions were then imaged by Z-series laser scanning confocal microscopy to obtain lesion volume using ImageJ software version 1.46. In all CNV studies, animals were randomized and treatments were blinded until all analysis was complete. All determinations were performed in at least six animals per group. In addition, gross retinal/choroidal structure and vascular patterns were examined for possible adverse effects of the test compound or vehicle. Mice received an intravitreal injection in the left eye of 0.5 μL in the OIR model and 1.0 μL in the CNV model while the right eye acted as the uninjected control. Groups of six animals received the following treatments. Ambion in Vivo siRNAs predesigned and prevalidated for targeting murine CRYAB (s64457), IRE1 (s95857), ATF6 (s105469), and scrambled were purchased from Life Technologies Corporation. These siRNAs incorporated additional chemical modifications for superior serum stability with in vivo applications and reduced susceptibility to degradation by ribonucleases. All siRNAs were 19mer to avoid the potential anti-angiogenic effect of 21mer or longer siRNAs acting via toll-like receptor 3.22Kleinman M.E. Yamada K. Takeda A. Chandrasekaran V. Nozaki M. Baffi J.Z. Albuquerque R.J. Yamasaki S. Itaya M. Pan Y. Appukuttan B. Gibbs D. Yang Z. Kariko K. Ambati B.K. Wilgus T.A. DiPietro L.A. Sakurai E. Zhang K. Smith J.R. Taylor E.W. Ambati J. Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.Nature. 2008; 452: 591-597Crossref PubMed Scopus (808) Google Scholar As previously determined, 1 and 2 μg per eye siRNA was delivered intravitreally at P12 in the OIR model or the time of laser injury in the CNV model. A total of 5 μmol/L of potential UPR pathway inhibitor, MG132, and 50 μmol/L 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) were delivered intravitreally at P12 in the OIR model or the time of laser injury in the CNV model. Saline or dimethyl sulfoxide vehicle controls were also included. A total of 5 ng per eye VEGF164 neutralizing antibody (anti-mouse VEGF164, AF-493-NA; R&D Systems) was delivered intravitreally at P12 in the OIR model or the time of laser injury in the CNV model. In some studies, the antibody was also given at 7 days after laser injury. Vehicle and 5 ng per eye of the appropriate Fc fragment (Jackson Immunoresearch, West Grove, PA) were included as controls. For combination therapy, 5 ng per eye of VEGF164 neutralizing antibody was delivered intravitreally, together with siRNA or UPR inhibitor at P12 in the OIR model or the time of laser injury in the CNV model. In some studies, the antibody was also given at 7 days after laser injury. For PCR assessment, 6-week-old female C57BL/6J mice received an intravitreal injection of 2 μg/μL siRNA at laser injury in the CNV model into the left eye, whereas the right eye served as an uninjected control. All siRNAs were as previously described. Mice were sacrificed at 2 days after treatment, eyes were enucleated, and the retina plus RPE/choroid were isolated. Total RNA was extracted with TRIzol total RNA isolation reagent (Invitrogen) and quantified using a Nanodrop Spectrophotometer (NanoDrop Products, Wilmington, DE). cDNA was synthesized from 1 μg total RNA from each sample using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA). cDNA at 1:10 dilution was used for quantitative RT-PCR (RT-qPCR) on a BioRad C1000 Thermal Cycler. mRNA expression was assessed using the RT2 qPCR Primer Assay from Qiagen (Valencia, CA), with the following primers: mouse Ern1 (PPM36937A-200), mouse Cryab (PPM03570F-200), and mouse Atf6 (PPM33057A-200). Final expression was normalized to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH): RT2 qPCR Primer Assay for mouse GAPDH (PPM02946E-200). Relative transcript abundance was determined by using the ΔΔCT or ΔCT method after normalization with GAPDH. All samples were run in triplicate. Error bars represent SEM. For immunohistochemistry (IHC), 6-week-old female C57BL/6J mice received an intravitreal injection of 2 μg/μL siRNA at laser injury and were sacrificed at 14 days after treatment. Eyes were enucleated, and the RPE/choroid/sclera were prepared and then blocked with 10% normal goat sera, plus 1% bovine serum albumin for 2 hours at room temperature. Mouse monoclonal anti-CRYAB, anti-ATF6, and rabbit polyclonal anti-IRE1 (Lifespan Biosciences, Seattle, WA) were diluted 1:50 and 1:200, respectively, in PBS with 1% normal goat sera plus 1% bovine serum albumin and incubated overnight at 4°C. Tissues received a secondary antibody conjugated with fluorescein isothiocyanate in 1:600 for 1 hour at room temperature in the dark. To visualize vessels, we used a vascular-specific dye, Ricinus Communis Agglutinin I, conjugated to tetramethylrhodamine isothiocyanate (TRITC), to label the whole flat mounts of RPE/choroid, which were incubated for 30 minutes at room temperature in 1:400 of 10 mmol/L HEPES, 150 mmol/L NaCl, and 0.1% Tween 20. The tissues were covered in aqueous VectaShield mounting medium (Vector Laboratories, Inc., Burlingame, CA) for observation on an Olympus DSU-Olympus IX81 confocal microscope (Olympus America, Inc., Center Valley, PA). All experiments were repeated at least three times. Results are expressed as means ± SEM. The Mann-Whitney test was used to determine statistical significance of the densitometry data of Western blot analysis. An unpaired two-tailed Student’s t-test was performed for the significance of the results of ELISA, in vitro tubule formation assay, in vivo CNV, and OIR models. Statistical analysis was performed using Prism 5 version 5.01 (GraphPad Software, Inc., La Jolla, CA) with P < 0.05 considered statistically significant. It has previously been reported that growth factors, hyperglycemia, and oxidative stress can activate the ER stress response.9Glembotski C.C. The role of the unfolded protein response in the heart.J Mol Cell Cardiol. 2008; 44: 453-459Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 10Malhotra J.D. Kaufman R.J. The endoplasmic reticulum and the unfolded protein response.Semin Cell Dev Biol. 2007; 18: 716-731Crossref PubMed Scopus (795) Google Scholar Western blot analysis demonstrated that exposure of microvascular endothelial cells to VEGF, high glucose, or H2O2 caused

Recently it has been shown that levels of circulating oxidized LDL immune complexes (ox-LDL-ICs) predict the development of diabetic retinopathy (DR). This study aimed to investigate whether ox-LDL-ICs are actually present in the diabetic retina, and to define their effects on human retinal pericytes versus ox-LDL. In retinal sections from people with type 2 diabetes, costaining for ox-LDL and IgG was present, proportionate to DR severity, and detectable even in the absence of clinical DR. In contrast, no such staining was observed in retinas from nondiabetic subjects. In vitro, human retinal pericytes were treated with native LDL, ox-LDL, and ox-LDL-IC (0–200 mg protein/l), and measures of viability, receptor expression, apoptosis, endoplasmic reticulum (ER) and oxidative stresses, and cytokine secretion were evaluated. Ox-LDL-IC exhibited greater cytotoxicity than ox-LDL toward retinal pericytes. Acting through the scavenger (CD36) and IgG (CD64) receptors, low concentrations of ox-LDL-IC triggered apoptosis mediated by oxidative and ER stresses, and enhanced inflammatory cytokine secretion. The data suggest that IC formation in the diabetic retina enhances the injurious effects of ox-LDL. These findings offer new insights into pathogenic mechanisms of DR, and may lead to new preventive measures and treatments. Recently it has been shown that levels of circulating oxidized LDL immune complexes (ox-LDL-ICs) predict the development of diabetic retinopathy (DR). This study aimed to investigate whether ox-LDL-ICs are actually present in the diabetic retina, and to define their effects on human retinal pericytes versus ox-LDL. In retinal sections from people with type 2 diabetes, costaining for ox-LDL and IgG was present, proportionate to DR severity, and detectable even in the absence of clinical DR. In contrast, no such staining was observed in retinas from nondiabetic subjects. In vitro, human retinal pericytes were treated with native LDL, ox-LDL, and ox-LDL-IC (0–200 mg protein/l), and measures of viability, receptor expression, apoptosis, endoplasmic reticulum (ER) and oxidative stresses, and cytokine secretion were evaluated. Ox-LDL-IC exhibited greater cytotoxicity than ox-LDL toward retinal pericytes. Acting through the scavenger (CD36) and IgG (CD64) receptors, low concentrations of ox-LDL-IC triggered apoptosis mediated by oxidative and ER stresses, and enhanced inflammatory cytokine secretion. The data suggest that IC formation in the diabetic retina enhances the injurious effects of ox-LDL. These findings offer new insights into pathogenic mechanisms of DR, and may lead to new preventive measures and treatments. Diabetic retinopathy (DR) remains a leading cause of vision loss in working-age adults. Early disease is characterized by vascular abnormalities, including pericyte loss, basement membrane thickening, microaneurysm formation, and capillary leakage (1Cogan D.G. Toussaint D. Kuwabara T. Retinal vascular patterns. IV. Diabetic retinopathy.Arch. Ophthalmol. 1961; 66: 366-378Crossref PubMed Scopus (507) Google Scholar, 2Kern T.S. Engerman R.L. Vascular lesions in diabetes are distributed non-uniformly within the retina.Exp. Eye Res. 1995; 60: 545-549Crossref PubMed Scopus (103) Google Scholar). Pericytes are critical to vascular integrity (3Hammes H.P. Pericytes and the pathogenesis of diabetic retinopathy.Horm. Metab. Res. 2005; 37: 39-43Crossref PubMed Scopus (187) Google Scholar), and their loss is considered an initiating event in DR. Dyslipidemia has been implicated in DR, and associations between plasma lipoprotein profiles and disease severity have been observed in large cohort studies (4Lloyd C.E. Klein R. Maser R.E. Kuller L.H. Becker D.J. Orchard T.J. The progression of retinopathy over 2 years: the Pittsburgh Epidemiology of Diabetes Complications (EDC) Study.J. Diabetes Complications. 1995; 9: 140-148Crossref PubMed Scopus (75) Google Scholar, 5Lyons T.J. Jenkins A.J. Zheng D. Lackland D.T. McGee D. Garvey W.T. Klein R.L. Diabetic retinopathy and serum lipoprotein subclasses in the DCCT/EDIC cohort.Invest. Ophthalmol. Vis. Sci. 2004; 45: 910-918Crossref PubMed Scopus (248) Google Scholar, 6van Leiden H.A. Dekker J.M. Moll A.C. Nijpels G. Heine R.J. Bouter L.M. Stehouwer C.D. Polak B.C. Blood pressure, lipids, and obesity are associated with retinopathy: the Hoorn study.Diabetes Care. 2002; 25: 1320-1325Crossref PubMed Scopus (325) Google Scholar), but were not of sufficient strength to define risk for individuals. The term “dyslipidemia” may include qualitative as well as quantitative alterations in plasma lipoproteins. The qualitative abnormalities include modification by oxidation, but oxidized LDL (ox-LDL) constitutes only a small fraction of total plasma LDL, ranging from 0.001% in healthy people to 5% in the presence of cardiovascular disease (7Holvoet P. De Keyzer D. Jacobs Jr, D.R. Oxidized LDL and the metabolic syndrome.Future Lipidol. 2008; 3: 637-649Crossref PubMed Scopus (86) Google Scholar). We therefore hypothesized that the dominant effects of lipoproteins in DR occur not in plasma, but rather after extravasation through leaking blood-retina barriers (BRBs) (8Wu M. Chen Y. Wilson K. Chirindel A. Ihnat M.A. Yu Y. Boulton M.E. Szweda L.I. Ma J-X. Lyons T.J. Intraretinal leakage and oxidation of LDL in diabetic retinopathy.Invest. Ophthalmol. Vis. Sci. 2008; 49: 2679-2685Crossref PubMed Scopus (80) Google Scholar, 9Song W. Barth J.L. Lu K. Yu Y. Huang Y. Gittinger C.K. Argraves W.S. Lyons T.J. Effects of modified low-density lipoproteins on human retinal pericyte survival.Ann. N. Y. Acad. Sci. 2005; 1043: 390-395Crossref PubMed Scopus (21) Google Scholar, 10Song W. Barth J.L. Yu Y. Lu K. Dashti A. Huang Y. Gittinger C.K. Argraves W.S. Lyons T.J. Effects of oxidized and glycated LDL on gene expression in human retinal capillary pericytes.Invest. Ophthalmol. Vis. Sci. 2005; 46: 2974-2982Crossref PubMed Scopus (32) Google Scholar, 11Diffley J.M. Wu M. Sohn M. Song W. Hammad S.M. Lyons T.J. Apoptosis induction by oxidized glycated LDL in human retinal capillary pericytes is independent of activation of MAPK signaling pathways.Mol. Vis. 2009; 15: 135-145PubMed Google Scholar, 12Wu M. Yang S. Elliott M.H. Fu D. Wilson K. Zhang J. Du M. Chen J. Lyons T. Oxidative and endoplasmic reticulum stresses mediate apoptosis induced by modified LDL in human retinal Müller cells.Invest. Ophthalmol. Vis. Sci. 2012; 53: 4595-4604Crossref PubMed Scopus (52) Google Scholar, 13Zhang S.X. Wang J.J. Dashti A. Wilson K. Zou M.H. Szweda L. Ma J.X. Lyons T.J. Pigment epithelium-derived factor mitigates inflammation and oxidative stress in retinal pericytes exposed to oxidized low-density lipoprotein.J. Mol. Endocrinol. 2008; 41: 135-143Crossref PubMed Scopus (63) Google Scholar, 14Du M. Wu M. Fu D. Yang S. Chen J. Wilson K. Lyons T.J. Effects of modified LDL and HDL on retinal pigment epithelial cells: a role in diabetic retinopathy?.Diabetologia. 2013; 56: 2318-2328Crossref PubMed Scopus (58) Google Scholar, 15Fu D. Wu M. Zhang J. Du M. Yang S. Hammad S.M. Wilson K. Chen J. Lyons T.J. Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy.Diabetologia. 2012; 55: 3128-3140Crossref PubMed Scopus (88) Google Scholar); the extent of this leakage may be more important than plasma concentrations of lipoproteins. We further hypothesized that extravasated intra-retinal lipoproteins undergo extensive modification (glycation, oxidation), rendering them cytotoxic (8Wu M. Chen Y. Wilson K. Chirindel A. Ihnat M.A. Yu Y. Boulton M.E. Szweda L.I. Ma J-X. Lyons T.J. Intraretinal leakage and oxidation of LDL in diabetic retinopathy.Invest. Ophthalmol. Vis. Sci. 2008; 49: 2679-2685Crossref PubMed Scopus (80) Google Scholar). Thus in DR, as in atherosclerosis (16Nishi K. Itabe H. Uno M. Kitazato K.T. Horiguchi H. Shinno K. Nagahiro S. Oxidized LDL in carotid plaques and plasma associates with plaque instability.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1649-1654Crossref PubMed Scopus (340) Google Scholar, 17Ylä-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar), extravascular modified LDL could play an important role in disease progression; but in contrast to the artery, extravasation of LDL in the retina does not occur unless BRB leakage is present, and thus particularly affects people with diabetes. Supporting these hypotheses, we demonstrated that apolipoprotein B and ox-LDL were present in diabetic human retinas, even before the development of clinical retinopathy, but were absent in nondiabetic retinas (8Wu M. Chen Y. Wilson K. Chirindel A. Ihnat M.A. Yu Y. Boulton M.E. Szweda L.I. Ma J-X. Lyons T.J. Intraretinal leakage and oxidation of LDL in diabetic retinopathy.Invest. Ophthalmol. Vis. Sci. 2008; 49: 2679-2685Crossref PubMed Scopus (80) Google Scholar). Consistent with a general neurovascular retinal insult in DR (18Antonetti D.A. Klein R. Gardner T.W. Diabetic retinopathy.N. Engl. J. Med. 2012; 366: 1227-1239Crossref PubMed Scopus (1156) Google Scholar), we also found evidence of a broad array of cytotoxic effects: “highly oxidized, glycated LDL” promoted oxidative stress, endoplasmic reticulum (ER) stress, inflammation and abnormal gene expression, and decreased viability, not only in retinal vascular cells (endothelial cells, pericytes), but also in Müller glia and retinal pigment epithelial cells (RPEs) (9Song W. Barth J.L. Lu K. Yu Y. Huang Y. Gittinger C.K. Argraves W.S. Lyons T.J. Effects of modified low-density lipoproteins on human retinal pericyte survival.Ann. N. Y. Acad. Sci. 2005; 1043: 390-395Crossref PubMed Scopus (21) Google Scholar, 10Song W. Barth J.L. Yu Y. Lu K. Dashti A. Huang Y. Gittinger C.K. Argraves W.S. Lyons T.J. Effects of oxidized and glycated LDL on gene expression in human retinal capillary pericytes.Invest. Ophthalmol. Vis. Sci. 2005; 46: 2974-2982Crossref PubMed Scopus (32) Google Scholar, 11Diffley J.M. Wu M. Sohn M. Song W. Hammad S.M. Lyons T.J. Apoptosis induction by oxidized glycated LDL in human retinal capillary pericytes is independent of activation of MAPK signaling pathways.Mol. Vis. 2009; 15: 135-145PubMed Google Scholar, 12Wu M. Yang S. Elliott M.H. Fu D. Wilson K. Zhang J. Du M. Chen J. Lyons T. Oxidative and endoplasmic reticulum stresses mediate apoptosis induced by modified LDL in human retinal Müller cells.Invest. Ophthalmol. Vis. Sci. 2012; 53: 4595-4604Crossref PubMed Scopus (52) Google Scholar, 13Zhang S.X. Wang J.J. Dashti A. Wilson K. Zou M.H. Szweda L. Ma J.X. Lyons T.J. Pigment epithelium-derived factor mitigates inflammation and oxidative stress in retinal pericytes exposed to oxidized low-density lipoprotein.J. Mol. Endocrinol. 2008; 41: 135-143Crossref PubMed Scopus (63) Google Scholar, 14Du M. Wu M. Fu D. Yang S. Chen J. Wilson K. Lyons T.J. Effects of modified LDL and HDL on retinal pigment epithelial cells: a role in diabetic retinopathy?.Diabetologia. 2013; 56: 2318-2328Crossref PubMed Scopus (58) Google Scholar, 15Fu D. Wu M. Zhang J. Du M. Yang S. Hammad S.M. Wilson K. Chen J. Lyons T.J. Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy.Diabetologia. 2012; 55: 3128-3140Crossref PubMed Scopus (88) Google Scholar). It is now well-known that modification of LDL may render it immunogenic, leading to the formation of LDL immune complexes (LDL-ICs) (19Truman J.P. Al Gadban M.M. Smith K.J. Jenkins R.W. Mayroo N. Virella G. Lopes-Virella M.F. Bielawska A. Hannun Y.A. Hammad S.M. Differential regulation of acid sphingomyelinase in macrophages stimulated with oxidized LDL and oxidized LDL immune complexes: role in phagocytosis and cytokine release.Immunology. 2012; 136: 30-45Crossref PubMed Scopus (38) Google Scholar). Recently, in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) cohort, we found that increased levels of circulating ox-LDL-ICs predicted risk for severe nonproliferative DR (NPDR) and proliferative DR (PDR) in type 1 diabetes (20Lopes-Virella M.F. Baker N.L. Hunt K.J. Lyons T.J. Jenkins A.J. Virella G. High concentrations of AGE-LDL and oxidized LDL in circulating immune complexes are associated with progression of retinopathy in type 1 diabetes.Diabetes Care. 2012; 35: 1333-1340Crossref PubMed Scopus (46) Google Scholar). Others have shown that plasma levels of antibodies to malondialdehyde-modified apolipoprotein B-100 are correlated with DR severity in type 2 diabetes (21Fredrikson G.N. Anand D.V. Hopkins D. Corder R. Alm R. Bengtsson E. Shah P.K. Lahiri A. Nilsson J. Associations between autoantibodies against apolipoprotein B-100 peptides and vascular complications in patients with type 2 diabetes.Diabetologia. 2009; 52: 1426-1433Crossref PubMed Scopus (45) Google Scholar). Further emphasizing the importance of ox-LDL-ICs, it is now established that in plasma, only a minor proportion of ox-LDL circulates free; 95% circulates in ICs (7Holvoet P. De Keyzer D. Jacobs Jr, D.R. Oxidized LDL and the metabolic syndrome.Future Lipidol. 2008; 3: 637-649Crossref PubMed Scopus (86) Google Scholar, 19Truman J.P. Al Gadban M.M. Smith K.J. Jenkins R.W. Mayroo N. Virella G. Lopes-Virella M.F. Bielawska A. Hannun Y.A. Hammad S.M. Differential regulation of acid sphingomyelinase in macrophages stimulated with oxidized LDL and oxidized LDL immune complexes: role in phagocytosis and cytokine release.Immunology. 2012; 136: 30-45Crossref PubMed Scopus (38) Google Scholar). For ox-LDL-ICs, as with ox-LDL, changes observed in plasma may provide only an indirect index of events in tissue. Thus in the retina, effects of ox-LDL-ICs could predominate over those of ox-LDL, and it is important to define whether IC formation alters the known toxicity of ox-LDL toward retinal cells. In the present study, we aimed to determine whether ox-LDL-ICs are indeed present in the diabetic retina, and to define their effects on human retinal pericytes versus ox-LDL. The study was approved by the Institutional Review Boards of the University of Oklahoma Health Sciences Center and the Medical University of South Carolina, and was conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from each living participant. Retinas were obtained postmortem from 15 type 2 diabetic and 5 nondiabetic humans through the National Disease Research Interchange (NDRI) (Philadelphia, PA) as described previously (8Wu M. Chen Y. Wilson K. Chirindel A. Ihnat M.A. Yu Y. Boulton M.E. Szweda L.I. Ma J-X. Lyons T.J. Intraretinal leakage and oxidation of LDL in diabetic retinopathy.Invest. Ophthalmol. Vis. Sci. 2008; 49: 2679-2685Crossref PubMed Scopus (80) Google Scholar). Subjects were grouped as follows: nondiabetic, diabetic without clinical DR, NPDR, and PDR (n = 5), based on the diagnosis provided by NDRI. Ages were similar across the groups (nondiabetic, 62.6 ± 14.9 years; diabetic without DR, 69.4 ± 9.9 years; NPDR, 70.6 ± 8.3 years; and PDR, 57 ± 6.5 years; mean ± SD, P > 0.05 by one-way ANOVA). The eyes were obtained and fixed in 10% neutral-buffered formalin within 12 h after death. For immunohistochemistry, retinal sections (5 μm) were incubated overnight with rabbit polyclonal anti-ox-LDL or goat polyclonal anti-human IgG antibody (Abcam, Cambridge, MA), followed by detection with fluorescence-conjugated anti-rabbit or -goat antibodies (Life Technologies, Carlsbad, CA) and confocal microscopy (Olympus, Japan) as described (8Wu M. Chen Y. Wilson K. Chirindel A. Ihnat M.A. Yu Y. Boulton M.E. Szweda L.I. Ma J-X. Lyons T.J. Intraretinal leakage and oxidation of LDL in diabetic retinopathy.Invest. Ophthalmol. Vis. Sci. 2008; 49: 2679-2685Crossref PubMed Scopus (80) Google Scholar). Absence of nonspecific tissue binding by secondary antibodies was confirmed. Native LDL (N-LDL) was isolated by sequential ultracentrifugation (density 1.019–1.063) of fresh plasma pooled from 4 to 6 fasted healthy volunteers; ox-LDL was prepared as before (12Wu M. Yang S. Elliott M.H. Fu D. Wilson K. Zhang J. Du M. Chen J. Lyons T. Oxidative and endoplasmic reticulum stresses mediate apoptosis induced by modified LDL in human retinal Müller cells.Invest. Ophthalmol. Vis. Sci. 2012; 53: 4595-4604Crossref PubMed Scopus (52) Google Scholar). For preparation of insoluble ox-LDL-ICs, human ox-LDL antibodies were isolated using a two-step protocol involving affinity chromatography with immobilized protein G (Protein G-Sepharose 4 Fast Flow; Amersham-Pharmacia Biotech, Piscataway, NJ) and fractionation by affinity chromatography in Sepharose-linked ox-LDL (22Virella G. Atchley D. Koskinen S. Zheng D. Lopes-Virella M.F. Group D.E.R. Proatherogenic and proinflammatory properties of immune complexes prepared with purified human oxLDL antibodies and human oxLDL.Clin. Immunol. 2002; 105: 81-92Crossref PubMed Scopus (96) Google Scholar). Isolated ox-LDL antibodies were centrifuged (90,000 g, 45 min) to eliminate aggregates, sterilized, and confirmed free of endotoxin using the Etoxate kit (detection limit 0.005–0.01 ng/ml; Sigma-Aldrich, St. Louis, MO). Subsequently, human ox-LDL-ICs were prepared (22Virella G. Atchley D. Koskinen S. Zheng D. Lopes-Virella M.F. Group D.E.R. Proatherogenic and proinflammatory properties of immune complexes prepared with purified human oxLDL antibodies and human oxLDL.Clin. Immunol. 2002; 105: 81-92Crossref PubMed Scopus (96) Google Scholar), with the ratio of ox-LDL and IgG antibody approximately 1:3.3 (w/w). Human retinal pericytes (Cambrex, Walkersville, MD) were cultured in EBM-2 medium (glucose 5.5 mM) supplemented with the EGM-2 SingleQuot kit (Lonza, Allendale, NJ). Cells of passages 3–9 (80–85% confluence) were pretreated with serum-free medium (SFM) for 18–24 h before being treated with lipoprotein preparations (50–200 mg protein/l). In some experiments, 50 mg/l monoclonal antibodies (Ancell Corp., Bayport, MN) were employed to block CD16, CD32, CD36, and CD64 receptors. Tunicamycin, 4-phenylbutyric acid (4-PBA), tauroursodeoxycholic acid (TUDCA), Tempol, and N-acetyl cysteine were obtained from Sigma-Aldrich. Pericyte viability was determined in 96-well plates (1 × 104 cells/well) using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Rockville, MD) (15Fu D. Wu M. Zhang J. Du M. Yang S. Hammad S.M. Wilson K. Chen J. Lyons T.J. Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy.Diabetologia. 2012; 55: 3128-3140Crossref PubMed Scopus (88) Google Scholar). Cultured pericytes were detached from the flask with trypsin, which was neutralized using a Lonza ReagentPack, per vendor's instruction (Lonza). Cells were then incubated with FITC-labeled anti-CD16, -CD32, -CD36, and -CD64 antibodies (R&D, Minneapolis, MN) for 25 min on wet ice and washed with PBS/2%BSA. Cells were resuspended in 300 μl fluorescent-activated cell sorting (FACS) solution, immediately followed by flow cytometry (FACS Calibur; BD Biosciences, San Jose, CA). Phosphatidylserine externalization was measured using an annexin V-FITC Apoptosis Detection Kit II (BD Pharmingen, Franklin Lakes, NJ) according to the manufacturer's manual. In brief, pericytes were trypsinized, washed twice with cold PBS, and resuspended in a binding buffer [10 mM HEPES/NaOH, (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2] at a density of 1 × 106 cells/ml. One hundred microliters of the cell suspension were transferred to a 5 ml tube to which 5 μl annexin V-FITC and 5 μl propidium iodide were added. After 20 min incubation at room temperature in the dark, an additional 400 μl of binding buffer was added, followed by immediate analysis by flow cytometry. Caspase-3 activity was measured by a caspase-3 activity assay kit (Cell Signaling Technology, Danvers, MA) per manufacturer's instructions. Briefly, extracted pericyte lysates containing 10 μg protein were added to a black 96-well plate containing 200 μl fluorogenic substrate solution [25 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10 mM DTT, 20 μM Ac-DEVD-AMC] and then incubated at 37°C for 2 h in the dark. Fluorescence was quantified by a VICTOR3 microplate reader (PerkinElmer, Waltham, MA), with excitation at 380 nm and emission at 460 nm. DiOC6(3) (Life Technologies) was used to evaluate the mitochondrial membrane potential (Δψm) changes (15Fu D. Wu M. Zhang J. Du M. Yang S. Hammad S.M. Wilson K. Chen J. Lyons T.J. Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy.Diabetologia. 2012; 55: 3128-3140Crossref PubMed Scopus (88) Google Scholar). Pericytes were harvested and resuspended in 400 m l PBS containing 20 nM DiOC6(3) followed by 15 min incubation at 37°C in the dark and then analyzed by flow cytometry (FACS Calibur; BD Biosciences). The results are shown as the proportion of cells exhibiting low Δψm as indicated by reduced DiOC6(3) uptake. Cells were homogenized (Complete Lysis Buffer; Roche, Indianapolis, IN) and protein concentrations determined (BCA assay; Pierce, Rutherford, IL). Protein (30 μg) was resolved by SDS-PAGE and blotted with antibodies against 3-nitrotyrosine, glutathione peroxidase 1, 78 kDa glucose-regulated protein (GRP78), phospho-inositol requiring enzyme 1α (p-IRE1α) (all from Abcam), phospho-protein kinase-like ER kinase (p-PERK), C/EBP-homologous protein (CHOP), activated caspase-3, and cleaved poly ADP ribose polymerase (PARP) (all from Cell Signaling Technology). Blots were stripped and reblotted with antibody against β-actin for standardization. Immunofluorescence for activating transcription factor 6 (ATF6) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed as previously (15Fu D. Wu M. Zhang J. Du M. Yang S. Hammad S.M. Wilson K. Chen J. Lyons T.J. Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy.Diabetologia. 2012; 55: 3128-3140Crossref PubMed Scopus (88) Google Scholar). In brief, pericytes were fixed (4% paraformaldehyde, 20 min), permeabilized, and incubated overnight at 4°C with primary antibody against ATF6 (Abcam), followed by the secondary antibody for 1 h. Apoptosis was assessed by the TUNEL assay (In-Situ-Cell-Death-Detection kit; Roche). Intracellular reactive oxygen species (ROS) were measured by the dichlorodihydrofluorescein method using CM-H2DCFDA (Invitrogen, Carlsbad, CA) as per manufacturer's instructions. Pericytes were washed, incubated with 2 μM CM-H2DCFDA in phenol red-free medium (37°C, 20 min), and fluorescence was measured (VICTOR3; PerkinElmer). Interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), pigment epithelium-derived factor (PEDF), and vascular endothelial growth factor (VEGF) were assayed in cell supernatants (DuoSet ELISA; R&D), per manufacturer's instructions. Data are expressed as means ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test (Prism 5; GraphPad, La Jolla, CA). P ≤ 0.05 was considered significant. Figure 1 shows the representative ox-LDL and IgG immunostaining in retinal sections from nondiabetic and three categories of type 2 diabetic subjects (no clinical DR, NPDR, PDR). No signal was detectable in nondiabetic retinas. Staining for ox-LDL (red) and IgG (green) was observed in all three diabetic groups, increasing with DR severity. Colocalization of ox-LDL and IgG was clearly seen in the photos merging the two stains, consistent with the presence of ox-LDL-ICs in diabetic retina. As shown in Fig. 2, both ox-LDL-ICs and ox-LDL decreased pericyte viability in a dose-dependent (0–200 mg/l) and time-dependent (0–48 h) manner. There was a significant leftward shift of the dose-response relationship for ox-LDL-ICs versus ox-LDL, indicating much higher potency of ox-LDL-ICs in triggering cell death. At 50 mg/l, ox-LDL-ICs elicited cell death much earlier that ox-LDL (6 vs. 48 h). To identify the receptors mediating ox-LDL-IC-induced toxicity, we examined the expression of CD36 (receptor for ox-LDL as well as other molecules and multi-molecular complexes), CD16 and CD32 (low-affinity IgG receptors, FcγRIII and FcγRII, respectively), and CD64 (high-affinity FcγRI). CD36 and CD64 were expressed on the pericyte surface, but neither CD16 nor CD32 was detectable (Fig. 3A). Ox-LDL-ICs, but not ox-LDL, induced CD64 upregulation versus SFM and N-LDL. To determine the role of CD36 and CD64, cells were pretreated with relevant blocking antibodies, and viability was determined. Both anti-CD36 and anti-CD64 attenuated ox-LDL-IC-induced cell death, and when combined, evidence suggesting additional protection was observed (P < 0.1 vs. anti-CD36 alone; Fig. 3B). Because 50 mg/l of ox-LDL was not lethal to pericytes over 24 h, this concentration was selected for additional studies. Flow cytometry showed that ox-LDL-IC dramatically increased apoptosis (annexin V-positive and propidium iodide-negative cells) versus SFM, N-LDL, or ox-LDL (Fig. 4A); apoptosis was attenuated by antagonism of CD36, CD64, or both combined (all P ≤ 0.05). There was a trend toward a greater effect by combined antagonism than by either alone (P = 0.06). TUNEL assay yielded similar results (Fig. 4B). Ox-LDL-ICs decreased Δψm (Fig. 4C, D). Ox-LDL-ICs also increased protein levels of both the cleaved PARP and activated caspase-3 (Fig. 4E), as well as caspase-3 activity (Fig. 4F). In contrast, for all endpoints, 50 mg/l ox-LDL produced minimal effects versus N-LDL. The data support apoptosis mediated by CD36 and CD64 receptors as the mechanism of pericyte death induced by ox-LDL-ICs, and demonstrate that ox-LDL-ICs have greater cytotoxicity than ox-LDL. Upon treatment with ox-LDL-ICs, pericytes expressed higher levels of GRP78, an ER stress chaperone, PERK phosphorylation, and CHOP, a pro-apoptotic factor activated in ER stress (Fig. 5A). ATF6 nuclear translocation was increased (Fig. 5B). These findings indicate increased ER stress; however, phosphorylation of IRE1α was unchanged. Tunicamycin (an ER stress inducer) was used to determine whether ER stress was sufficient to induce apoptosis. After 12 h exposure, increased GRP78 and CHOP confirmed ER stress (Fig. 5C), while caspase-3 activation and PARP cleavage confirmed apoptosis (Fig. 5D). The ER stress inhibitors, 4-PBA and TUDCA, were also employed prior to ox-LDL-IC challenge (Fig. 5E–H). They attenuated ER stress (Fig. 5E), reduced apoptosis (Fig. 5F), and mitigated mitochondrial dysfunction (Fig. 5G, H). Again, CD36 and CD64 blockade inhibited apoptosis and improved mitochondrial function (Fig. 5F–H). The data support a role for ER stress in ox-LDL-IC-induced apoptosis, implicating CHOP. Ox-LDL-ICs significantly increased intracellular ROS versus N- or ox-LDL in a time-dependent fashion (Fig. 6A). The increase was attenuated by pretreatment with the antioxidant Tempol, and by blockade of CD36 and CD64. Ox-LDL-ICs also increased levels of 3-nitrotyrosine-positive proteins, and decreased glutathione peroxidase 1, whereas N- and ox-LDL did not (Fig. 6B). Antioxidants Tempol and N-acetyl cysteine decreased ER stress markers (Fig. 6C, D), suggesting that oxidative stress is upstream of ER stress. Again, blockade of CD36/CD64 was protective. Secretion of cytokines related to inflammation, oxidative stress, and angiogenesis was determined (Fig. 7). Compared with SFM, ox-LDL-ICs significantly increased cellular secretion of IL-6 and sICAM-1, decreased PEDF, and had no effect on sVCAM-1 or VEGF. Ox-LDL affected only IL-6, while N-LDL was without effect. We present the first evidence for the presence/formation of ox-LDL-ICs in human diabetic retinas. Immunostaining for ox-LDL, detectable prior to the onset of clinical DR, confirmed earlier observations (8Wu M. Chen Y. Wilson K. Chirindel A. Ihnat M.A. Yu Y. Boulton M.E. Szweda L.I. Ma J-X. Lyons T.J. Intraretinal leakage and oxidation of LDL in diabetic retinopathy.Invest. Ophthalmol. Vis. Sci. 2008; 49: 2679-2685Crossref PubMed Scopus (80) Google Scholar), while costaining for IgG supported the concept of ox-LDL-IC formation in the retina. We therefore compared effects of ox-LDL-ICs versus ox-LDL (and N-LDL) on human retinal capillary pericytes in vitro. We found that ox-LDL-ICs exerted greater toxicity toward retinal capillary pericytes than ox-LDL; low concentrations, at which ox-LDL itself was almost without effect, induced oxidative and ER stresses, apoptosis, increased secretion of inflammatory cytokines, and reduced secretion of a key anti-angiogenic factor, PEDF. The scavenger receptor CD36 and Fcγ receptor I (CD64) were implicated. Previously, we demonstrated the presence of intra-retinal apolipoprotein B and ox-LDL, attributing it to vascular leakage (no LDL was detectable in the absence of diabetes) (8Wu M. Chen Y. Wilson K. Chirindel A. Ihnat M.A. Yu Y. Boulton M.E. Szweda L.I. Ma J-X. Lyons T.J. Intraretinal leakage and oxidation of LDL in diabetic retinopathy.Invest. Ophthalmol. Vis. Sci. 2008; 49: 2679-2685Crossref PubMed Scopus (80) Google Scholar). Such leakage is likely: in diabetes the BRB becomes permeable to particles that are much larger than LDL (23Qaum T. Xu Q. Joussen A.M. Clemens M.W. Qin W. Miyamoto K. Hassessian H. Wiegand S.J. Rudge J. Yancopoulos G.D. et al.VEGF-initiated blood-retinal barrier breakdown in early diabetes.Invest. Ophthalmol. Vis. Sci. 2001; 42: 2408-2413PubMed Google Scholar). Intra-retinal ox-LDL was associated with apoptosis, and in proliferative DR, with macrophage infiltration (8Wu M. Chen Y. Wilson K. Chirindel A. Ihnat M.A. Yu Y. Boulton M.E. Szweda L.I. Ma J-X. Lyons T.J. Intraretinal leakage and oxidation o

Abstract Ocular neovascularization underlies major blinding eye diseases such as “wet” age‐related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo . FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fech m1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2-/y -Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage- c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2-/y -Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy. Stem Cells 2018;36:1430-1440.

Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in elderly people, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver X receptors (LXRs), encoded by the nuclear receptor subfamily 1 group H members 2 and 3 (NR1H3 and NR1H2), are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice lacking LXR presented with isoform-dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform resulted in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 was associated with ocular lipoidal degeneration. LXR activation not only ameliorated lipid accumulation and oxidant-induced injury in RPE cells but also decreased ocular inflammatory markers and lipid deposition in a mouse model, thereby providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD.

Retinal homeostasis is under both diurnal and circadian regulation. We sought to investigate the diurnal expression of autophagy proteins in normal rodent retina and to determine if this is impaired in diabetic retinopathy. C57BL/6J mice and Bio-Breeding Zucker (BBZ) rats were maintained under a 12h/12h light/dark cycle and eyes, enucleated over a 24 h period. Eyes were also collected from diabetic mice with two or nine-months duration of type 1 diabetes (T1D) and Bio-Breeding Zucker diabetic rat (BBZDR/wor rats with 4-months duration of type 2 diabetes (T2D). Immunohistochemistry was performed for the autophagy proteins Atg7, Atg9, LC3 and Beclin1. These autophagy proteins (Atgs) were abundantly expressed in neural retina and endothelial cells in both mice and rats. A differential staining pattern was observed across the retinas which demonstrated a distinctive diurnal rhythmicity. All Atgs showed localization to retinal blood vessels with Atg7 being the most highly expressed. Analysis of the immunostaining demonstrated distinctive diurnal rhythmicity, of which Atg9 and LC3 shared a biphasic expression cycle with the highest level at 8:15 am and 8:15 pm. In contrast, Beclin1 revealed a 24-h cycle with the highest level observed at midnight. Atg7 was also on a 24-h cycle with peak expression at 8:15am, coinciding with the first peak expression of Atg9 and LC3. In diabetic animals, there was a dramatic reduction in all four Atgs and the distinctive diurnal rhythmicity of these autophagy proteins was significantly impaired and phase shifted in both T1D and T2D animals. Restoration of diurnal rhythmicity and facilitation of autophagy protein expression may provide new treatment strategies for diabetic retinopathy.

To determine whether lipofuscin is detrimental to lysosomal and antioxidant function in cultured human retinal pigment epithelial (RPE) cells.Isolated lipofuscin granules were fed to confluent RPE cultures and the cells maintained in basal medium for 7 days. Parallel cultures were established that did not receive lipofuscin. Cultures were either exposed to visible light (390-550 nm) at an irradiance of 2.8 mW/cm(2) or maintained in the dark at 37 degrees C for up to 24 hours. Cells were subsequently assessed for cell viability, lysosomal enzyme activity, and antioxidant capacity.There was no loss of cell viability during the first 3 hours of light exposure, whereas a 10% loss of viability was observed in lipofuscin-fed cultures after 6 hours' exposure to light. Activities of acid phosphatase, N-acetyl-beta-glucuronidase, and cathepsin D were decreased by up to 50% in lipofuscin-fed cells exposed to light compared with either unfed cells or cells maintained in the dark. There was also a decrease in the antioxidant potential of RPE cells. Catalase and superoxide dismutase activities decreased by up to 60% and glutathione levels by 28% in light-exposed lipofuscin-fed cells compared with unfed cells or cells maintained in the dark.Lipofuscin has the capacity to reduce the efficacy of the lysosomal and antioxidant systems in RPE cells that may play an important role in retinal ageing and the development of age-related macular degeneration.

To determine whether aging is accompanied by changes in aerobic photoreactivity of retinal pigment epithelial (RPE) melanosomes isolated from human donors of different ages, and to compare the photoreactivity of aged melanosomes with that of RPE lipofuscin.Human RPE pigment granules were isolated from RPE cells pooled into groups according to the age of the donors. Photoreactivity was determined by blue-light-induced oxygen uptake and photogeneration of reactive oxygen species. Short-lived radical intermediates were detected by spin-trapping, hydrogen peroxide by an oxidase electrode, singlet oxygen by cholesterol assay, and lipid hydroperoxides by iodometric assay.Blue-light photoexcitation of melanosomes resulted in age-related increases in both oxygen uptake and the accumulation of superoxide anion spin adducts. The efficiencies of these processes, however, were still significantly lower than that induced by photoexcited lipofuscin. During irradiation of melanosomes, a substantial amount of oxygen was converted into hydrogen peroxide, whereas for lipofuscin, hydrogen peroxide accounted for not more than 3% of oxygen consumed. In contrast to lipofuscin, photoexcited melanosomes did not substantially increase the rate of oxidative reactions in the presence of polyunsaturated lipids or albumin. However, oxygen uptake was significantly elevated in the presence of ascorbate. Thus, the rate of photo-induced oxygen uptake in samples containing both ascorbate and melanosomes approached that observed in lipofuscin samples.Blue-light-induced photoreactivity of melanosomes increases with age, perhaps providing a source of reactive oxygen species and leading to depletion of vital cellular reductants, which, together with lipofuscin, may contribute to cellular dysfunction.

In this study the activities of two lysosomal enzymes in retinal pigment epithelial (RPE) cells isolated from three regions of the human fundus were examined: the macula, the nasal midzone, and the periphery.The results obtained showed that the activities of acid phosphatase and cathepsin D were significantly higher in the RPE cells derived from the macular region when compared with those in the periphery.The values for the midzone appeared to be intermediate between the other two regions.Furthermore, the overall activity ofboth enzymes increased as a function ofage.

The purpose of this study was to determine whether an age-related increase in photoreactivity of human retinal melanosomes (MS) can cause phototoxicity to retinal pigment epithelium (RPE) cells. MS were isolated post mortem from young (20-30 years, young human melanosomes [YHMs]) and old (60-90 years, old human melanosomes [OHMs]) human eyes and from young bovine eyes (bovine melanosomes [BMs]). Confluent cultured ARPE-19 cells were fed equivalent numbers of OHMs or BMs and accumulated similar amounts of melanin as determined by electron paramagnetic resonance assay. Cells with and without MS were either maintained in the dark or exposed to blue light for up to 96 h and assessed for alterations in cell morphology, cell viability and lysosomal integrity. Incubation of cells in dark in the presence of internalized MS or irradiation of cells with blue light in the absence or presence of BMs did not significantly affect cell viability. However, exposures to blue light in the presence of OHMs resulted in abnormal cell morphology, up to approximately 75% decrease in mitochondrial activity, loss of lysosomal pH and cell death. OHMs contained significantly less melanin than YHMs, supporting the hypothesis that melanin undergoes degradation during RPE aging. Our results demonstrate that aged MS can be phototoxic to human RPE cells and support a contributing role of MS in RPE aging and in the pathogenesis of age-related macular degeneration.

Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of aged or damaged intracellular organelles and their delivery to lysosomes for degradation. Autophagy begins with the formation of membranes, arising in part from the endoplasmic reticulum, that elongate and fuse engulfing cytoplasmic constituents into a classic double-membrane bound nascent autophagosome. These early autophagosomes undergo a stepwise maturation process to form the late autophagosome or amphisome that ultimately fuses with a lysosome. Efficient autophagy is dependent on an equilibrium between the formation and elimination of autophagosomes; thus, a deficit in any part of this pathway will cause autophagic dysfunction. Autophagy plays a role in aging and age-related diseases. However, few studies of autophagy in retinal disease have been reported.

Journal of Cellular and Molecular MedicineVolume 12, Issue 3 p. 781-795 Open Access γ-Secretase: a multifaceted regulator of angiogenesis Michael E. Boulton, Corresponding Author Michael E. Boulton Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, TX, USA *Correspondence to: Michael E. BOULTON, PhD,PO Box 100235, Anatomy and Cell Biology, University ofFlorida, Gainesville, FL 32610-0235, USA.Tel.: 352-273-8471Fax: 352-846-1248E-mail: [email protected]Search for more papers by this authorJun Cai, Jun Cai Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, TX, USASearch for more papers by this authorMaria B. Grant, Maria B. Grant Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USASearch for more papers by this author Michael E. Boulton, Corresponding Author Michael E. Boulton Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, TX, USA *Correspondence to: Michael E. BOULTON, PhD,PO Box 100235, Anatomy and Cell Biology, University ofFlorida, Gainesville, FL 32610-0235, USA.Tel.: 352-273-8471Fax: 352-846-1248E-mail: [email protected]Search for more papers by this authorJun Cai, Jun Cai Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, TX, USASearch for more papers by this authorMaria B. Grant, Maria B. Grant Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USASearch for more papers by this author First published: 21 May 2008 https://doi.org/10.1111/j.1582-4934.2008.00274.xCitations: 41 Guest Editor: N.I. Moldovan AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract • Introduction • Regulated intramembrane proteolysis • γ-Secretase – Structure – Receptor cleavage • Is there more to γ-Secretase than regulated intramembrane proteolysis? – Receptor translocation – Presenilin-binding proteins – Phosphorylation • Role of γ-Secretase in angiogenesis – Notch – Vascular endothelial growth factor receptor-1 (VEGFR-1) – Insulin-like growth factor-I receptor (IGF-1R) – ErbB4 – Cadherins – Amyloid precursor protein (APP) – Other substrates • γ-Secretase as a therapeutic target • γ-Secretase, what next? Physiological angiogenesis is essential for development, homeostasis and tissue repair but pathological neovascularization is a major feature of tumours, rheumatoid arthritis and ocular complications. Studies over the last decade have identified γ-secretase, a presenilin-dependent protease, as a key regulator of angiogenesis through: (i) regulated intramembrane proteolysis and transmembrane cleavage of receptors (e.g. VEGFR-1, Notch, ErbB-4, IGFI-R) followed by translocation of the intracellular domain to the nucleus, (ii) translocation of full length membrane-bound receptors to the nucleus (VEGFR-1), (iii) phosphorylation of membrane bound proteins (VEGFR-1 and ErbB-4), (iv) modulation of adherens junctions (cadherin) and regulation of permeability and (v) cleavage of amyloid precursor protein to amyloid-β which is able to regulate the angiogenic process. The γ-secretase-induced translocation of receptors to the nucleus provides an alternative intracellular signalling pathway, which acts as a potent regulator of transcription. γ-secretase is a complex composed of four different integral proteins (presenilin, nicastrin, Aph-1 and Pen-2), which determine the stability, substrate binding, substrate specificity and proteolytic activity of γ-secretase. This seeming complexity allows numerous possibilities for the development of targeted γ-secretase agonists/antagonists, which can specifically regulate the angiogenic process. This review will consider the structure and function of γ-secretase, the growing evidence for its role in angiogenesis and the substrates involved, γ-secretase as a therapeutic target and future challenges in this area. Introduction Angiogenesis is one of the most crucial biological processes, which ensures the formation and physiological function of all organs and virtually all tissues in the body [1]. However, aberrant angiogenesis is a major pathological feature of tumours, diabetic retinopathy, rheumatoid arthritis and age-related macular degeneration. Pathology of the vascular system involves the endothelium, which comprises the innermost layer of all blood vessels, cardiac valves and several other body cavities. The endothelial cells are responsible for maintaining vessel patency, preventing thrombosis, and relaxation and contraction of vessels. The migration, proliferation and organization of endothelial cells and their precursors into tubules defines the process of angiogenesis, whether it be during development or during adult reparative or pathological processes [2]. Furthermore, the classic perception that adult angiogenesis develops solely from existing endothelial cells has been challenged by us and others who have shown that hematopoiet-ic stem cells (HSCs) can contribute up to 26% of the endothelial cells in a new vessel [3–5]. It is now quite evident that there is a plethora of pro- and anti-angiogenic factors that regulate the vasculature and are involved in the development and progression of neovascularization [6, 7]. Furthermore, the spatio-temporal balance of these pro- and anti-angiogenic factors is critical in determining whether vascular home-ostasis or pathology occurs [8]. The collective evidence suggests that the vascular endothelial growth factor (VEGF) family is critical for angiogenesis [6, 7, 9]. Increasing VEGF in animal models promotes neovascularization and this can be reversed by neutralizing VEGF or its receptors [10–12]. VEGF is hypoxia-inducible and thus is dramatically up-regulated by the hypoxic environment associated with tumours and diabetic retinopathy [9, 12, 13]. Moreover, treatment of patients with tumours or choroidal neovascularization with VEGF inhibitors such as Avastin or Lucentis significantly reduces the aberrant angiogenesis resulting in the regression of tumours and, in the case of choroidal neovascularization, vessel regression and improved vision [11, 14, 15]. However, many other factors including Delta/Notch, insulin-like growth factor-I (IGF-I), fibroblast growth factor (FGF), angiopoietin and placenta growth factor (PlGF) are also, given the correct tissue environment, strongly proangiogenic. It is also important to note that these factors are constitutively expressed in most tissues under physiological conditions and thus must play an important role in homeostasis. Their pro-angiogenic potential is regulated by a fine balance of anti-angiogenic factors including pigment epithelium-derived factor (PEDF), IGF-binding protein-3 (IGFBP-3) [16] and thrombospondin [2]. When we think of receptor activation by these angiogenic factors we typically associate this with tyrosine kinase activation and downstream intracellular signalling driving a cascade of protein phosphorylation events [17]. However, it has long been recognized, and often dismissed, that full length receptors and receptor fragments are present within the cytosol and nucleus [18, 19]. It is now apparent that these translocated receptors provide an alternative signalling pathway, bind to transcription factors and associate with adherens junctions to regulate intercellular permeability [19]. Furthermore, despite the presence of numerous membrane receptors (e.g. VEGFR-1, VEGFR-2, CXCR4) on HSCs little is known about their down stream signalling [5]. This review will focus on the intracellular translocation of receptors in angiogenesis and will carefully examine the contribution of γ-secretase and its active proteolytic component, presenilin, which plays a critical role in this process. Regulated intramembrane proteolysis Over the last decade regulated intramembrane proteolysis (RIP) has been revealed as a novel, but highly conserved mechanism in cell signalling (reviewed in [20–22]). RIP results in the release of extracellular/luminal and/or cytoplasmic domains from transmembrane proteins. These cleaved fragments have been shown to act as biological effectors at other sites within the cell. For example, the intracellular domain of Notch released by RIP translocates to the nucleus where it acts as a transcriptional activator [23] (Fig. 1). RIP is mediated by at least three distinct families of evolutionarily conserved intramembrane proteases, which cleave substrates usually within their transmembrane domains [22]:(1) the presenilin-type aspartyl proteases, including the presenilin-dependent γ-secretase and the signal peptide peptidase (SPP) that cleave tyrosine kinase receptors and the HLA-E epitope [24–25]; (2) the site-2 protease (S2P) family, zinc-metalloproteases that cleave and activate sterol regulatory element binding proteins (SREBPs) [26]; (3) the rhomboid serine proteases that cleave transmembrane ligand substrates including the main EGF ligand Spitz [27]. Presenilin-dependent γ-secretase and rhomboids are believed to cleave only type-I membrane substrates (single pass transmembrane proteins with a cytoplasmic C-terminus and an extracellular or luminal N-terminus), while S2P and SPP cleave type-II membrane proteins (the N- and C-termini in type II proteins are the reverse orientation of type-I proteins). Cleavage of multipass proteins is under debate but there is recent evidence that this might occur with the CXCR4 receptor [28]. Figure 1Open in figure viewerPowerPoint Role of γ-secretase in growth factor receptor signalling. Ligand binding induces ectodomain shedding of the receptor allowing for the second intramembrane cleavage that releases the active cytoplasmic domain, which in the case of Notch translocates to the nucleus. Modified from Landman and Kim [22]. γ-Secretase Structure γ-secretase is a complex composed of four different integral membrane proteins: presenilin (PS), nicastrin, Aph-1, and Pen-2 (Fig. 2) [29, 30]. The most studied component of the γ-secretase complex is presenilin, which is an integral enzyme in the cleavage of amyloid precursor protein and contributes to the accumulation of amyloid-β peptide in Alzheimer's disease. Activation of PS is dependent on its endoproteolysis into an N-terminal fragment (NTF) and C-terminal fragment (CTF) [30, 31]. Nicastrin has recently been described as 'the gatekeeper of the γ-secretase complex'[32]. The extracellular orientated domain of nicastrin is essential for substrate recognition by the γ-secretase complex and nicastrin binding to the substrate is required before presenilin can exert its proteolytic activity. This extracellular domain of nicastrin usually binds to specific amino terminal residues of the transmembrane substrate. Thus nicastrin facilitates presenilin-dependent RIP of the transmembrane fragment [33]. Of the two other proteins, which constitute γ-secretase, Aph-1 is believed to be a scaffolding protein and Pen-2 appears to regulate PS activity. Assembly of the γ-secretase complex begins in the endoplasmic reticulum and is concluded after translocation of the four proteins to the cell membrane [30]. Figure 2Open in figure viewerPowerPoint Components and assembly of the γ-secretase complex. (A) γ-secretase is composed of four different integral membrane proteins; presenilin, nicastrin, Aph-1 and Pen-2. Presenilin undergoes endoproteolysis into an N-terminal fragment (NTF) and C-terminal fragment (CTF) that remain associated. (B) Model for how the components of γ-secretase are arranged within the active protease complex. Modified from Wolfe [30]. Although γ-secretase is ubiquitously expressed in a wide range of cell types which themselves express multiple substrates ranging from signalling receptors to junctional complexes there is clear evidence of substrate specificity. However, exactly how this specificity is achieved remains unclear [34]. Six variants of γ-secretase exist based upon different combinations of the two PS and three different Aph-1 proteins. Furthermore, the complex contains a number of different binding, docking and active enzyme sites that exhibit varying degrees of substrate specificity (see Wolfe [30]). There also appears to be allosteric modulation of γ-secretase activity through binding of extrinsic factors to protein components in the complex (see Wolfe [30]). Thus the extrinsic components, in combination with core complexes, design functional variants of γ-secretase that have substrate specificity. Even when active γ-secretase is present in the endothelial cell membrane, transmembrane receptors are not cleaved until they bind to their respective ligand which presumably results in a change in receptor conformation that makes either a docking or cleavage site available on the receptor for γ-secretase. Although a great deal of progress has been made in identifying the components of the γ-secretase complex, the endogenous regulatory mechanism of γ-secretase expression/activity is only now being elucidated. We have recently demonstrated that the potent anti-angiogenic inhibitor pigment epithelial-derived factor (PEDF) promotes translocation of PS and nicastrin from the cell membrane and up-regulates γ-secretase activity [31]. It is conceivable that this involves kinase-dependent pathways since Kim and colleagues [35] have shown that γ-secretase is endogenously regulated via the extracellular signal regulated MAP kinase (ERK) MAPK pathway. Information on the transcriptional regulation of the proteins of the γ-secretase complex is limited but there is evidence that the PS-1 promoter has Ets binding domains [36], that both PS and Pen-2 are transcriptionally regulated by cyclic AMP-response element binding protein (CREB) [37] and ubiquilin can regulate the post-transcriptional modification of PS, Pen-2 and nicastrin [38]. Substrate cleavage Presenilin-dependent regulated intramembrane proteolysis has been extensively studied in the context of Notch signalling. Cleavage of transmembrane proteins by presenilin leads to the generation of biologically active protein fragments that signal at various locations within the cell including the nucleus and the adherens junctions (reviewed in [22]). RIP of membrane receptors usually requires two sequential proteolytic cleavages, carried out by different proteases. The first cleavage occurs at the cell surface, and usually leads to the shedding of the protein's extracellular or luminal domain. The initiation of this first cleavage is normally in response to ligand binding to the receptor presumably causing a conformational change and exposing a cleavage site [26, 39, 40]. This cleavage of the ectodomain is typically carried out by proteases of the disintegrin and metalloprotease (ADAM) families, whose active site domain is located in the extracellular/luminal space [22, 41]. This primary cleavage shortens the ectodomain (usually to less than 30 amino acids), which allows the transmembrane cleavage to occur via the action of γ-secretase. The active cytoplasmic domain, which is released subsequently, translocates within the cell where it can either (1) locate to the nucleus where it serves to regulate gene expression through an association with DNA bound cofactors, as reported for Notch and ErbB-4 (reviewed in [42, 43]) or (2) bind to cytsolic proteins and regulate their action (e.g. p75NTR[44] and E-cadherin [45]). Thus, γ-secretase-dependent proteolysis can regulate intracellular signalling pathways via two major mechanisms: elimination of critical domains of the membrane receptors or generation of bioactive intracellular domains. γ-secretase-dependent proteolysis can cleave a number of membrane receptors (see Parks and Curtis) [46]) including ErbB4 [41] to modulate proliferation and differentiation, CD44 that regulates transcriptional activity [47], p75 neurotrophin receptor to regulate cell survival and transcription [48], E-cadherin to change permeability [45], LDL-receptor to regulate transcription [49], VEGFR-1 to regulate angiogenesis [31] and IGF-IR with as yet undetermined functions and Tie 1 [50, 51]. Is there more to γ-secretase than regulated intramembrane proteolysis? Receptor translocation Growth factor receptors localized to the plasma membrane are not restricted to the cell surface. Tyrosine kinase receptors can, usually following binding of their concomitant ligand, be rapidly internalized through clathrin-coated pits and enter the endocytic pathway from which they are degraded in lysosomes or be recycled back to the cell surface [19]. However, there is accumulating evidence that membrane bound receptors have an alternative signalling pathway, which involves either the intact full length receptor or a cleaved cytoplasmic domain fragment being translocated to the nucleus. Full length EGFR, FGFR, VEGFR-1 and VEGFR2 appear to accumulate in the nucleus as an intact receptor [18, 31, 52, 53]. Cytosolic fragments of Notch, ErbB-4 and VEGFR-1 have all been localized to the nucleus. γ-secretase appears critical for the transmembrane cleavage of receptors since γ-secretase inhibitors prevent cleavage and subsequent accumulation of receptors in the nucleus. However, we have recently demonstrated that γ-secretase can also regulate the nuclear translocation of intact receptors [31]. Although, the mechanism by which this occurs has yet to be elucidated. The nuclear localization of membrane bound receptors appears to regulate gene expression either by (i) acting directly as a transcription factor (FGFR-1 acts as a transcription factor at the FGF-2 promoter) [54], (ii) binding to and regulating the activity of transcription factors, (iii) induction of early response and cell cycle genes [55] or (iv) co-transport of other molecules into the nucleus (ErbB-1 has been reported to transport the tyrosine phosphorylated transcription factor STAT-3 from the cytosol into the nucleus) [56]. Presenilin-binding proteins Presenilin/γ-secretase has been shown to date to interact with over 30 proteins but the significance of this association remains unclear (reviewed in [46]. The functions of the diverse range of binding proteins include vesicle trafficking [57], apoptotic regulation and proteosomal degradation [58], ubiquitination [59], endosome recycling [58] and calcium regulation [60]. It is highly likely that many of these proteins will regulate presenilin levels/activity or the stability of the γ-secretase complex. For example, the calcium-binding protein calsenelin can promote γ-secretase-mediated cleavage of Notch and APP [60]. However, it is possible that presenilin binding can lead to a functional change in the bound protein, possibly through phosphorylation. Phosphorylation There is some suggestion in the literature that PS regulates the phosphorylation state of a variety of proteins including retinoblastoma protein [61], Src [62], ErbB-4 [63, 64] and VEGFR-1 [31]. Whether this is a direct or indirect phosphorylation is unclear but PS1 is known to be able to activate PI3K leading to downstream phosphorylation/dephosphorylation [65, 66]. There is also evidence that presenilin can bind and phosphorylate proteins both alone and as part of the γ-secretase complex. PS-1 is able to promote cell survival by activating the PI3K/Akt pathway [5, 40], phosphorylating glycogen synthase kinase-3 (GSK-3) and suppressing GSK-3-dependent phosphorylation of tau [65]. The function of PS-1 was not affected by γ-secretase inhibition. However, Cai and colleagues [31] observed that γ-secretase is able to regulate phosphorylation of VEGFR-1. γ-secretase induction greatly reduced VEGFR-1 phosphorylation and phosphorylation was least when γ-secretase was induced in combination with VEGF. This could be blocked by inhibition of γ-secretase. It would thus appear that presenilin can mediate protein function either alone or as part of the γ-secretase complex. Role of γ-secretase in angiogenesis We and others have identified a potential role for γ-secretase in vasculogenesis and angiogenesis [31, 67–72]. Abnormal blood vessel development occurs in mice lacking presenilin-1 [68]. Presenilin-1 controls the growth and differentiation of endothelial progenitor cells [73]. An Ets domain is located on the PS promoter and Ets is known to be a key transcriptional regulator of vasculogenesis and angiogenesis [74]. Furthermore, the transcriptional elements Ets and CREB are known to regulate expression of PS and these elements are themselves regulated by a variety of growth factors including VEGF [74, 75]. Aph1A (one of the three Aph1 isoforms that contribute to the stability of the γ-secretase complex) knockout mice failed to develop an organized vascular system [70]. We have shown that γ-secretase is able to regulate VEGFR-2-induced vascular permeability and angiogenesis in retinal microvascular endothelial cells via VEGFR-1 cleavage and translocation of the C-terminal domain and full length VEGFR-1 [31]. A role for γ-secretase in retinal neovascularization is further supported by the observation that PS expression is up-regulated in the OIR model of retinopathy of prematurity [67]. Notch, a γ-secretase regulated receptor, is an important modulator of endothelial behavior. Furthermore, there is crosstalk between Notch and VEGF receptors and Notch is known to regulate both VEGF and VEGFR expression in a variety of vascular and nonvascular cells. Notch Notch receptor signalling, unlike many other transmembrane signalling receptors that use protein phosphorylation cascades to transmit intracellular signals, relies on cleavage of its intracellular domain and subsequent translocation to bind directly with downstream transcriptional regulators [20]. Notch undergoes a typical regulated intramembrane proteolysis. Upon ligand binding to Delta or Jagged, the ectodomain is cleaved by TACE/ADAM metalloproteinases. The resultant membrane-anchored c-terminal fragment is subsequently further cleaved by γ-secretase to liberate the Notch intracellular domain, which translocates to the nucleus. The best characterized Notch targets are transcriptional repressors of the Hes and Hey families that act by negatively regulating expression of target genes such as tissue-specific transcriptional activators [71, 76, 77]. Although Notch is classically considered to be a regulator of cell differentiation and pattern formation there is considerable evidence in the literature to support its role in developmental, adult and pathological angiogenesis [71]. Of the Notch receptors, only Notch 1 and Notch 4 are expressed in vascular endothelial cells [78]. There is a close association between Notch and VEGF. Treatment of endothelial cells with VEGF increases γ-secretase activity, Notch 1 cleavage and Hes-1 expression while inhibition of γ-secretase leads to decreased angiogenesis and blocks VEGF-induced endothelial cell proliferation, migration and survival. For example, both Notch and its ligand Delta-like 4 (Dll4) are induced in vascular endothelial cells by VEGF [79], Notch activation down-regulates VEGFR-2 expression on vascular endothelial cells [77] and Notch activation can regulate VEGF expression [80]. γ-secretase is also critical for cell-autonomous notch signalling and regulates endothelial cell branching and proliferation during vascular tubulogenesis [69]. In the last few years, it has become apparent that the activation of Notch by Dll4 regulates the formation of appropriate numbers of tip cells to control vessel sprouting and branching [81]. Inhibition of Notch signalling using γ-secretase inhibitors, inactivation of one allele of Dll4 or genetic deletion of Notch 1 all promoted increased numbers of tip cells. VEGF is essential for the induction of endothelial tip cells and appears to be able to up-regulate Dll4 which is also increased under hypoxia [72]. Furthermore, Dll4 is able to reduce VEGFR-2 expression in endothelial cells thus providing a negative feedback loop for VEGF-induced angiogenesis. Dll4 overexpressing cells show significant induction of Hey-2, which is one of the transcription factors that mediates the Dll4 induced Notch activation [72]. γ-secretase inhibition blocked Dll4 activation of Notch and reconstituted VEGFR-2 expression. It also appears that Dll4/Notch 4 interactions lead to an up-regulation of ephrin B2 (a regulator of cell segregation) and down-regulation of its receptor EphB4, which was blocked by γ-secre-tase inhibition [82]. Presenilin processing of ephrinB itself regulates EphB-induced Src phosphorylation and signalling [62]. It now appears that Dll4 and Notch can regulate multiple angiogenic pathways in HUVEC including VEGF, PlGF, FGF and HGF as well as facilitating up-regulation of VEGFR-1 allowing a continued response to PlGF [83]. Thurston et al. report a paradox for Dll4 activation of Notch, which leads to more tumour vessels but less tumour growth [84]. Thus Dll4, Notch and γ-secretase all appear to play a critical role in VEGF-induced angiogenesis. Vascular endothelial growth factor receptor-1 (VEGFR-1) Although VEGFR-2 has long been considered the major effector in VEGF-induced angiogenesis, there is a growing body of evidence that VEGFR-1 is a potent negative regulator of VEGFR-2 [12]. Mice expressing the VEGFR-1 extracellular and transmembrane domains but lacking the tyrosine kinase domain (VEGFR-1 TK(–) mice) develop an essentially normal vasculature [85], which suggests that the VEGFR-1 tyrosine kinase domain appears to be dispensable and that VEGFR-1 may signal via an alternative pathway or pathways. This process may also in part be dependent on the extracellular matrix since Nozaki and colleagues [86] observed that choroidal neovascularization induced by injury was increased by excess VEGF-A before injury but was suppressed by VEGF-A after injury. This antiangiogenic effect was mediated via VEGFR-1 activation that was silenced by secreted protein, acidic and rich in cysteine (SPARC). Thus, the mechanism by which VEGFR-1 supports angiogenesis is complex and likely involves several different mechanisms with different levels of specificity and controls. We have recently reported that γ-secretase is expressed in retinal microvascular endothelial cells and is able to elicit regulated intramembrane proteolysis of VEGFR-1 as well as translocation and phosphorylation of the full length receptor [31]. The translocation of the cleaved intracellular domain of VEGFR-1 as well as full length VEGFR-1 acts as a potent negative regulator of VEGFR-2 driven neovascularization. Pigment epithelial-derived factor (PEDF), a potent anti-angiogenic factor, induces a greater than 8-fold increase in γ-secretase activity in microvascular endothelial cells. This increase was not due to up-regulated gene expression but reflected mobilization of endogenous γ-secretase constituents to the plasma membrane and an increase in the ratio of the active PS-1 C-terminal fragment (CTF) to inactive full length within 30 min. Once complex formation was complete, both PS-1 and nicastrin co-localized to membrane-bound VEGFR-1 but not VEGFR-2. From studies in other signalling systems, we believe that PS-1 is bound to nicastrin, which in turn binds to VEGFR1. We conclude from these observations that VEGFR-1 will have sequence domains for both nicastrin binding and PS-1 cleavage. Computational modelling studies show that the VEGFR-1 transmembrane domain is highly conserved and contains a valine site ('cvaatlfwllltlf'), which could serve as a substrate for PS-1. γ-secretase induction in the presence of VEGF resulted in the appearance of an intracellular cleaved 80 kD C-terminal domain of VEGFR-1 within 1 hr after induction. Interestingly, even though we could induce the formation of an active γ-secretase complex in the plasma membrane, VEGF binding to VEGFR-1 was essential before cleavage occured leading us to hypothesize that a change in VEGFR-1 conformation occurs which in turn exposes the conserved valine residue to PS-1. A challenging thought is that the 80 kD C-terminal fragments of VEGFR-1 released into the cytosol could act to inhibit VEGFR-2 phosphorylation and that some remnant 100 kD N-terminal domains are released extracellularly to involve sequestration of the VEGF similar to the soluble form of VEGFR-1. Subcellular fractionation detected full length 180 kD VEGFR-1 in the membrane, cytoskeletal and the nuclear fractions plus an additional 80 kD (cleaved) C-terminal domain of VEGFR-1 was observed in the cytosol fraction of γ-secretase-induced cells. The cleavage and translocation of the 80 kD (cleaved) C-terminal domain of VEGFR-1 was blocked by γ-secretase inhibition. The presence of the N-terminal (extra-cellular) domain of VEGFR-1 confirmed the presence of full length VEGFR-1 in membrane, cytoskeletal, and nuclear fractions, but not in the cytosolic fraction. γ-secretase induction also regulated the phosphorylation state of VEGFR-1 and its translocated domains. Immunoprecipitated phosphorylated proteins and Western blotting for VEGFR-1 demonstrated that VEGF induced an increase in autophosphorylation of VEGFR-1 compared to control with bands at 250 and 180 kD in whole cell lysates. γ-secretase induction greatly reduced VEGFR-1 phosphorylation in both the 250 and 180 kD bands and dephosphorylation was greatest'when γ-secretase was induced by PEDF in combination with VEGF. Analysis of the subcellular fractions demonstrated that VEGF induced an increase in auto-phosphorylation of full length VEGFR-1 in mem

Aging of the human retina is characterized by progressive pathology, which can lead to vision loss. This progression is believed to involve reactive metabolic intermediates reacting with constituents of Bruch's membrane, significantly altering its physiochemical nature and function. We aimed to replace a myriad of techniques following these changes with one, Raman spectroscopy. We used multiplexed Raman spectroscopy to analyze the age-related changes in 7 proteins, 3 lipids, and 8 advanced glycation/lipoxidation endproducts (AGEs/ALEs) in 63 postmortem human donors. We provided an important database for Raman spectra from a broad range of AGEs and ALEs, each with a characteristic fingerprint. Many of these adducts were shown for the first time in human Bruch's membrane and are significantly associated with aging. The study also introduced the previously unreported up-regulation of heme during aging of Bruch's membrane, which is associated with AGE/ALE formation. Selection of donors ranged from ages 32 to 92 yr. We demonstrated that Raman spectroscopy can identify and quantify age-related changes in a single nondestructive measurement, with potential to measure age-related changes in vivo. We present the first directly recorded evidence of the key role of heme in AGE/ALE formation.

Dysregulation of circadian rhythmicity is identified as a key factor in disease pathogenesis. Circadian rhythmicity is controlled at both a transcriptional and post-transcriptional level suggesting the role of microRNA (miRNA) and double-stranded RNA (dsRNA) in this process. Endonuclease Dicer controls miRNA and dsRNA processing, however the role of Dicer in circadian regulation is not known. Here we demonstrate robust diurnal oscillations of Dicer expression in central and peripheral clock control systems including suprachiasmatic nucleolus (SCN), retina, liver, and bone marrow (BM). The Dicer oscillations were either reduced or phase shifted with aging and Type 2 diabetes. The decrease and phase shift of Dicer expression was associated with a similar decrease and phase shift of miRNAs 146a and 125a-5p and with an increase in toxic Alu RNA. Restoring Dicer levels and the diurnal patterns of Dicer-controlled miRNA and RNA expression may provide new therapeutic strategies for metabolic disease and aging-associated complications.

Therapeutic efficacy is routinely assessed by measurement of lesion size using flatmounted choroids and confocal microscopy in the laser-induced choroidal neovascularization (L-CNV) rodent model. We investigated whether optical coherence tomography (OCT) quantification, using an ellipsoid volume measurement, was comparable to standard ex vivo evaluation methods for this model and whether this approach could be used to monitor treatment-related lesion changes.Bruch's membrane was ruptured by argon laser in the dilated eyes of C57BL/6J mice, followed by intravitreal injections of anti-VEGF164 or vehicle, or no injection. In vivo OCT images were acquired using Micron III or InVivoVue systems at 7, 10, and/or 14 days post-laser and neovascular lesion volume was calculated as an ellipsoid. Subsequently, lesion volume was compared to that calculated from confocal Z-stack images of agglutinin-stained choroidal flatmounts.Ellipsoid volume measurement of orthogonal 2-dimensional OCT images obtained from different imaging systems correlated with ex vivo lesion volumes for L-CNV (Spearman's ρ=0.82, 0.75, and 0.82 at days 7, 10, and 14, respectively). Ellipsoid volume calculation allowed temporal monitoring and evaluation of CNV lesions in response to antivascular endothelial growth factor treatment.Ellipsoid volume measurements allow rapid, quantitative use of OCT for the assessment of CNV lesions in vivo. This novel method can be used with different OCT imaging systems with sensitivity to distinguish between treatment conditions. It may serve as a useful adjunct to the standard ex vivo confocal quantification, to assess therapeutic efficacy in preclinical models of CNV, and in models of other ocular diseases.

Background— Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. Methods and Results— Angiogenesis was examined in Cd82 -null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82 -null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82 -null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82 -null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. Conclusions— Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief. Stem Cells 2017;35:1303-1315.

In this study, the role of CX3CR1 in the progression of diabetic retinopathy (DR) was investigated. The retinas of wild-type (WT), CX3CR1 null (CX3CR1gfp/gfp, KO), and heterozygous (CX3CR1+/gfp, Het) mice were compared in the presence and absence of streptozotocin (STZ)-induced diabetes. CX3CR1 deficiency in STZ-KO increased vascular pathology at 4 months of diabetes, as a significant increase in acellular capillaries was observed only in the STZ-KO group. CX3CR1 deficiency and diabetes had similar effects on retinal neurodegeneration measured by an increase in DNA fragmentation. Retinal vascular pathology in STZ-KO mice was associated with increased numbers of monocyte-derived macrophages in the retina. Furthermore, compared to STZ-WT, STZ-KO mice exhibited increased numbers of inflammatory monocytes in the bone marrow and impaired homing of monocytes to the spleen. The induction of retinal IL-10 expression by diabetes was significantly less in KO mice, and when bone marrow-derived macrophages from KO mice were maintained in high glucose, they expressed significantly less IL-10 and more TNF-α in response to LPS stimulation. These findings support that CX3CR1 deficiency accelerates the development of vascular pathology in DR through increased recruitment of proinflammatory myeloid cells that demonstrate reduced expression of anti-inflammatory IL-10. • CX3CR1 deletion in STZ-diabetic mice accelerated the onset of diabetic retinopathy (DR). • The early onset of DR was associated with increased retinal cell apoptosis. • The early onset of DR was associated with increased recruitment of bone marrow-derived macrophages to the retina. • Bone marrow-derived macrophages from CX3CR1 KO diabetic mice expressed more TNF-α and less IL-10. • The role of IL-10 in protection from progression of DR is highlighted.

purpose. Glaucoma is presumed to result in the selective loss of retinal ganglion cells. In many neural systems, this loss would initiate a cascade of transneuronal degeneration. The quantification of changes in neuronal populations in the middle layers of the retina can be difficult with conventional histologic techniques. A method was developed based on multiphoton imaging of 4′,6′-diamino-2-phenylindole (DAPI)–stained tissue to quantify neuron loss in postmortem human glaucomatous retinas. methods. Retinas from normal and glaucomatous eyes fixed in 4% paraformaldehyde were incubated at 4°C overnight in DAPI solution. DAPI-labeled neurons at different levels of the retina were imaged by multiphoton confocal microscopy. Algorithms were developed for the automated identification of neurons in the retinal ganglion cell layer (RGCL), inner nucleus layer (INL), and outer nuclear layer (ONL). results. In glaucomatous retinas, the mean density of RGCs within 4 mm eccentricity was reduced by approximately 45%, with the greatest RGC loss occurring in a region that corresponds to the central 6° to 14° of vision. Significant neuron loss in the INL and ONL was also seen at 2 to 4 mm and 2 to 3 mm eccentricities, respectively. The ratios of neuron densities in the INL and ONL relative to the RGCL (INL/RGC and ONL/RGC, respectively) were found to increase significantly at 3 to 4 mm eccentricity. conclusions. The data confirm that the greatest neuronal loss occurs in the RGCL in human glaucoma. Neuronal loss was also observed in the outer retinal layers (INL and ONL) that correlated spatially with changes in the RGCL. Further work is necessary to confirm whether these changes arise from transneuronal degeneration.

We have reported previously that pigment epithelium-derived factor (PEDF) can, via γ-secretase-mediated events, inhibit VEGF-induced angiogenesis in microvascular endothelial cells by both (a) cleavage and intracellular translocation of a C-terminal fragment of VEGF receptor-1 (VEGFR1) and (b) inhibition of VEGF-induced phosphorylation of VEGFR1. Using site-direct mutagenesis and transfection of wild type and mutated receptors into endothelial cells, we showed that transmembrane cleavage of VEGFR1 occurs at valine 767 and that a switch from valine to alanine at this position prevented cleavage and formation of a VEGFR1 intracellular fragment. Using siRNA to selectively knock down protein-tyrosine phosphatases (PTPs) in endothelial cells, we demonstrated that vascular endothelial PTP is responsible for dephosphorylation of activated VEGFR1. PEDF up-regulation of full-length presenilin 1 (Fl.PS1) facilitated the association of vascular endothelial PTP and VEGFR1. Knockdown of Fl.PS1 prevented dephosphorylation of VEGFR1, whereas up-regulation of Fl.PS1 stimulated VEGFR1 dephosphorylation. Fl.PS1 associated with VEGFR1 within 15 min after PEDF treatment. In conclusion, we determined the PEDF-mediated events responsible for VEGFR1 signaling and identified full-length presenilin as a critical adaptor molecule in the dephosphorylation of VEGFR1. This greater understanding of the regulation of VEGFR1 signaling will help identify novel anti-VEGF therapeutic strategies. We have reported previously that pigment epithelium-derived factor (PEDF) can, via γ-secretase-mediated events, inhibit VEGF-induced angiogenesis in microvascular endothelial cells by both (a) cleavage and intracellular translocation of a C-terminal fragment of VEGF receptor-1 (VEGFR1) and (b) inhibition of VEGF-induced phosphorylation of VEGFR1. Using site-direct mutagenesis and transfection of wild type and mutated receptors into endothelial cells, we showed that transmembrane cleavage of VEGFR1 occurs at valine 767 and that a switch from valine to alanine at this position prevented cleavage and formation of a VEGFR1 intracellular fragment. Using siRNA to selectively knock down protein-tyrosine phosphatases (PTPs) in endothelial cells, we demonstrated that vascular endothelial PTP is responsible for dephosphorylation of activated VEGFR1. PEDF up-regulation of full-length presenilin 1 (Fl.PS1) facilitated the association of vascular endothelial PTP and VEGFR1. Knockdown of Fl.PS1 prevented dephosphorylation of VEGFR1, whereas up-regulation of Fl.PS1 stimulated VEGFR1 dephosphorylation. Fl.PS1 associated with VEGFR1 within 15 min after PEDF treatment. In conclusion, we determined the PEDF-mediated events responsible for VEGFR1 signaling and identified full-length presenilin as a critical adaptor molecule in the dephosphorylation of VEGFR1. This greater understanding of the regulation of VEGFR1 signaling will help identify novel anti-VEGF therapeutic strategies.

Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT(1A) receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT(1A) receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 µM H(2)O(2) was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H(2)O(2)-treated cells compared to controls and protected against H(2)O(2)-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT(1A) agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT(1A) receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.

With the advent of many-core computer architectures such as GPGPUs from NVIDIA and AMD, and more recently Intel’s Xeon Phi, ensuring performance portability of HPC codes is potentially becoming more complex. In this work we have focused on one important application area — structured grid codes — and investigated techniques for ensuring performance portability across a diverse range of different, high-end many-core architectures. We chose three codes to investigate: a 3D lattice Boltzmann code (D3Q19 BGK), the CloverLeaf hydrodynamics mini application from Sandia’s Mantevo benchmark suite, and ROTORSIM, a production-quality structured grid, multiblock, compressible finite-volume CFD code. We have developed OpenCL versions of these codes in order to provide cross-platform functional portability, and compared the performance of the OpenCL versions of these structured grid codes to optimized versions on each platform, including hybrid OpenMP/MPI/AVX versions on CPUs and Xeon Phi, and CUDA versions on NVIDIA GPUs. Our results show that, contrary to conventional wisdom, using OpenCL it is possible to achieve a high degree of performance portability, at least for structured grid applications, using a set of straightforward techniques. The performance portable code in OpenCL is also highly competitive with the best performance using the native parallel programming models on each platform.

Paperdry', patients accumulate extracellular lipid and protein filled deposits below the retinal pigment epithelial (RPE) cell layer [1,5,9], which normally serves as crucial support to the overlying neural retina and forms part of the outer blood retina barrier [10].These deposits can lead to dysfunction and atrophy of RPE cells, which along with loss of photoreceptors and choroidal endothelial cells, are major steps in AMD progression towards severe vision deterioration associated with the 'late dry' sub-type known as geographic atrophy [1,3,11].Advanced exudative or 'wet' AMD is characterized by endothelial invasion www.aging-us.com

Disruption of circadian regulation was recently shown to cause diabetes and metabolic disease. We have previously demonstrated that retinal lipid metabolism contributed to the development of diabetic retinopathy. The goal of this study was to determine the effect of diabetes on circadian regulation of clock genes and lipid metabolism genes in the retina and retinal endothelial cells (REC). Diabetes had a pronounced inhibitory effect on the negative clock arm with lower amplitude of the period (per) 1 in the retina; lower amplitude and a phase shift of per2 in the liver; and a loss of cryptochrome (cry) 2 rhythmic pattern in suprachiasmatic nucleus (SCN). The positive clock arm was increased by diabetes with higher amplitude of circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 (bmal1) and phase shift in bmal1 rhythmic oscillations in the retina; and higher bmal1 amplitude in the SCN. Peroxisome proliferator-activated receptor (PPAR) α exhibited rhythmic oscillation in retina and liver; PPARγ had lower amplitude in diabetic liver; sterol regulatory element-binding protein (srebp) 1c had higher amplitude in the retina but lower in the liver in STZ- induced diabetic animals. Both of Elongase (Elovl) 2 and Elovl4 had a rhythmic oscillation pattern in the control retina. Diabetic retinas lost Elovl4 rhythmic oscillation and had lower amplitude of Elovl2 oscillations. In line with the in vivo data, circadian expression levels of CLOCK, bmal1 and srebp1c had higher amplitude in rat REC (rREC) isolated from diabetic rats compared with control rats, while PPARγ and Elovl2 had lower amplitude in diabetic rREC. In conclusion, diabetes causes dysregulation of circadian expression of clock genes and the genes controlling lipid metabolism in the retina with potential implications for the development of diabetic retinopathy.

Retinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow–derived CD34+ cells and vascular wall–derived endothelial colony–forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.

To study the behaviour of residual lens epithelial cells after capsulorhexis and expression of material from the isolated lens.Human and bovine lens capsules were isolated, sterile non-toxic silicone rings inserted, and the preparations placed in organ culture for up to 12 weeks. Cell coverage of the posterior lens capsule was recorded and the capsules were examined, both pre- and post-coverage, for (a) proliferative activity and (b) cytoskeletal components.After a lag period outgrowth was observed across the posterior capsule. The rate of cell coverage was dependent upon both species and the presence or absence of serum. The proliferative activity of the cells was greatest at or near the leading edge and decreased once covered. Wrinkles became visible in the posterior capsule during the late stages of precoverage and increased in both number and complexity. All cells on both the human and bovine posterior capsules contained F-actin and vimentin and the majority were immunolabelled for alpha-smooth muscle actin (alpha-SMA).This model exhibits many of the in vivo characteristics of the lens capsule after extracapsular surgery and may prove useful in further elucidating the cellular mechanisms of posterior capsule opacification.

Opa3 mRNA is expressed in all tissues examined to date, but currently the function of the OPA3 protein is unknown. Intriguingly, various mutations in the OPA3 gene lead to two similar diseases in humans: autosomal dominant inherited optic atrophy and cataract (ADOAC) and a metabolic condition; type 3-methylglutaconic aciduria (MGA). Early onset bilateral optic atrophy is a common characteristic of both disorders; retinal ganglion cells are lost and visual acuity is impaired from an early age. In order to investigate the function of the OPA3 protein, we have generated a novel ENU-induced mutant mouse carrying a missense mutation in the OPA3 gene. The heterozygous mutation in exon 2, causes an amino acid change p.L122P (c.365T>C), which is predicted to alter tertiary protein structure. In the heterozygous state, the mice appear uncompromised however; in the homozygous state mice display some of the features of MGA. Visual function is severely reduced, consistent with significant loss of retinal ganglion cells and degeneration of axons in the optic nerve. In the homozygous optic nerve, there was evidence of increased mitochondrial activity, as demonstrated by the increased presence of mitochondrial marker Cytochrome C Oxidase (COX) histochemistry. Mice homozygous for the opa3(L122P) mutation also display a severe multi-systemic disease characterized by reduced lifespan (majority dying before 4 months), decreased weight, dilated cardiomyopathy, extrapyramidal dysfunction and gross neuro-muscular defects. All of these defects are synonymous with the phenotypic characteristics of Type III MGA found in humans. This model will be of major importance for future studies of the specific function of the OPA3 gene.

The ability to control the differentiation of adult hematopoietic stem cells (HSCs) would promote development of new cell-based therapies to treat multiple degenerative diseases. Systemic injection of NaIO₃ was used to ablate the retinal pigment epithelial (RPE) layer in C57Bl6 mice and initiate neural retinal degeneration. HSCs infected <i>ex vivo</i> with lentiviral vector expressing the RPE-specific gene <i>RPE65</i> restored a functional RPE layer, with typical RPE phenotype including coexpression of another RPE-specific marker, CRALBP, and photoreceptor outer segment phagocytosis. Retinal degeneration was prevented and visual function, as measured by electroretinography (ERG), was restored to levels similar to that found in normal animals. None of the controls (no HSCs, HSCs alone and HSCs infected with lentiviral vector expressing <i>LacZ</i>) showed these effects. <i>In vitro</i> gene array studies demonstrated that infection of HSC with RPE65 increased adenylate cyclase mRNA. <i>In vitro</i> exposure of HSCs to a pharmacological agonist of adenylate cyclase also led to <i>in vitro</i> differentiation of HSCs to RPE-like cells expressing pigment granules and the RPE-specific marker, CRALBP. Our data confirm that expression of the cell-specific gene <i>RPE65</i> promoted fate determination of HSCs toward RPE for targeted tissue repair, and did so in part by activation of adenylate cyclase signaling pathways. Expression by HSCs of single genes unique to a differentiated cell may represent a novel experimental paradigm to influence HSC plasticity, force selective differentiation, and ultimately lead to identification of pharmacological alternatives to viral gene delivery.

Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.

Abstract All‐ trans ‐retinal (AtRal) can accumulate in the retina as a result of excessive exposure to light. The purpose of this study was to compare cytotoxicity of AtRal and photodegraded AtRal (dAtRal) on cultured human retinal pigment epithelial cells in dark and upon exposure to visible light. AtRal was degraded by exposure to visible light. Cytotoxicity was monitored by imaging of cell morphology, propidium iodide staining of cells with permeable plasma membrane and measurements of reductive activity of cells. Generation of singlet oxygen photosensitized by AtRal and dAtRal was monitored by time‐resolved measurements of characteristic singlet oxygen phosphorescence. Photodegradation of AtRal resulted in a decrease in absorption of visible light and accumulation of the degradation products with absorption maximum at ∼330 nm. Toxicity of dAtRal was concentration‐dependent and was greater during irradiation with visible light than in dark. DAtRal was more cytotoxic than AtRal both in dark and during exposure to visible light. Photochemical properties of dAtRal indicate that it may be responsible for the maximum in the action spectra of retinal photodamage recorded in animals. In conclusion, photodegradation products of AtRal may impose a significant threat to the retina and therefore their roles in retinal pathology need to be explored.

We aimed to develop an anti-angiogenic model for breast cancer by combining (1) siRNA-based therapy delivered by self-complementary adeno-associated virus serotype 2 (scAAV2) vectors to target tumor vasculature, and (2) noninvasive monitoring to tumor response to anti-angiogenesis by photoacoustic (PA) imaging. scAAV2 vector containing 7 surface exposed tyrosine to phenylanine capsid mutations was able to transduce microvascular endothelial cells with high efficiency. siRNAs against UPR (unfolded protein response)-IRE1α, XBP-1, ATF6 significantly inhibited breast cancer-induced angiogenesis in vitro by inhibiting endothelial cell survival. PA imaging showed that knockdown of UPR proteins greatly reduced tumor angiogenesis in vivo in breast cancer models.

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.

<h3>AIM</h3> To determine the spatial and temporal changes in the staining pattern of the VEGF receptors FLT-1, KDR, and the putative receptor FLT-4 during the pathogenesis of diabetic retinopathy. <h3>METHODS</h3> Immunohistochemical localisation of VEGF receptors, using antibodies against FLT-1, FLT-4, and KDR, was carried out on specimens of normal human retina (n=10), diabetic retinas (a) with no overt retinopathy (n=12), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n=5), (c) with active proliferative retinopathy (n=6), and (d) with no residual proliferative retinopathy after scatter photocoagulation therapy (n=14), and surgically excised diabetic fibrovascular membranes (n=11). The degree and pattern of immunostaining was recorded. <h3>RESULTS</h3> FLT-1 staining was apparent in the retinas from both non-diabetic and diabetic retinas; weak to moderate staining was generally confined to the inner nuclear layer, the ganglion cell layer, and the retinal vessels during all stages of the disease process. Staining of the retinal vessels was raised in diabetic tissue compared with non-diabetic tissue. The preretinal vessels of the diabetic subjects stained moderately to intensely for FLT-1. In contrast with FLT-1 staining minimal immunostaining for KDR was demonstrated in the non-diabetic eyes and the unlasered eyes; however, weak staining for KDR was observed in the inner nuclear layer and the ganglion cell layer of the unlasered eyes with diabetic changes. In those retinas with preretinal neovascularisation KDR immunoreactivity was moderate to intense in the intra- and preretinal vessels. However, in the excised membranes, where the vessels may have been in a quiescent state, the levels of KDR were weak to moderate. After apparently successful laser treatment KDR staining was reduced in the intraretinal vessels. Minimal FLT-4 staining was observed throughout normal eyes while weak to moderate FLT-4 staining was generally confined to the inner nuclear layer and the ganglion cell layer of the unlasered diabetic eyes. Weak to moderate levels of FLT-4 staining were observed in the intraretinal vessels except after apparently successful laser treatment where reduced levels of staining were observed. Weak to moderate staining was observed in the preretinal vessels. <h3>CONCLUSIONS</h3> This study supports a role for FLT-1, KDR, and possibly FLT-4 in the pathogenesis of diabetic retinopathy; however, their specific roles in the progression of the disease may differ.

Abstract Spectral signal intensities, especially in ‘real‐world’ applications with nonstandardized sample presentation due to uncontrolled variables/factors, commonly require additional spectral processing to normalize signal intensity in an effective way. In this study, we have demonstrated the complexity of choosing a normalization routine in the presence of multiple spectrally distinct constituents by probing a dataset of Raman spectra. Variation in absolute signal intensity (90.1% of total variance) of the Raman spectra of these complex biological samples swamps the variation in useful signals (9.4% of total variance), degrading its diagnostic and evaluative potential. Using traditional spectral band choices, it is shown that normalization results are more complex than generally encountered in traditionally designed sample sets investigating limited chemical species. We demonstrate that no choice of a single band proves to be appropriate for predicting all the reference parameters, instead requiring a tailored normalization routine for each parameter. Of the reference parameters studied in the chosen system, signals from pathogenic adducts in ocular tissues called advanced glycation endproducts were most prominent when normalizing about the 1550–1690 cm −1 region of the spectrum (17.5% of total variance, compared with 0.3% for unnormalized), while prediction of pentosidine and gender were optimized by normalization about the 1570 ( R 2 = 0.97 vs 0.57 for unnormalized) and 1003 cm −1 (p &lt; 0.0000001 vs p &lt; 0.01 for unnormalized) bands, respectively. The data obtained point to the extreme sensitivity of multivariate analysis to signal intensity normalization. Some general guidelines for making appropriate band choices are given, including the use of peak‐finding routines. Copyright © 2008 John Wiley &amp; Sons, Ltd.

<h3>Aim:</h3> To determine if retinal ganglion cell (RGC) loss influences the loss of surrounding RGCs to generate clustered patterns of cell death in human glaucoma. It is hypothesised that retinal ganglion cell loss accelerates the loss of surrounding cells to generate, at a local, cellular scale, clustered patterns of retinal of RGC death. The absence of these interactions would result in a diffuse pattern RGC loss. <h3>Method:</h3> Six glaucomatous retinas (67–83 years old) and six age-matched control retinas (61–89 years old) were prepared as wholemounts and stained by 4′,6-diamidino-2-phenylindole (DAPI) solution (3 μg/ml in PBS). An area corresponding to central 14° of the visual field was imaged. The nearest-neighbour distribution was determined for cells in both normal and glaucomatous RGCL. <h3>Results:</h3> Clustered RGC loss in human glaucoma was observed on a background of diffuse loss. The mean nearest-neighbour distance (NND) of the glaucomatous retinas was significantly higher than with controls (p&lt;0.001). The distribution of NND in glaucomatous retinas was skewed to the higher values with a higher positive kurtosis relative to controls. The quantitative analysis of the pattern of cell loss is supported by the visual inspection of the patterns of cell loss. <h3>Discussion:</h3> The nearest-neighbour analysis is consistent with the presence of two patterns of cell loss in the RGCL in glaucoma. While the diffuse of cell loss can account for an overall reduction in the RGC population, an additional non-random pattern is consistent with the hypothesis that RGC loss has a local influence on the viability of surrounding cells.

To evaluate 3 approaches, both cellular and acellular, to improve the healing of laser in situ keratomileusis flaps in bovine corneas.School of Optometry and Vision Sciences and Cardiff Institute of Tissue Engineering and Repair, Cardiff University, Cardiff, United Kingdom.Experimental study.Laser in situ keratomileusis-like flaps were created in bovine corneas, and the flap bed was treated with tumor necrosis factor-α, interleukin-1α, Fas ligand, transforming growth factor-β(1), or activated stromal fibroblasts. In separate experiments, flaps were created and repositioned. The corneas were then crosslinked using ultraviolet-A (UVA) light. All samples were then placed in organ culture for up to 4 weeks. Untreated samples acted as controls.All treatments increased the adherence of the stromal flap. This was achieved at the expense of corneal clarity except in the case of crosslinking (CXL). In this case, the flap adhesion force immediately increased while the cornea remained clear. The force then decreased gradually during organ culture, although it remained at twice the level of the control corneas after 3 weeks in culture.The results suggest that riboflavin-UVA CXL is a hopeful approach for increasing the adherence strength of corneal flaps while keeping the cornea clear. Further studies are necessary to confirm the durability of the strengthening effect and to exclude serious late complications.No author has a financial or proprietary interest in any material or method mentioned.

Previously, we showed that insulin growth factor (IGF)-1 binding protein-3 (IGFBP-3), independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR). The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB) integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1) and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP). IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO) release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab). Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB) stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs). NO release was neither associated with an increase in intracellular calcium nor decreased by Ca(2+)/calmodulin-dependent protein kinase II (CamKII) blockade; however, dephosphorylation of eNOS-Thr(495) was observed. Phosphatidylinositol 3-kinase (PI3K) activity and Akt-Ser(473) phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate vasorelaxation via activation of SRB1. This response is IGF-1- and calcium-independent, but requires PI3K/Akt activation, suggesting that IGFBP-3 has novel protective effects on retinal and systemic vasculature and may be a therapeutic candidate for ocular complications such as diabetic retinopathy.

In response to ischemia, retinal neuronal cells express nerve growth factor (NGF), which can be proangiogenic. Endothelial progenitor cells (EPCs) can participate with the resident vasculature to promote angiogenesis. We postulated that NGF may stimulate CD34⁺ EPCs to convert to an angiogenic phenotype.Human CD34⁺ cells and human retinal endothelial cells (HRECs) were used to examine the effect of NGF on key steps associated with neovascularization. CD34⁺ cells and HRECs were stimulated with NGF (1 to 4 pM) for 24, 48, and 72 hours. Cell migration was measured using a modified Boyden chamber assay. Expression of the receptor for the cytokine stromal derived growth factor 1 (SDF-1), CXCR-4, was assessed by flow cytometry. In vitro angiogenesis was tested using a three-dimensional (3D) extracellular matrix with HRECs/CD34⁺ cell cocultures. NGF receptor activation was assessed by western analysis.NGF promoted proliferation of CD34⁺ cells but not HRECs. Pretreatment of CD34⁺ cells with NGF increased CXCR-4 expression in CD34⁺ cells, resulting in enhanced migration to SDF-1 (P < 0.0001). The enhanced tubule-forming effect of NGF in HRECs was further potentiated by coculture with NGF-pretreated CD34⁺ cells (P < 0.01). The beneficial effect of NGF was blocked (P < 0.0001) by the ERK inhibitor PD98059. In both CD34⁺ and HRECs, NGF increased phosphorylation of neurotrophic tyrosine kinase receptor type 1 (TrkA) receptor by ERK1 activation (P < 0.01).Our in vitro results suggest that NGF released from ischemic nerves in vivo may contribute to the "angiogenic switch" by stimulating the angiogenic behavior of CD34⁺ cells while minimally affecting resident retinal endothelial cells.

<h3>Aim</h3> Retinal cell remodelling has been reported as a consistent feature of ageing. However, the degree to which this results in transretinal degeneration is unclear. To address this, the authors used multiphoton microscopy to quantify retinal degeneration in post-mortem human eyes of two age groups. <h3>Methods</h3> Retinas from six young subjects (18–33 years old) and six older subjects (74–90 years old) were prepared as wholemount preparations. All retinas were stained with 4,6-diamidino-2-phenylindole and imaged by multiphoton confocal microscopy to quantify neuron densities in the retinal ganglion cell layer (RGCL), inner nuclear layer (INL) and outer nuclear layer (ONL). Neurons were counted using automated cell identification algorithms. All retinas were imaged hydrated to minimise tissue artefacts. <h3>Results</h3> In both groups, 56% of the area within the central 4 mm eccentricity and 27% of the area with eccentricity between 4 mm and 7 mm were imaged. Compared with young subjects, the peak RGCL neuron loss in the aged subjects (25.5%) was at 1 mm eccentricity. INL and ONL neuron densities significantly decreased at 1–2 mm eccentricity (8.7%) and 0.5–4 mm eccentricity (15.6%) respectively (P &lt;0.05). The reduction in neuron density in the INL corresponded, spatially, to the region with the greatest neuron loss in the RGCL and ONL. <h3>Conclusions</h3> This is the first study to correlate neurodegeneration in different populations of cells in the ageing retinas. These data confirm that the greatest neuronal loss occurs in the RGCL and ONL in human ageing retinas, whereas the INL is relatively preserved.

The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling.There are currently no FDA approved small molecules for treating these blinding eye diseases.Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches.Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037.SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain.sEH levels and activity are upregulated in the eyes of a choroidal neovascularization (CNV) mouse model.sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization.Known sEH inhibitors delivered intraocularly suppressed CNV.Thus, by dissecting a bioactive compound's mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.

Bone marrow stem and progenitor cells can differentiate into a range of non-hematopoietic cell types, including retinal pigment epithelium (RPE)-like cells. In this study, we programmed bone marrow-derived cells (BMDCs) ex vivo by inserting a stable RPE65 transgene using a lentiviral vector. We tested the efficacy of systemically administered RPE65-programmed BMDCs to prevent visual loss in the superoxide dismutase 2 knockdown (Sod2 KD) mouse model of age-related macular degeneration. Here, we present evidence that these RPE65-programmed BMDCs are recruited to the subretinal space, where they repopulate the RPE layer, preserve the photoreceptor layer, retain the thickness of the neural retina, reduce lipofuscin granule formation, and suppress microgliosis. Importantly, electroretinography and optokinetic response tests confirmed that visual function was significantly improved. Mice treated with non-modified BMDCs or BMDCs pre-programmed with LacZ did not exhibit significant improvement in visual deficit. RPE65-BMDC administration was most effective in early disease, when visual function and retinal morphology returned to near normal, and less effective in late-stage disease. This experimental paradigm offers a minimally invasive cellular therapy that can be given systemically overcoming the need for invasive ocular surgery and offering the potential to arrest progression in early AMD and other RPE-based diseases.

Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR + CD56 + APLNR + (KNA + ) expression. KNA + cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA + cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice ( db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA + cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA + cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor–derived KNA + cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide “proof of principle” that KNA + cells restore perfusion and correct vascular dysfunction in db/db mice.

Eight eyes exhibited massive proliferation of lens epithelial remnants following Nd-YAG posterior capsulotomy. All eyes had pre-existing retinal pathology. Six had undergone vitrectomy (four for proliferative diabetic retinopathy) before extracapsular cataract extraction with posterior chamber intraocular lens implantation. The other two eyes had familial exudative vitreoretinopathy or retinopathy of prematurity, respectively. Five eyes required removal of the lens proliferations via a pars plana approach. High levels of growth factors in the posterior segment associated with proliferative disorders of the retina may play a role in lens cell proliferation.

To undertake a qualitative and quantitative analysis in three dimensions of the human retinal vasculature.Fixed and excised whole retinas were permeabilised and subjected to immunofluorescent staining for blood vessel components followed by confocal laser scanning microscopy. Single projection and stereoimages were constructed using computer software. XZ sections through the retina were constructed and the vasculature analysed using appropriate software.Immunofluorescent staining with no discontinuities was present in vessels of all sizes, the confocal images of the capillary network being free of out of focus blur at all depths. Quantitative analysis of XZ sections confirmed the qualitative impression of sharp delineation of the deep retinal capillary plexus, an absence of laminar arrangement of capillaries within the inner retina, and a truncated cone of capillaries around the foveal avascular zone (FAZ) wherein the superficial capillaries approached the FAZ more closely than those in the deeper retina.Immunofluorescent staining of the retina and confocal laser scanning microscopy were shown to be useful in analysing accurate three dimensional reconstructions of the normal retinal vasculature without affecting overall tissue architecture.

Corneal collagen ultrastructural changes occur during the healing process. The present study was designed to compare collagen ultrastructural changes after trephine wounding or flap creation. Bovine corneas were injured and maintained in organ culture for up to 4 weeks. Samples were removed from culture at 0, 1, 2, 3 and 4 weeks and snap frozen in liquid N2. X-ray scattering was used to measure changes in collagen interfibrillar spacing, intermolecular spacing and fibrillar diameter. Some samples were fixed in 10% Neutral Buffered Formalin solution and wax embedded for immunohistochemistry to monitor myofibroblast differentiation in corneal flaps. Swelling effects (i.e. changes in interfibrillar spacing) were more severe in trephined corneas than in those with stromal flaps. Collagen fibrillar diameter remained normal in the periphery of injured corneas, but increased significantly in areas within and around the wound in trephined samples and in the epithelial incision site in corneal flaps. Intermolecular spacing was unchanged in all samples. In the flaps, αSMA expression was only detected in an area adjacent to the epithelial plug, and cell numbers gradually increased during the culture. We conclude that stromal swelling is more rapid for trephine-wounded corneas than in stromal flaps, indicating that the intensity of the corneal healing response depends on the type of injury.

To examine the effect of free insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), independent of the effect of insulin-like growth factors, in modulating retinal vascular permeability.We assessed the ability of a form of IGFBP-3 that does not bind IGF-1 (IGFBP-3NB), to regulate the blood retinal barrier (BRB) using two distinct experimental mouse models, laser-induced retinal vessel injury and vascular endothelial growth factor (VEGF)-induced retinal vascular permeability. Additionally, in vitro studies were conducted. In the animal models, BRB permeability was quantified by intravenous injection of fluorescein labeled serum albumin followed by digital confocal image analysis of retinal flat-mounts. Claudin-5 and vascular endothelial-cadherin (VE-cadherin) localization at interendothelial junctions was studied by immunofluorescence. In vitro changes in transendothelial electrical resistance (TEER) and flux of fluorescent dextran in bovine retinal endothelial monolayers (BREC) were measured after IGFBP-3NB treatment. Acid (ASMase) and neutral (NSMase) sphingomyelinase mRNA levels and activity were measured in mouse retinas.Four days postinjury, laser-injured mouse retinas injected with IGFBP-3NB plasmid demonstrated reduced vascular permeability compared with retinas of laser-injured mouse retinas injected with control plasmid. IGFBP-3NB administration resulted in a significant decrease in laser injury-associated increases in ASMase and NSMase mRNA and activity when compared with laser alone treated mice. In vivo, intravitreal injection of IGFBP-3NB reduced vascular leakage associated with intravitreal VEGF injection. IGFBP-3NB partially restored VEGF-induced in vivo permeability and dissociation of claudin-5 and VE-cadherin at junctional complexes. When IGFBP-3NB was applied basally to bovine retinal endothelial cells (BREC) in vitro, TEER increased and macromolecular flux decreased.Intravitreal administration of IGFBP-3NB preserves junctional integrity in the presence of VEGF or laser injury by reducing BRB permeability in part by modulating sphingomyelinase levels.

Journal Article Non-dimensional analysis of retinal microaneurysms: critical threshold for treatment Get access Elishai Ezra, Elishai Ezra Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel Search for other works by this author on: Oxford Academic Google Scholar Eliezer Keinan, Eliezer Keinan Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel Search for other works by this author on: Oxford Academic Google Scholar Yossi Mandel, Yossi Mandel Hansen Experimental Physics Laboratory, Stanford University, Stanford, California, USA Search for other works by this author on: Oxford Academic Google Scholar Michael E. Boulton, Michael E. Boulton Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, USA Search for other works by this author on: Oxford Academic Google Scholar Yaakov Nahmias Yaakov Nahmias Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, IsraelDepartment of Cell and Developmental Biology, The Hebrew University of Jerusalem, Jerusalem, Israel E-mail: ynahmias@cs.huji.ac.il Search for other works by this author on: Oxford Academic Google Scholar Integrative Biology, Volume 5, Issue 3, March 2013, Pages 474–480, https://doi.org/10.1039/c3ib20259c Published: 31 January 2013 Article history Received: 24 October 2012 Accepted: 04 January 2013 Published: 31 January 2013

The ability to effectively deliver genetic material to vascular endothelial cells remains one of the greatest unmet challenges facing the development of gene therapies to prevent diseases with underlying vascular etiology, such as diabetes, atherosclerosis, and age-related macular degeneration. Herein, we assess the effectiveness of an rAAV2-based capsid mutant vector (Y272F, Y444F, Y500F, Y730F, T491V; termed QuadYF+TV) with strong endothelial cell tropism at transducing the vasculature after systemic administration. Intravenous injection of QuadYF+TV resulted in widespread transduction throughout the vasculature of several major organ systems, as assessed by in vivo bioluminescence imaging and postmortem histology. Robust transduction of lung tissue was observed in QuadYF+TV-injected mice, indicating a role for intravenous gene delivery in the treatment of chronic diseases presenting with pulmonary complications, such as α1-antitrypsin deficiency. The QuadYF+TV vector cross-reacted strongly with AAV2 neutralizing antibodies, however, indicating that a targeted delivery strategy may be required to maximize clinical translatability.

The current understanding of the molecular pathogenesis of diabetic retinopathy does not provide a mechanistic link between early molecular changes and the subsequent progression of the disease. In this study, we found that human diabetic retinas overexpressed TRIB3 and investigated the role of TRIB3 in diabetic retinal pathobiology in mice. We discovered that TRIB3 controlled major molecular events in early diabetic retinas via HIF1α-mediated regulation of retinal glucose flux, reprogramming cellular metabolism, and governing of inflammatory gene expression. These early molecular events further defined the development of neurovascular deficit observed in mice with diabetic retinopathy. TRIB3 ablation in the streptozotocin-induced mouse model led to significant retinal ganglion cell survival and functional restoration accompanied by a dramatic reduction in pericyte loss and acellular capillary formation. Under hypoxic conditions, TRIB3 contributed to advanced proliferative stages by significant upregulation of GFAP and VEGF expression, thus controlling gliosis and aberrant vascularization in oxygen-induced retinopathy mouse retinas. Overall, our data reveal that TRIB3 is a master regulator of diabetic retinal pathophysiology that may accelerate the onset and progression of diabetic retinopathy to proliferative stages in humans and present TRIB3 as a potentially novel therapeutic target for diabetic retinopathy.

To determine whether endothelial cell loss of human corneas stored in organ culture before transplantation is due to apoptosis.The corneal endothelium of human corneas, stored in organ culture at 34 degrees C for varying periods of time, were analyzed for the presence of apoptotic cells using the TdT-mediated dUTP nick-end labeling (TUNEL) technique. Corneal endothelial cell apoptosis was confirmed by Hoechst staining and immunolabeling with anti-caspase 3 active antibody.Apoptotic cells were identified in the corneal endothelium of human organ cultured corneas: their number and distribution demonstrated a close correlation with corneal folding and overall quality of the corneal endothelium. TUNEL-positive labeling of cells was confirmed as apoptotic by the presence of morphologic nuclear alterations identified by Hoechst staining and the presence of immunostaining for caspase-3 activity. Corneal endothelial cell apoptosis was independent of cause of donor death, death to enucleation time, and death to culture times.Corneal endothelial cell apoptosis appears to determine the suitability of a cornea for transplantation.

To determine vascular endothelial growth factor C (VEGF-C) expression in retinal endothelial cells, its antiapoptotic potential and its putative role in diabetic retinopathy.Cultured retinal endothelial cells and pericytes were exposed to tumour necrosis factor (TNF)alpha and VEGF-C expression determined by reverse transcriptase-polymerase chain reaction. Secreted VEGF-C protein levels in conditioned media from endothelial cells were examined by western blotting analysis. The ability of VEGF-C to prevent apoptosis induced by TNFalpha or hyperglycaemia in endothelial cells was assessed by flow cytometry. The expression of VEGF-C in diabetic retinopathy was studied by immunohistochemistry of retinal tissue.VEGF-C was expressed by both vascular endothelial cells and pericytes. TNFalpha up regulated both VEGF-C and vascular endothelial growth factor receptor-2 (VEGFR)-2 expression in endothelial cells in a dose-dependent manner, but had no effect on VEGFR-3. Flow cytometry results showed that VEGF-C prevented endothelial cell apoptosis induced by TNFalpha and hyperglycaemia and that the antiapoptotic effect was mainly via VEGFR-2. In pericytes, the expression of VEGF-C mRNA remained stable on exogenous TNFalpha treatment. VEGF-C immunostaining was increased in retinal vessels in specimens with diabetes compared with retinal specimens from controls without diabetes.In retinal endothelial cells, TNFalpha stimulates the expression of VEGF-C, which in turn protects endothelial cells from apoptosis induced by TNFalpha or hyperglycaemia via VEGFR-2 and thus helps sustain retinal neovascularisation.

To investigate the role of poly(ADP-ribose)-polymerase (PARP) in protecting against oxidative (H(2)O(2)) and alkylation (MMS) damage to the nDNA and mtDNA genomes of the retinal pigment epithelium (RPE). We further hypothesized that PARP ribosylation enzymatic activity is required to facilitate efficient nDNA and mtDNA repair to enable the RPE to survive chronic oxidative stress exposure.Cellular sensitivity to H(2)O(2) and MMS was determined by the MTT and LDH assays. PARP ribosyl(ation) activity was inhibited by supplementation of 3-aminobenzamide (competitive PARP inhibitor). The susceptibility and repair capacities of nuclear and mitochondrial genomes were assessed by quantitative PCR and PARP activity assessed using an enzyme assay.This study demonstrated that cells lacking ribosyl(ation) activity had a significantly lower lesion repair capacity in both nDNA and mtDNA (p < 0.05), which culminated in reduced cell viability after H(2)O(2) exposure only (p < 0.05). Furthermore, the mtDNA demonstrated a significantly greater sensitivity compared to nDNA to both oxidative and alkylation damage (p < 0.05).PARP activity has an important role in providing the RPE with the high oxidative tolerance required for this cell type to survive the constant reactive oxygen species attack in vivo for several decades.

The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE.

Abstract Ocular neovascular diseases including neovascular age-related macular degeneration (nvAMD) are widespread causes of blindness. Patients’ non-responsiveness to currently used biologics that target vascular endothelial growth factor (VEGF) poses an unmet need for novel therapies. Here, we identify protein arginine methyltransferase 5 (PRMT5) as a novel therapeutic target for nvAMD. PRMT5 is a well-known epigenetic enzyme. We previously showed that PRMT5 methylates and activates a proangiogenic and proinflammatory transcription factor, the nuclear factor kappa B (NF-κB), which has a master role in tumor progression, notably in pancreatic ductal adenocarcinoma and colorectal cancer. We identified a potent and specific small molecule inhibitor of PRMT5, PR5-LL-CM01, that dampens the methylation and activation of NF-κB. Here for the first time, we assessed the antiangiogenic activity of PR5-LL-CM01 in ocular cells. Immunostaining of human nvAMD sections revealed that PRMT5 is highly expressed in the retinal pigment epithelium (RPE)/choroid where neovascularization occurs, while mouse eyes with laser induced choroidal neovascularization (L-CNV) showed PRMT5 is overexpressed in the retinal ganglion cell layer and in the RPE/choroid. Importantly, inhibition of PRMT5 by PR5-LL-CM01 or shRNA knockdown of PRMT5 in human retinal endothelial cells (HRECs) and induced pluripotent stem cell (iPSC)-derived choroidal endothelial cells (iCEC2) reduced NF-κB activity and the expression of its target genes, such as tumor necrosis factor α (TNF-α) and VEGF-A. In addition to inhibiting angiogenic properties of proliferation and tube formation, PR5-LL-CM01 blocked cell cycle progression at G 1 /S-phase in a dose-dependent manner in these cells. Thus, we provide the first evidence that inhibition of PRMT5 impedes angiogenesis in ocular endothelial cells, suggesting PRMT5 as a potential therapeutic target to ameliorate ocular neovascularization.

To evaluate whether transfection of human retinal endothelial cells (HRECs) with plasmids expressing ribozymes designed to specifically cleave the mRNA and reduce expression of either vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1), or VEGF receptor-2 (VEGFR-2), or insulin-like growth factor-I receptor (IGF-IR) modulates occludin expression in high glucose-treated cells.Hammerhead ribozymes that specifically cleave the human VEGFR-1, VEGFR-2, and IGF-IR mRNAs were developed and tested in vitro to determine ribozyme kinetics and cleavage specificity. HRECs grown in normal (5.5 mM) and high (25 mM) glucose medium were transfected with plasmids expressing VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes. VEGF and IGF-I levels were measured in conditioned medium of HREC exposed to high glucose conditions, and the effect of varying glucose concentration on VEGFR-1 and VEGFR-2 phosphorylation was examined. The amount of the tight junction protein occludin was determined by western analysis, and the protein was localized by immunohistochemistry.Exposure of HRECs to high glucose resulted in increased VEGF and IGF-I expression as well as VEGFR-2 but not VEGFR-1 phosphorylation. Immunocytochemistry and western analysis revealed that HRECs exposed to high glucose had reduced occludin staining and protein expression, respectively. Transfection of HRECs exposed to high glucose with either VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes prevented the downregulation of occludin protein expression.Our studies support that activation of VEGFR-1, VEGFR-2, and IGF-IR by high glucose contributes to disruption of tight junctions by decreasing occludin expression and may be important in the pathogenesis of blood-retinal barrier dysfunction in diabetic retinopathy.

To investigate the effect of EGF, IGF-1, and VEGF on ARPE19 cell proliferation and differentiation.The gene expression of RPE-specific differentiation and proliferation markers and the transcriptional and translational activity of beta-catenin signaling markers were measured by flow cytometry and RT-PCR.The data showed a significant decrease in RPE65, CRALBP, and cytokeratin 18 in ARPE-19 cells stimulated with EGF and IGF-1. In addition, a significant decrease in GSK-3beta and beta-catenin was observed that was paralleled by an increase in cyclin D1 expression. Cell cycle studies revealed an increase in ARPE cells in the S-G(2)/M-phase after treatment with EGF or IGF-1. VEGF, on the other hand, led to a reduction in cyclin D1 and to an increase in GSK 3beta and beta-catenin expression which was paralleled by an increase in RPE-specific differentiation markers.The data demonstrate the induction of proliferation by EGF and IGF-1 and upregulation of the beta-catenin signaling pathway in ARPE-19 cells. The data suggest that activation of the beta-catenin signaling pathway may be key in activating ARPE-19 cells by different growth factors.

Abstract The purpose of this study was to determine the effects of increasing concentration of ascorbate alone and in combinations with α‐tocopherol and zeaxanthin on phototoxicity to the retinal pigment epithelium. ARPE‐19 cells were exposed to rose bengal and visible light in the presence and absence of antioxidants. Toxicity was quantified by an assay of cell‐reductive activity. A 20 min exposure to visible light and photosensitizer decreased cell viability to ca 42%. Lipophilic antioxidants increased viabilities to ca 70%, 61% and 75% for α‐tocopherol, zeaxanthin and their combination, respectively. Cell viabilities were ca 70%, 56% and 5% after exposures in the presence of 0.35, 0.7 and 1.4 m m ascorbate, respectively. A 45 min exposure increased cell death to ca 74% and &gt;95% in the absence and presence of ascorbate, respectively. In the presence of ascorbate, zeaxanthin did not significantly affect phototoxicity. α‐Tocopherol and its combination with zeaxanthin enhanced protective effects of ascorbate, but did not prevent from ascorbate‐mediated deleterious effects. In conclusion, there is a narrow range of concentrations and exposure times where ascorbate exerts photoprotective effects, exceeding which leads to ascorbate‐mediated increase in photocytotoxicity. Vitamin E and its combination with zeaxanthin can enhance protective effects of ascorbate, but do not ameliorate its deleterious effects.