APOPTOSIS AND ANGIOFIBROSIS IN DIABETIC TRACTIONAL MEMBRANES AFTER VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITION: Results of a Prospective Trial. Report No. 2.

PURPOSE: We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy. METHODS: Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed. RESULTS: High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF. CONCLUSION: Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.

A Novel HDL-Mimetic Peptide HM-10/10 Protects RPE and Photoreceptors in Murine Models of Retinal Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD. Oxidative stress plays a key role in the development of AMD. We generated a chimeric high-density lipoprotein (HDL), mimetic peptide named HM-10/10, with anti-oxidant properties and investigated its potential for the treatment of retinal disease using cell culture and animal models of RPE and photoreceptor (PR) degeneration. Treatment with HM-10/10 peptide prevented human fetal RPE cell death caused by tert-Butyl hydroperoxide (tBH)-induced oxidative stress and sodium iodate (NaIO3), which causes RPE atrophy and is a model of geographic atrophy in mice. We also show that HM-10/10 peptide ameliorated photoreceptor cell death and significantly improved retinal function in a mouse model of N-methyl-N-nitrosourea (MNU)-induced PR degeneration. Our results demonstrate that HM-10/10 protects RPE and retina from oxidant injury and can serve as a potential therapeutic agent for the treatment of retinal degeneration.

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition.

Subretinal fibrosis is an end stage of neovascular age-related macular degeneration, characterized by fibrous membrane formation after choroidal neovascularization. An initial step of the pathogenesis is an epithelial-mesenchymal transition (EMT) of retinal pigment epithelium cells. αB-crystallin plays multiple roles in age-related macular degeneration, including cytoprotection and angiogenesis. However, the role of αB-crystallin in subretinal EMT and fibrosis is unknown. Herein, we showed attenuation of subretinal fibrosis after regression of laser-induced choroidal neovascularization and a decrease in mesenchymal retinal pigment epithelium cells in αB-crystallin knockout mice compared with wild-type mice. αB-crystallin was prominently expressed in subretinal fibrotic lesions in mice. In vitro, overexpression of αB-crystallin induced EMT, whereas suppression of αB-crystallin induced a mesenchymal-epithelial transition. Transforming growth factor-ß2-induced EMT was further enhanced by overexpression of αB-crystallin but was inhibited by suppression of αB-crystallin. Silencing of αB-crystallin inhibited multiple fibrotic processes, including cell proliferation, migration, and fibronectin production. Bone morphogenetic protein 4 up-regulated αB-crystallin, and its EMT induction was inhibited by knockdown of αB-crystallin. Furthermore, inhibition of αB-crystallin enhanced monotetraubiquitination of SMAD4, which can impair its nuclear localization. Overexpression of αB-crystallin enhanced nuclear translocation and accumulation of SMAD4 and SMAD5. Thus, αB-crystallin is an important regulator of EMT, acting as a molecular chaperone for SMAD4 and as its potential therapeutic target for preventing subretinal fibrosis development in neovascular age-related macular degeneration.

Targeting of exon VI-skipping human RGR-opsin to the plasma membrane of pigment epithelium and co-localization with terminal complement complex C5b-9.

PURPOSE: Rare mutations in the human RGR gene lead to autosomal recessive retinitis pigmentosa or dominantly inherited peripapillary choroidal atrophy. Here, we analyze a common exon-skipping isoform of the human retinal G protein-coupled receptor opsin (RGR-d) to determine differences in subcellular targeting between RGR-d and normal RGR and possible association with abnormal traits in the human eye. METHODS: The terminal complement complex (C5b-9), vitronectin, CD46, syntaxin-4, and RGR-d were analyzed in human eye tissue from young and old donors or in cultured fetal RPE cells by means of immunofluorescent labeling and high-resolution confocal microscopy or immunohistochemical staining. RESULTS: We observed that RGR-d is targeted to the basolateral plasma membrane of the RPE. RGR-d, but not normal RGR, is expressed in cultured human fetal RPE cells in which the protein also trafficks to the plasma membrane. In young donors, the amount of RGR-d protein in the basolateral plasma membrane was much higher than that in the RPE cells of older subjects. In older donor eyes, the level of immunoreactive RGR-d within RPE cells was often low or undetectable, and immunostaining of RGR-d was consistently strongest in extracellular deposits in Bruch's membrane. Double immunofluorescent labeling in the basal deposits revealed significant aggregate and small punctate co-localization of RGR-d with C5b-9 and vitronectin. CONCLUSIONS: RGR-d may escape endoplasmic reticulum-associated degradation and in contrast to full-length RGR, traffick to the basolateral plasma membrane, particularly in younger subjects. RGR-d in the plasma membrane indicates that the protein is properly folded, as misfolded membrane proteins cannot otherwise sort to the plasma membrane. The close association of extracellular RGR-d with both vitronectin and C5b-9 suggests a potential role of RGR-d-containing deposits in complement activation.

SIRT1 mediated inhibition of VEGF/VEGFR2 signaling by Resveratrol and its relevance to choroidal neovascularization.

SIRT1, a NAD(+) -dependent histone deacetylase, has been shown to act as a key regulator of angiogenesis. The purpose of this study was to determine the effects of resveratrol (RSV, a SIRT1 activator) on the vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway and to establish its relevance to choroidal neovascularization (CNV), a blinding complication of age-related macular degeneration. Western blot and ELISA assay showed that RSV inhibited hypoxia-inducible factor (HIF)-1α accumulation and VEGF secretion induced by cobalt chloride (CoCl2) through SIRT1 in human retinal pigment epithelial (hRPE) cells. Furthermore, RSV down-regulated VEGFR2 phosphorylation and activation induced by VEGF in endothelial cells via SIRT1. Thus, the inhibitory effect of RSV on the HIF-1α/VEGF/VEGFR2 signaling axis is mediated, at least in part, through SIRT1. The results suggest that targeting SIRT1 could have therapeutic potential for the treatment of CNV.

Ceramide inhibits connective tissue growth factor expression by human retinal pigment epithelial cells.

Connective tissue growth factor (CTGF) is known to be involved in retinal fibrotic disorders. We used human retinal pigment epithelial cells (HRPE), which play critical roles in retinal fibrosis, to examine the expression of CTGF and its regulation by ceramide and TGF-ß. Real-time PCR analysis showed downregulation of CTGF mRNA by C2 ceramide and upregulation by TGF-ß. C2 ceramide also inhibited constitutive and TGF-ß-enhanced CTGF secretion by HRPE cells. Predominant secretion (>80% of total) of CTGF from the apical side was observed in highly polarized HRPE cells. Fumonosin, an inhibitor of ceramide synthesis, stimulated CTGF secretion while 4HPR, an activator of ceramide synthesis, downregulated CTGF secretion. Based on these results demonstrating ceramide regulation of CTGF secretion by HRPE, we suggest that ceramide may have therapeutic potential for the treatment of retinal fibrotic diseases by inhibiting CTGF production.

Paraoxonase 2 Deficiency Causes Mitochondrial Dysfunction in Retinal Pigment Epithelial Cells and Retinal Degeneration in Mice.

Although AMD is a complex disease, oxidative stress is a crucial contributor to its development, especially in view of the higher oxygen demand of the retina. Paraoxonase 2 (PON2) is a ubiquitously and constitutively expressed antioxidant protein that is found intracellularly associated with mitochondrial membranes and modulates mitochondrial ROS production and function. The contribution of PON2 to AMD has not been studied to date. In this study, we examined the role of PON2 in AMD utilizing both in vitro and in vivo models of AMD with emphasis on mitochondrial function. Mitochondrial localization and regulation of PON2 following oxidative stress were determined in human primary cultured retinal pigment epithelium (hRPE) cells. PON2 was knocked down in RPE cells using siRNA and mitochondrial bioenergetics were measured. To investigate the function of PON2 in the retina, WT and PON2-deficient mice were administered NaIO3 (20 mg/kg) intravenously; fundus imaging, optical coherence tomography (OCT), electroretinography (ERG) were conducted; and retinal thickness and cell death were measured and quantified. In hRPE, mitochondrial localization of PON2 increased markedly with stress. Moreover, a time-dependent regulation of PON2 was observed following oxidative stress, with an initial significant increase in expression followed by a significant decrease. Mitochondrial bioenergetic parameters (basal respiration, ATP production, spare respiratory capacity, and maximal respiration) showed a significant decrease with oxidative stress, which was further exacerbated in the absence of PON2. NaIO3 treatment caused significant retinal degeneration, retinal thinning, and reduced rod and cone function in PON2-deficient mice when compared to WT mice. The apoptotic cells and active caspase 3 significantly increased in PON2-deficient mice treated with NaIO3, when compared to WT mice. Our investigation demonstrates that deficiency of PON2 results in RPE mitochondrial dysfunction and a decline in retinal function. These findings imply that PON2 may have a beneficial role in retinal pathophysiology and is worthy of further investigation.

Characterization and contribution of RPE senescence to Age-related macular degeneration in Tnfrsf10 knock out mice.

Background: Retinal pigment epithelial cells (RPE) play vital role in the pathogenesis of age-related macular degeneration (AMD). Our laboratory has shown that RPE cellular senescence contributed to the pathophysiology of experimental AMD, and SASP members are involved in this process. Recently, we presented confirmatory evidence to earlier GWAS studies that dysregulation of tumor necrosis factor receptor superfamily 10A (TNFRSF10A) dysregulation leads to AMD development and is linked to RPE dysfunction. This study aims to investigate the contribution of RPE senescence to AMD pathophysiology using TNFRSF10A silenced human RPE (hRPE) cells and Tnfrsf10 KO mice. Methods: Sub-confluent primary hRPE cells and TNFRSF10A silenced hRPE were exposed to stress-induced premature senescence with H2O2 (500 µM, 48h), and senescence-associated markers (ßgal, p16, and p21) were analyzed by RT-PCR and WB analysis. The effect of H2O2-induced senescence in non-silenced and silenced hRPE on OXPHOS and glycolysis was determined using Seahorse XF96 analyzer. Male C57BL/6J Tnfrsf10 KO ( Tnfrsf10 -/- ) mice were used to study the regulation of senescence by TNFRSF10A in vivo . Expression of p16 and p21 in control and KO mice of varying ages were determined by RT-PCR, WB, and immunostaining analysis. Results: The senescence-associated p16 and p21 showed a significant ( p < 0.01) upregulation with H2O2 induction at the gene (1.8- and 3-fold) and protein (3.2- and 4-fold) levels in hRPE cells. The protein expression of p16 and p21 was further significantly increased by co-treatment with siRNA ( p < 0.05 vs. H2O2). Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (pmol/min/total DNA) increased with senescence induction by H2O2 for 48h in control RPE, and knockdown of TNFRSF10A caused a further increase in OCR and ECAR. In addition, co-treatment with PKC activator significantly improved all parameters. Similarly, in vivo studies showed upregulation of p16 and p21 by RT-PCR, WB, and immunostaining analysis in RPE/choroid of Tnfrsf10 KO mice. When subjected to examination across distinct age groups, namely young (1-3 months), middle (6-9 months), and old (12-15 months) mice, a discernible age-related elevation in the expression of p16 and p21 was observed. Conclusions: Our findings suggest that TNRSF10A is a regulator of regulates in RPE senescence. Further work on elucidating pathways of senescence will facilitate the development of new therapeutic targets for AMD.

Over-expression of BMP4 inhibits experimental choroidal neovascularization by modulating VEGF and MMP-9.

Bone morphorgenetic protein (BMP)-4 has been shown to play a pivotal role in eye development; however, its role in mature retina or ocular angiogenic diseases is unclear. Activating downstream Smad signaling, BMP4 can be either pro-angiogenic or anti-angiogenic, depending on the context of cell types and associated microenvironment. In this study, we generated transgenic mice over-expressing BMP4 in retinal pigment epithelial (RPE) cells (Vmd2-Bmp4 Tg mice), and used the laser-induced choroidal neovascularization (CNV) model to study the angiogenic properties of BMP4 in adult eyes. Vmd2-Bmp4 Tg mice displayed normal retinal histology at 10 weeks of age when compared with age-matched wildtype mice. Over-expression of BMP4 in RPE in the transgenic mice was confirmed by real-time PCR and immunostaining. Elevated levels of Smad1,5 phosphorylation were found in BMP4 transgenic mice compared to wildype mice. Over-expression of BMP4 was associated with less severe CNV as characterized by fluorescein angiography, CNV volume measurement and histology. While control mice showed increased levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 after laser injury, Vmd2-Bmp4 Tg showed no increase in either VEGF or MMP-9. Further, we found that TNF-induced MMP-9 secretion in vitro was reduced by pretreatment of RPE cells with BMP4. The inhibition of MMP-9 was Smad-dependent because BMP4 failed to repress TNF-induced MMP-9 expression when Smad1,5 was silenced by siRNA. In summary, our studies identified an anti-angiogenic role for BMP4 in laser-induced CNV, mediated by direct inhibition of MMP-9 and indirect inhibition of VEGF.

alphaB-crystallin regulation of angiogenesis by modulation of VEGF.

alphaB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (alphaB-crystallin(-/-)) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in alphaB-crystallin(-/-) retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both alphaB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed alphaB-crystallin binding to VEGF-A but not transforming growth factor-beta in cultured retinal pigment epithelial (RPE) cells. alphaB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. alphaB-crystallin(-/-) RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and alphaB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with alphaB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in alphaB-crystallin(-/-) than wild-type mice. Proteasomal inhibition in alphaB-crystallin(-/-) mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for alphaB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target.

Transcriptional regulation of bone morphogenetic protein 4 by tumor necrosis factor and its relationship with age-related macular degeneration.

Bone morphogenetic protein-4 (BMP4) may be involved in the molecular switch that determines which late form of age-related macular degeneration (AMD) an individual develops. BMP4 expression is high in retinal pigment epithelium (RPE) cells in late, dry AMD patients, while BMP4 expression is low in the wet form of the disease, characterized by choroidal neovascularization (CNV). Here, we sought to determine the mechanism by which BMP4 is down-regulated in CNV. BMP4 expression was decreased within laser-induced CNV lesions in mice at a time when tumor necrosis factor (TNF) expression was high (7 d postlaser) and was reexpressed in RPE when TNF levels declined (14 d postlaser). We found that TNF, an important angiogenic stimulus, significantly down-regulates BMP4 expression in cultured human fetal RPE cells, ARPE-19 cells, and RPE cells in murine posterior eye cup explants. We identified two specificity protein 1 (Sp1) binding sites in the BMP4 promoter that are required for basal expression of BMP4 and its down-regulation by TNF. Through c-Jun NH(2)-terminal kinase (JNK) activation, TNF modulates Sp1 phosphorylation, thus decreasing its affinity to the BMP4 promoter. The down-regulation of BMP4 expression by TNF in CNV and mechanisms established might be useful for defining novel targets for AMD therapy.

Oxidative Stress and Lipid Accumulation Augments Cell Death in LDLR-Deficient RPE Cells and Ldlr-/- Mice.

Lipid peroxidation from oxidative stress is considered a major contributor to age-related macular degeneration (AMD). The retina is abundant with circulating low-density lipoproteins (LDL), which are taken up by LDL receptor (LDLR) in the RPE and Müller cells. The purpose of this study is to investigate the role of LDLR in the NaIO3-induced model of dry AMD. Confluent primary human RPE (hRPE) and LDLR-silenced ARPE-19 cells were stressed with 150 µM tert-butyl hydroperoxide (tBH) and caspase 3/7 activation was determined. WT and Ldlr-/- mice were administered NaIO3 (20 mg/kg) intravenously. On day 7, fundus imaging, OCT, ERG, and retinal thickness were measured. Histology, TUNEL, cleaved caspase 3 and lipid accumulation were assessed. Treatment of hRPE with tBH markedly decreased LDLR expression. Caspase 3/7 activation was significantly increased in LDLR-silenced ARPE-19 cells treated with tBH. In Ldlr-/- mice, NaIO3 administration resulted in significant (a) retinal thinning, (b) compromised photoreceptor function, (c) increased percentage of cleaved caspase 3 positive and apoptotic cells, and (d) increased lipid droplet accumulation in the RPE, Bruch membrane, choroid, and sclera, compared to WT mice. Our findings imply that LDLR loss leads to lipid accumulation and impaired retinal function, which may contribute to the development of AMD.

Mechanisms of Epithelial-Mesenchymal Transition and Prevention of Dispase-Induced PVR by Delivery of an Antioxidant αB Crystallin Peptide.

Proliferative Vitreoretinopathy (PVR) is a refractory retinal disease whose primary pathogenesis involves the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. At present, there is no effective treatment other than surgery for PVR. The purpose of this study was to investigate the effect of αB crystallin peptide (αBC-P) on EMT in PVR. We have previously shown that this peptide is antiapoptotic and regulates RPE redox status. Subconfluent primary human RPE (hRPE) cells were stimulated by TGFß2 (10 ng/mL) with or without αBC-P (50 or 75 µg/mL) for 48 h and expression of EMT/mesenchymal to epithelial transition (MET) markers was determined. Mitochondrial ROS (mtROS) generation in hRPE cells treated with TGFß2 was analyzed. The effect of TGFß2 and αBC-P on oxidative phosphorylation (OXPHOS) and glycolysis in hRPE was studied. RPE cell migration was also assessed. A PVR-like phenotype was induced by intravitreal dispase injection in C57BL/6J mice. PVR progression and potential therapeutic efficiency of αBC-Elastin-like polypeptides (ELP) was studied using fundus photography, OCT imaging, ERG, and histologic analysis of the retina. αSMA, E-cadherin, Vimentin, Fibronectin and, RPE65, and CTGF were analyzed on Day 28. Additionally, the amount of VEGF-A in retinal cell lysates was measured. The EMT-associated αSMA, Vimentin, SNAIL and SLUG showed a significant upregulation with TGFß2, and their expression was significantly suppressed by cotreatment with αBC-P. The MET-associated markers, E-cadherin and Sirt1, were significantly downregulated by TGFß2 and were restored by αBC-P. Incubation of hRPE with TGFß2 for 24 h showed a marked increase in mitochondrial ROS which was noticeably inhibited by αBC-ELP. We also showed that after TGFß2 treatment, SMAD4 translocated to mitochondria which was blocked by αBC-ELP. Mitochondrial oxygen consumption rate increased with TGFß2 treatment for 48 h, and αBC-P co-treatment caused a further increase in OCR. Glycolytic functions of RPE were significantly suppressed with αBC-P (75 µg/mL). In addition, αBC-P significantly inhibited the migration from TGFß2 treatment in hRPE cells. The formation of proliferative membranes was suppressed in the αBC-ELP-treated group, as evidenced by fundus, OCT, and H&E staining in dispase-induced PVR in mice. Furthermore, ERG showed an improvement in c-wave amplitude. In addition, immunostaining showed significant suppression of αSMA and RPE65 expression. It was also observed that αBC-ELP significantly reduced the expression level of vimentin, fibronectin, and CTGF. Our findings suggest that the antioxidant αBC-P may have therapeutic potential in preventing PVR by reversing the phenotype of EMT/MET and improving the mitochondrial function in RPE cells.

Blocking Ocular Sympathetic Activity Inhibits Choroidal Neovascularization.

Purpose: To investigate how modulating ocular sympathetic activity affects progression of choroidal neovascularization (CNV), a hallmark feature of wet age-related macular degeneration (AMD). Methods: In the first of two studies, Brown Norway rats underwent laser-induced CNV and were assigned to one of the following groups: daily eye drops of artificial tears (n = 10; control group); daily eye drops of the ß-adrenoreceptor agonist isoproterenol (n = 10); daily eye drops of the ß-adrenoreceptor antagonist propranolol (n = 10); sympathetic internal carotid nerve (ICN) transection 6 weeks prior to laser-induced CNV (n = 10). In the second study, rats underwent laser-induced CNV followed by ICN transection at different time points: immediately after the laser injury (n = 6), 7 days after the laser injury (n = 6), and sham surgery 7 days after the laser injury (n = 6; control group). All animals were euthanized 14 days after laser application. CNV development was quantified with fluorescein angiography and optical coherence tomography (in vivo), as well as lesion volume analysis using 3D confocal reconstruction (postmortem). Angiogenic growth factor protein levels in the choroid were measured with ELISA. Results: In the first study, blocking ocular sympathetic activity through pharmacological or surgical manipulation led to a 75% or 70% reduction in CNV lesion volume versus the control group, respectively (P < 0.001). Stimulating ocular sympathetic activity with isoproterenol also led to a reduction in lesion volume, but only by 27% versus controls (P < 0.05). VEGF protein levels in the choroid were elevated in the three treatment groups (P < 0.01). In the second study, fluorescein angiography and CNV lesion volume analysis indicated that surgically removing the ocular sympathetic supply inhibited progression of laser-induced CNV, regardless of whether ICN transection was performed on the same day or 7 days after the laser injury. Conclusion: Surgical and pharmacological block of ocular sympathetic activity can inhibit progression of CNV in a rat model. Therefore, electrical block of ICN activity could be a potential bioelectronic medicine strategy for treating wet AMD.

Neutrophils compromise retinal pigment epithelial barrier integrity.

We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE). The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [3H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP-) 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P < .05). Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P < .05) and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.

A comparison of hypoxia-inducible factor-α in surgically excised neovascular membranes of patients with diabetes compared with idiopathic epiretinal membranes in nondiabetic patients.

PURPOSE: The purpose of this study was to first determine whether hypoxia-inducible factor-1α (HIF-1 α) was detectable in diabetic preretinal membranes and to compare the presence of HIF-1α in fibrovascular proliferative diabetic retinopathy membranes with nondiabetic, idiopathic, epiretinal membranes. METHODS: Twelve patients with proliferative diabetic retinopathy membranes requiring pars plana vitrectomy and nine nondiabetic patients with idiopathic epiretinal membranes requiring pars plana vitrectomy underwent excision of these membranes. Immunohisto-chemical staining for the presence of HIF-1α was performed on the excised membranes. The degree of staining for HIF-1α (1+, 2+, and 3+ scale) and the cellular location of staining were determined for each specimen. Institutional Review Board approval and informed consent were obtained for all patients. RESULTS: Eleven of 12 (92%) diabetic preretinal membranes were positive for HIF-1α, and most had intense (2+ to 3+) cytoplasmic staining with occasional focal nuclear positivity. Five of 9 (55%) nondiabetic epiretinal membranes were positive for HIF-1α with significantly weaker cytoplasmic staining (1+ to 2+) with occasional focal punctuate nuclear staining. CONCLUSION: Hypoxia-inducible factor-1α is found more often and more intensely in diabetic preretinal membranes compared with nondiabetic idiopathic epiretinal membranes.

Transporter-Mediated Mitochondrial GSH Depletion Leading to Mitochondrial Dysfunction and Rescue with αB Crystallin Peptide in RPE Cells.

: Mitochondrial glutathione (mGSH) is critical for cell survival. We recently reported the localization of OGC (SLC25A11) and DIC (SLC25A10) in hRPE. Herein, we investigated the suppression of OGC and DIC and the effect of αB crystallin chaperone peptide co-treatment on RPE cell death and mitochondrial function. Non-polarized and polarized human RPE were co-treated for 24 h with phenyl succinic acid (PS, 5 mM) or butyl malonic acid (BM, 5 mM) with or without αB cry peptide (75 µg/mL). mGSH levels, mitochondrial bioenergetics, and ETC proteins were analyzed. The effect of mGSH depletion on cell death and barrier function was determined in polarized RPE co-treated with PS, OGC siRNA or BM and αB cry peptide. Inhibition of OGC and DIC resulted in a significant decrease in mGSH and increased apoptosis. mGSH depletion significantly decreased mitochondrial respiration, ATP production, and altered ETC protein expression. αB cry peptide restored mGSH, attenuated apoptosis, upregulated ETC proteins, and improved mitochondrial bioenergetics and biogenesis. mGSH transporters exhibited differential polarized localization: DIC (apical) and OGC (apical and basal). Inhibition of mGSH transport compromised barrier function which was partially restored by αB cry peptide. Our findings suggest mGSH augmentation by its transporters may be a valuable approach in AMD therapy.

Characterization and Regulation of Carrier Proteins of Mitochondrial Glutathione Uptake in Human Retinal Pigment Epithelium Cells.

Purpose: To characterize two mitochondrial membrane transporters 2-oxoglutarate (OGC) and dicarboxylate (DIC) in human RPE (hRPE) and to elucidate their role in the regulation of mitochondrial glutathione (mGSH) uptake and cell death in oxidative stress. Methods: The localization of OGC and DIC proteins in confluent hRPE, polarized hRPE monolayers and mouse retina was assessed by immunoblotting and confocal microscopy. Time- and dose-dependent expression of the two carriers were determined after treatment of hRPE with H2O2, phenyl succinate (PS), and butyl malonate (BM), respectively, for 24 hours. The effect of inhibition of OGC and DIC on apoptosis (TUNEL), mGSH, and mtDNA was determined. Silencing of OGC by siRNA knockdown on RPE cell death was studied. Kinetics of caspase 3/7 activation with OGC and DIC inhibitors and effect of cotreatment with glutathione monoethyl ester (GSH-MEE) was determined using the IncuCyte live cell imaging. Results: OGC and DIC are expressed in hRPE mitochondria and exhibited a time- and dose-dependent decrease with stress. Pharmacologic inhibition caused a decrease in OGC and DIC in mitochondria without changes in mtDNA and resulted in increased apoptosis and mGSH depletion. GSH-MEE prevented apoptosis through restoration of mGSH. OGC siRNA exacerbated apoptotic cell death in stressed RPE which was inhibited by increased mGSH from GSH-MEE cotreatment. Conclusions: Characterization and mechanism of action of two carrier proteins of mGSH uptake in RPE are reported. Regulation of OGC and DIC will be of value in devising therapeutic strategies for retinal disorders such as AMD.

Sympathetic Effects of Internal Carotid Nerve Manipulation on Choroidal Vascularity and Related Measures.

Purpose: To investigate specific effects of denervation and stimulation of the internal carotid nerve (ICN) on the choroid and retina. Methods: Female Sprague Dawley rats underwent unilateral ICN transection (n = 20) or acute ICN electrical stimulation (n = 7). Rats in the denervation group were euthanized 6 weeks after nerve transection, and eyes were analyzed for changes in choroidal vascularity (via histomorphometry) or angiogenic growth factors and inflammatory markers (via ELISA). Rats in the stimulation group received acute ICN electrical stimulation with a bipolar cuff electrode over a range of stimulus amplitudes, frequencies, and pulse widths. Choroidal blood flow and pupil diameter were monitored before, during, and after stimulation. Results: Six weeks after unilateral ICN transection, sympathectomized choroids exhibited increased vascularity, defined as the percentage of choroidal surface area occupied by blood vessel lumina. Vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein levels in denervated choroids were 61% and 124% higher than in contralateral choroids, respectively. TNF-α levels in denervated retinas increased by 3.3-fold relative to levels in contralateral retinas. In animals undergoing acute ICN electrical stimulation, mydriasis and reduced choroidal blood flow were observed in the ipsilateral eye. The magnitude of the reduction in blood flow correlated positively with stimulus frequency. Conclusions: Modulation of ICN activity reveals a potential role of the ocular sympathetic system in regulating endpoints related to neovascular diseases of the eye.

N-(4-hydroxyphenyl) retinamide augments laser-induced choroidal neovascularization in mice.

PURPOSE: To evaluate the effect of N-4-hydroxyphenyl retinamide (4-HPR) on experimental laser-induced choroidal neovascularization (CNV) and on the expression and secretion of relevant growth factors by cultured human retinal pigment epithelial (RPE) cells. METHODS: CNV was induced by laser photocoagulation in C57BL/6 mice. 4-HPR (0.2 or 1 mg) or vehicle, was injected intraperitoneally twice daily for 14 days. Plasma and tissue levels of 4-HPR were measured by HPLC. CNV was evaluated by fluorescein angiography, histology, and quantitative confocal analysis of isolectin B4 histochemistry on days 7 and 14. Induction of apoptosis and expression and secretion of growth factors was studied in 4-HPR-treated RPE cultures. RESULTS: Mice treated with 4-HPR exhibited time- and dose-dependent increases in plasma and tissue 4-HPR levels. CNV lesions showed increased volume with increased vascular leakage and contained fewer lesion-associated RPE in treated versus untreated mice. Treatment of nonpolarized RPE cultures with 4-HPR in the presence of serum resulted in RPE apoptosis; however, apoptosis was minimal in similarly treated highly polarized RPE. Treatment of RPE cells with 4-HPR resulted in the upregulation of VEGF-A and -C (P < 0.05) and Ang-1 (P < 0.01) mRNA and increased secretion of VEGF-A and -C (P < 0.05), whereas pigment epithelium-derived growth factor (PEDF) and thrombospondin (TSP)-1 mRNA expression and secretion were downregulated (P < 0.05). CONCLUSIONS: 4-HPR increases lesion size and leakage in laser-induced CNV and is associated with the upregulation of key proangiogenic factors and the downregulation of antiangiogenic factors. Consistent with the preferential loss of RPE in CNV lesions in vivo, 4-HPR induces apoptosis of nonpolarized RPE in the presence of serum.