7-Ketocholesterol Promotes Retinal Pigment Epithelium Senescence and Fibrosis of Choroidal Neovascularization via IQGAP1 Phosphorylation-Dependent Signaling.

Accumulation of 7-ketocholesterol (7KC) occurs in age-related macular degeneration (AMD) and was found previously to promote fibrosis, an untreatable cause of vision loss, partly through induction of endothelial-mesenchymal transition. To address the hypothesis that 7KC causes mesenchymal transition of retinal pigment epithelial cells (RPE), we exposed human primary RPE (hRPE) to 7KC or a control. 7KC-treated hRPE did not manifest increased mesenchymal markers, but instead maintained RPE-specific proteins and exhibited signs of senescence with increased serine phosphorylation of histone H3, serine/threonine phosphorylation of mammalian target of rapamycin (p-mTOR), p16 and p21, ß-galactosidase labeling, and reduced LaminB1, suggesting senescence. The cells also developed senescence-associated secretory phenotype (SASP) determined by increased IL-1ß, IL-6, and VEGF through mTOR-mediated NF-κB signaling, and reduced barrier integrity that was restored by the mTOR inhibitor, rapamycin. 7KC-induced p21, VEGF, and IL-1ß were inhibited by an inhibitor of protein kinase C. The kinase regulates IQGAP1 serine phosphorylation. Furthermore, after 7KC injection and laser-induced injury, mice with an IQGAP1 serine 1441-point mutation had significantly reduced fibrosis compared to littermate control mice. Our results provide evidence that age-related accumulation of 7KC in drusen mediates senescence and SASP in RPE, and IQGAP1 serine phosphorylation is important in causing fibrosis in AMD.

Retinopathy of prematurity protection conferred by uteroplacental insufficiency through erythropoietin signaling in an experimental Murine Model.

BACKGROUND: Recent clinical studies suggest that preeclampsia, characterized by uteroplacental insufficiency (UPI) and infant intrauterine growth restriction (IUGR), may be protective against retinopathy of prematurity (ROP) in preterm infants. Experimental models of UPI/IUGR have found an association of erythropoietin (EPO) with less severe oxygen-induced retinopathy (OIR); however, it is unclear if EPO/EPO receptor (EPOR) signaling was involved. We hypothesized that maternal UPI and resultant infant IUGR would protect against features of ROP through EPO/EPOR signaling. METHODS: We compared transgenic mice with hypoactive EPOR signaling (hWtEPOR) to littermate wild-type mice (mWtEpoR) in a novel combined model of IUGR and ROP. Thromboxane A2 (TXA2) was infused into pregnant C57Bl/6J dams to produce UPI/IUGR; postnatal pups and their foster dams were subjected to a murine OIR model. RESULTS: Following hyperoxia, hematocrits were similar between littermate wild-type (mWtEpoR) TXA2/OIR and vehicle/OIR pups. mWtEpoR TXA2/OIR had increased serum EPO, retinal EPO and VEGF, and decreased avascular retinal area (AVA) compared to vehicle/OIR pups. In comparison to the mWtEpoR TXA2/OIR pups, AVA was not reduced in hWtEPOR TXA2/OIR pups. CONCLUSION: Our findings provide biologic evidence that UPI/OIR-induced endogenous EPOR signaling confers protection against hyperoxia-induced vascular damage that may be related to pathophysiology in ROP. IMPACT: Maternal preeclampsia and infant growth restriction confer retinovascular protection against high oxygen-induced damage through endogenous erythropoietin signaling.

Role of Erythropoietin Receptor Signaling in Macrophages or Choroidal Endothelial Cells in Choroidal Neovascularization.

Erythropoietin (EPO) has been proposed to reduce the progression of atrophic age-related macular degeneration (AMD) due to its potential role in neuroprotection. However, overactive EPO receptor (EPOR) signaling increased laser-induced choroidal neovascularization (CNV) and choroidal macrophage number in non-lasered mice, which raised the question of whether EPOR signaling increased CNV through the recruitment of macrophages to the choroid that released pro-angiogenic factors or through direct angiogenic effects on endothelial cells. In this study, we addressed the hypothesis that EPOR signaling increased CNV by direct effects on macrophages or endothelial cells. We used tamoxifen-inducible macrophage-specific or endothelial cell-specific EPOR knockout mice in the laser-induced CNV model, and cultured choroidal endothelial cells isolated from adult human donors. We found that macrophage-specific knockout of EPOR influenced laser-induced CNV in females only, whereas endothelial-specific knockout of EPOR reduced laser-induced CNV in male mice only. In cultured human choroidal endothelial cells, knockdown of EPOR reduced EPO-induced signal transducer and activator of transcription 3 (STAT3) activation. Taken together, our findings suggest that EPOR signaling in macrophages or choroidal endothelial cells regulates the development of CNV in a sex-dependent manner. Further studies regarding the role of EPO-induced signaling are required to assess EPO safety and to select or develop appropriate therapeutic approaches.

The Effects of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase and Erythropoietin, and Their Interactions in Angiogenesis: Implications in Retinopathy of Prematurity.

Retinopathy of prematurity (ROP) is a leading cause of vision impairment and blindness in premature infants. Oxidative stress is implicated in its pathophysiology. NADPH oxidase (NOX), a major enzyme responsible for reactive oxygen species (ROS) generation in endothelial cells, has been studied for its involvement in physiologic and pathologic angiogenesis. Erythropoietin (EPO) has gained interest recently due to its tissue protective and angiogenic effects, and it has been shown to act as an antioxidant. In this review, we summarize studies performed over the last five years regarding the role of various NOXs in physiologic and pathologic angiogenesis. We also discuss the effect of EPO in tissue and vasoprotection, and the intersection of EPO and NOX-mediated oxidative stress in angiogenesis and the pathophysiology of ROP.