Changes in components of the neurovascular unit in the retina in a rat model of retinopathy of prematurity.

An impairment of cellular interactions between the elements of the neurovascular unit contributes to the onset and/or progression of retinal diseases. The present study aims to examine how elements of the neurovascular unit are altered in a rat model of retinopathy of prematurity (ROP). Neonatal rats were treated subcutaneously with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8 to induce ROP. Morphological assessments were performed of blood vessels, astrocytes and neuronal cells in the retina. Aggressive angiogenesis, tortuous arteries and enlarged veins were observed in the retinal vasculature of KRN633-treated (ROP) rats from P14 to P28, compared to age-matched control (vehicle-treated) animals. Morphological abnormalities in the retinal vasculature showed a tendency toward spontaneous recovery from P28 to P35 in ROP rats. Immunofluorescence staining for glial fibrillary acidic protein and Pax2 (astrocyte markers) revealed that morphological changes to and a reduction in the number of astrocytes occurred in ROP rats. The developmental cell death was slightly accelerated in ROP rats; however, no visible changes in the morphology of retinal layers were observed on P35. The abnormalities in astrocytes might contribute, at least in part, to the formation of abnormal retinal blood vessels and the pathogenesis of ROP.

Knockout of αA-crystallin inhibits ocular neovascularization.

PURPOSE: The present study aimed to investigate the effects of αA-crystallin on pathologic ocular neovascularization. METHODS: Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and αA-crystallin-knockout (CRYAA[-/-]) mice were used for the in vivo study. αA-crystallin was knocked down in HUVECs by using a specific small interfering RNA (siRNA), and the effects of αA-crystallin knockdown on proliferation, migration, tube formation, and apoptosis were evaluated. Enzyme-linked immunosorbent assays were performed to investigate extracellular concentrations of vascular endothelial growth factor (VEGF). In vivo mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) were generated by using CRYAA(-/-) mice. The nonperfused area in the OIR model was measured in flat-mounted retinas, and angiogenesis resulting in CNV was evaluated in retinal sections. Western blot analysis was performed to investigate the phosphorylation status of vascular endothelial growth factor receptor 2 (VEGFR2, high affinity receptor for VEGF), AKT, PLCγ1, FAK, Src, p42/p44MAPK, p38MAPK, caspase-3, and caspase-9 in cultured HUVECs, as well as in the OIR and CNV models. RESULTS: The CRYAA siRNA not only induced HUVEC apoptosis but also inhibited exogenous and endogenous VEGF-induced cell activities, including proliferation, migration, and tube formation. Pathologic neovascularization was attenuated in the CRYAA(-/-) murine OIR and CNV models. Both in vitro and in vivo, the inhibition of angiogenesis was mediated by the suppression of VEGF secretion and the inhibition of the VEGFR2 signaling pathway; VEGFR2, AKT, PLCγ1, FAK, Src, p42/p44 MAPK, and p38 MAPK all showed reduced phosphorylation levels. In addition, CRYAA knockout led to increased levels of cleaved caspase-9 and caspase-3. CONCLUSIONS: Knockout of αA-crystallin inhibited pathologic neovascularization through the VEGF and VEGFR2 signaling pathways both in vitro and in vivo. These results suggest that αA-crystallin could be a novel pharmaceutical target for the prevention of ocular neovascularization.

Matrix metalloproteinase activity creates pro-angiogenic environment in primary human retinal pigment epithelial cells exposed to complement.

PURPOSE: Mechanistic studies have shown that inflammation, complement activation, extracellular matrix (ECM) turnover, growth factor imbalance, and oxidative stress are fundamental components of age-related macular degeneration (AMD). Matrix metalloproteinases (MMPs) mediate ECM turnover but also process various bioactive molecules. Here, we tested whether complement attack on RPE monolayers changes MMP secretion and activation, thereby altering the availability of growth factors in the extracellular space. METHODS: Human embryonic RPE monolayers with stable transepithelial resistance (TER) were established. Complement activation was induced with H2O2 and normal human serum. MMP-2/9, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) protein, and mRNA levels were analyzed by Western blotting, ELISA, and real-time PCR; activity of MMP-2/9 by gelatin zymography. RESULTS: Complement activation resulted in a loss of TER, which required transient membrane attack complex formation, activation of the alternative pathway, and VEGF secretion and signaling. Despite the generation of reactive oxygen species, cellular integrity or intracellular adenosine triphosphate (ATP) levels were unaffected. However, expression of MMP-2/9 and their protease activity was elevated. Inhibition of MMP-2/9 activity increased PEDF and decreased VEGF levels in the apical and basal supernatants but had no effect on their expression levels. VEGF levels in the supernatant correlated with the level TER reduction. CONCLUSIONS: These studies suggest that complement activation, by altering the expression and activation of MMPs, has the ability to generate a proangiogenic environment by altering the balance between VEGF and PEDF. Our findings link reported results that have been associated with AMD pathogenesis; oxidative stress; complement activation; VEGF/PEDF ratio; and MMP activity.

Alteration of developmental and pathological retinal angiogenesis in angptl4-deficient mice.

Proper vessel maturation, remodeling of endothelial junctions, and recruitment of perivascular cells is crucial for establishing and maintaining vessel functions. In proliferative retinopathies, hypoxia-induced angiogenesis is associated with disruption of the vascular barrier, edema, and vision loss. Therefore, identifying factors that regulate vascular maturation is critical to target pathological angiogenesis. Given the conflicting role of angiopoietin-like-4 (ANGPTL4) reported in the current literature using gain of function systems both in vitro and in vivo, the goal of this study was to characterize angiogenesis, focusing on perinatal retinal vascularization and pathological circumstances in angpl4-deficient mice. We report altered organization of endothelial junctions and pericyte coverage, both leading to impaired angiogenesis and increased vascular leakage that were eventually caught up, suggesting a delay in vessel maturation. In a model of oxygen-induced retinopathy, pathological neovascularization, which results from tissue hypoxia, was also strongly inhibited in angptl4-deficient mice. This study therefore shows that ANGPTL4 tunes endothelial cell junction organization and pericyte coverage and controls vascular permeability and angiogenesis, both during development and in pathological conditions.