Michael H. Davies

MCP-1 deficiency delays regression of pathologic retinal neovascularization in a model of ischemic retinopathy.

PURPOSE The present study investigates whether retinal neovascularization (NV) and apoptosis are altered in MCP-1-deficient ((-/-)) mice in the OIR model. METHODS Postnatal day (P) 7 MCP-1(-/-) and C57BL/6 (B6) mice were exposed to 75% oxygen for 5 days and then recovered in room air. Immunostaining was performed to localize macrophages/microglia within retinal whole mounts and cross-sections. Retinopathy was qualitatively assessed in FITC-dextran-perfused retinas, and preretinal NV was quantified on P17, P21, and P24. TUNEL analysis was used to compare apoptosis between B6 and MCP-1(-/-) mice. RESULTS MCP-1(-/-) and B6 mice revealed normal vascular development in room air controls and similar vaso-obliteration in oxygen-exposed mice on P12. MCP-1(-/-) mice exhibited significantly reduced vascular tuft-associated F4/80(+) cells compared with B6 mice. FITC-dextran-perfused retinas exhibited prominent neovascular tufts on P17, and quantification of preretinal nuclei revealed no significant differences between MCP-1(-/-) and B6 mice. In contrast, on P21 and P24, MCP-1(-/-) mice exhibited significant increases in preretinal neovascular nuclei compared with B6 controls. These increases in NV in the MCP-1(-/-) mice were associated with a significant reduction in vascular tuft apoptosis. CONCLUSIONS The results demonstrate that the absence of MCP-1 does not alter normal retinal vascular development. Furthermore, MCP-1(-/-) mice exhibit a similar neovascular response on P17. However, the reduction in tuft-associated macrophages/microglia in the MCP-1(-/-) mice correlates with reduced vascular tuft apoptosis and delayed regression of retinal NV. These findings suggest that macrophages/microglia may contribute to tuft regression through their proapoptotic properties.

Increased expression of chemokine KC, an interleukin-8 homologue, in a model of oxygen-induced retinopathy.

Purpose: The purpose of this study was to determine the retinal expression of angiogenic chemokines/cytokines in a mouse model of oxygen-induced retinopathy. Methods: C57BL/6 (B6) mice were exposed to 75% oxygen from postnatal day 7 (P7) to P12 and then recovered in room air. Reverse transcription–polymerase chain reaction (RT-PCR) was used to determine relative mRNA levels of KC, macrophage inflammatory protein-2 (MIP-2), interleukin-1α (IL-1α), and interferon gamma (IFN-γ). Immunohistochemistry was used to localize KC in the retina. IL-1α was also injected into the vitreous of mouse eyes, and KC expression was examined by RT-PCR, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. Results: KC expression at both the mRNA and protein levels was increased in P14, P17, and P21 of hyperoxia-injured eyes. KC immunoreactivity was localized along the nerve fiber layer and in radial Müller cell processes. IL-1α mRNA was modestly increased in hyperoxia-injured eyes on P14 and P17. INF-γ mRNA was not detected in the retina. Adult mouse eyes injected with IL-1α demonstrated increased levels of both KC mRNA and protein, with KC immunoreactivity localized to Müller cell processes. Conclusions: Oxygen-induced injury to the developing retina results in the induction of the CXC chemokine KC at both the mRNA and protein levels during the peak time points of neovascularization, suggesting a possible role in the pathogenesis of retinopathy of prematurity.

Soluble Ephrin-B2 Mediates Apoptosis in Retinal Neovascularization and in Endothelial Cells

PURPOSE EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. METHODS C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 microL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). RESULTS Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2+/-VEGF compared to IgG control+/-VEGF (P<0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. CONCLUSIONS These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.

Altered Retinal Neovascularization in TNF Receptor–Deficient Mice

Purpose: Tumor necrosis factor alpha (TNF-α) has been shown to play an integral role in inflammation, apoptosis, and angiogenesis. We induced retinopathy in tumor necrosis factor receptor–deficient mice (TNFR-) in order to examine the role TNF-α plays in the pathogenesis of retinopathy of prematurity. Methods: On postnatal day (P) 7, TNFR-knockout mice and their congenic controls, B6129JF1 (B6129) mice, were exposed to 75% oxygen for up to 5 days and then allowed to recover in room air. Retinopathy was qualitatively assessed by examining fluorescein (FITC) angiography. Furthermore, retinal vascular changes were quantified by immunolabeling retinal vessels in cross sections with an anti–type IV collagen antibody. Disease pathology was quantified by counting preretinal neovascular nuclei. TUNEL analysis was performed to determine if TNFR-mice exhibited a reduced number of apoptotic cells after oxygen-induced retinopathy. Results: FITC-perfused retinas qualitatively demonstrated similar degrees of vascular development and vaso-obliteration on P12 in the room air and hyperoxia-exposed TNFR- and B6129 mice. On P17, the hyperoxia-exposed TNFR- and B6129 mice qualitatively appeared to develop a similar degree of retinal neovascularization. However, FITC-perfused retinal flat mounts on P21 suggested that the hyperoxia-exposed TNFR-mice had a prolonged neovascular response compared to the hyperoxia-exposed B6129 mice. Type IV collagen staining revealed delayed development of the deep intraretinal vessels in the TNFR-room control mice and hyperoxia-exposed TNFR-mice, as compared with B6129 controls. On P17, the average number of preretinal nuclei was similar between the hyperoxia-exposed TNFR-mice and B6129 mice. However, on P21, the neovascularization in the B6129 mice had regressed (3.9 ± 0.57, preretinal nuclei), whereas neovascularization in the TNFR-mice remained prominent (25.6 ± 6.3, preretinal nuclei). On P21, the B6129 mice exhibited increased apoptosis in preretinal vascular tufts as compared with TNFR- mice. Conclusions: TNFR- mice had both an altered development of the intraretinal vessels and altered angiogenic response after hyperoxia. Therefore, absence of the TNF-α pathway appears to disrupt the local microenvironment promoting angiogenesis in the deep retinal vascular network, as well as altering tuft regression by modifying endothelial cell apoptosis.

Altered vascular expression of EphrinB2 and EphB4 in a model of oxygen-induced retinopathy

EphrinB2 ligands and EphB4 receptors are expressed on endothelial cells (EC) of arteries and veins, respectively, and are essential for vascular development. To understand how these molecules regulate retinal neovascularization (NV), we evaluated their expression in a model of oxygen‐induced retinopathy (OIR). EphrinB2 and EphB4 were expressed on arterial and venous trunks, respectively, and on a subset of deep capillary vessels. EphB4 expression was reduced following hyperoxia, while ephrinB2 expression remained unaltered. In addition, a subset of EphB4‐positive veins regressed in a caspase‐3‐dependent manner during hyperoxia. Arteriovenous malformations were also observed with loss of arterial‐venous boundaries. Finally, both ephrinB2 and EphB4 were expressed on a subset of neovascular tufts following hyperoxia. These data confirm the contribution of ECs from both venous and arterial origins to the development of retinal NV. Developmental Dynamics 239:1695–1707, 2010.

TRAIL-deficient mice exhibit delayed regression of retinal neovascularization.

While it is well established that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cell types, the role of TRAIL in regulation of retinal neovascularization (NV) has not been described. Here we determined the role of TRAIL in retinal NV during oxygen-induced retinopathy using TRAIL deficient ((-/-)) mice. TRAIL and its receptor, DR5, were expressed in wild-type retinas at all time points evaluated (postnatal days 12, 17, 21, 24) during oxygen-induced retinopathy and in age-matched room air control animals. Localization of TRAIL(+) cells within the neovascular tufts of hyperoxia- exposed wild-type mice suggested TRAIL plays a role in oxygen-induced retinopathy. Retinal vascular development appeared normal in the TRAIL(-/-) mice, except for a small but significant difference in the capillary-free zone surrounding major arteries. A minimal difference in avascularity was observed at postnatal day 12 in the retinas of TRAIL(-/-) mice after hyperoxia-exposure compared with wild-type mice, suggesting that TRAIL does not play a major role in the vaso-obliterative phase of oxygen-induced retinopathy. However, at the peak of NV, TRAIL(-/-) mice had a significant increase in retinal neovascularization. In addition, when NV naturally regresses in wild-type mice, TRAIL(-/-) mice continued to display significantly high levels of NV. This was attributed to a significant decrease in neovascular tuft cells undergoing apoptosis in TRAIL(-/-) mice. Together, these data strongly suggest that TRAIL plays a role in the control of retinal NV.