Hypoxia-Induced Annexin A2 Expression Regulates Oxygen-Induced Retinopathy.

Abstract 471

Annexin A2 (A2), a Ca²⁺-dependent membrane binding protein that usually complexes with a S100 protein, p11, regulates fibrinolysis by serving as an endothelial cell co-receptor for plasminogen and tissue plasminogen activator to promote plasmin generation. Compared to A2^+/+ mice, A2^-/- mice have reduced post-hyperoxic retinal neovascularization in the model of oxygen-induced retinopathy (OIR) that mimics human retinopathy of prematurity. Here, we sought to determine the mechanism by which A2 contributes to the pathogenesis of OIR. A2^+/+ mouse pups (P7) were placed with their dams in a high O₂ (75%) chamber for 5 consecutive days (until P12), and then rapidly shifted to room air (21% O₂) for several more days. Mouse retinas were isolated over a range of time points for analysis of neovascularization, A2 protein and mRNA expression, fibrin deposition, cell proliferation and apoptosis assays. In post-hyperoxic A2^+/+ mice, we noted extensive retinal neovascularization along with a more than 10-fold increase of VEGFA compared to retinas of mice maintained in room air. Both A2 protein and mRNA levels increased 2-4-fold in mouse retinas with OIR in comparison with expression of either CD31 or VE-cadherin 5. p11 and hypoxia-inducible factor-1α (HIF-1α) protein, but not mRNA levels, also increased 2-4-fold during OIR. Because increased A2 expression was predominantly associated with retinal neovessels, we examined the response of cultured endothelial cells to hypoxia, and observed a 2-4-fold increase in A2 protein and mRNA. Although HIF-1α stabilization was not affected by A2 deficiency, upregulation of A2 by hypoxia was HIF-1α dependent. Expression of A6, on the other hand, was not upregulated by hypoxia. Neither erythropoietin (EPO) nor VEGFA signaling changed A2 expression. In addition, hypoxic endothelial cells translocated 50% more A2 protein to the cell surface, generated 30% more plasmin, and migrated twice as efficiently. While stability of A2 mRNA was not changed by hypoxia-related stimuli, we observed direct binding between HIF-1α and the A2 promoter by electrophoretic mobility shift assay, and a 3-4-fold increased A2 promoter activity in the presence of HIF-1α and HIF-1β by luciferase reporter assay. Promoter activation was abrogated when the HIF-1α binding site within the A2 promoter was mutated. By immunofluorescent staining of post-hyperoxic P17 retinal sections, we found that infiltration of endothelial cells and pericytes was 2-fold greater in A2^+/+ mice as compared to A2^-/- mice. In addition, there was significantly less fibrin deposition, more macrophage recruitment, less apoptosis and greater cell proliferation in P17 post-hyperoxic retinas of A2^+/+ mice as compared to A2^-/- mice. In conclusion, annexin A2 is upregulated by hypoxia through direct HIF-1α-mediated transcriptional regulation. Upregulated A2 appears to enhance plasmin generation, facilitate directed migration of vascular endothelial cells, and enable the recruitment of supporting cells (e.g. pericytes) and inflammatory cells (e.g. macrophages). It also reduces fibrin deposition, inhibits apoptosis, and promotes cell proliferation, thereby promoting pathologic oxygen-induced retinal neovascularization.