Hypoxia Induces the Expression of Membrane Type 1–Matrix Metalloproteinase in Retinal Glial Cells

Abstract

Abstract: : Purpose: Membrane type 1–matrix metalloproteinase (MT1–MMP) is known to be the main activator of proMMP–2. We previously reported that activation of proMMP–2 by MT1–MMP expressed in glial cells might be involved in the pathogenesis of proliferative diabetic retinopathy (PDR) through degrading the internal limiting membrane. The purpose of this study is to investigate whether hypoxia, which is one of the main pathological features in PDR, induces the expression of MT1–MMP in retinal glial cells. Methods: Retinal glial cells isolated from rabbit according to the methods as described previously, were incubated under hypoxic (1% O₂ concentration) and normoxic condition (20% O₂). Expression of MT1–MMP mRNA in the glial cell cultures was assessed by real–time polymerase chain reaction (PCR). Also, mRNA expression of MMP–2, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)–1 and –2 was analyzed by conventional reverse transcriptional PCR. Protein levels of MT1–MMP were determined by enzyme immunoassay (EIA) and immunoblotting. Immunohistochemical staining was performed to demonstrate the localization of MT1–MMP on the glial cell surface. Moreover, to investigate whether VEGF is a mediator of hypoxic induction of MT1–MMP, neutralization assay was conducted using mouse monoclonal anti–VEGF antibody. Results: Real–time PCR demonstrated that MT1–MMP mRNA was upregulated in the retinal glial cells exposed to hypoxia in a time–dependent manner. Hypoxic stimulation of glial cells for 24 hours increased the level of MT1–MMP mRNA by 2.3–fold as compared with the cells cultured under normoxic condition. Besides, in response to hypoxia, mRNA expression of VEGF and VEGFR–2, but not MMP–2 and VEGFR–1, is elevated in retinal glial cells. Hypoxic induction of MT1–MMP protein was also confirmed by EIA and immunoblotting. Immunohistochemical study showed that MT1–MMP was localized on elongated cell processes of cultured glial cells. In neutralization assay, it was revealed that anti–VEGF antibody inhibited hypoxic induction of MT1–MMP in retinal glial cells. Conclusions: These results implicated that hypoxia is capable of inducing the MT1–MMP and VEGF expression in retinal glial cells, and hypoxia–induced expression of MT1–MMP is, at least in part, mediated by VEGF. Current data supports our previous hypothesis that MT1–MMP expressed in glial cells might be involved in the formation of fibrovascular tissues in PDR.

Keywords: retinal glia • hypoxia • ischemia