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L. Notari, A. Miller, A. Martinez, J. Amaral, J. Meihua, G. Robison, L.E. H. Smith, S. Becerra; Pigment Epithelium–derived Factor Is a Substrate of MMPS: Implications for Downregulation in Hypoxia . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1012.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: PEDF, a protein secreted by the retinal pigment epithelium (RPE), acts on retinal survival and angiogenesis. Because hypoxia and VEGF upregulate matrix metalloproteinases (MMPs), their effects on PEDF proteolysis were explored. Methods: Hypoxia in RPE was induced in an in vivo mouse model for Retinopathy of Prematurity, and in cultured monkey cells by exposure to low oxygen and chemical hypoxia mimetics. mRNA levels were determined by RT–PCR. Proteolytic activities were determined by gelatin zymography and DQ–gelatin degradation solution assays. MMP–2 and MMP–9 were detected by western blotting. PEDF proteolysis was assayed in solution by SDS–PAGE followed by Coomassie blue staining or immunostaining. Retinal cell survival was assayed in R28 retinal cells. Ex vivo chick embryonic aortic vessel sprouting assay and directed in vivo angiogenesis assays were performed. Results: PEDF levels in the RPE/choroid of the ROP model significantly decreased when tissue hypoxia was induced. In cultured RPE cells, hypoxia decreased PEDF levels in the media, with no significant change for PEDF mRNA. Conversely, hypoxia increased PEDF proteolysis, gelatinolytic activities of ∼57–kDa and ∼86–kDa zymogens, and MMP–2 immunoreactivities in a dose–dependent fashion. BHK cells secreted PEDF–degrading activities as well as ∼57–kDa zymogens and MMP–2 immunoreactivities. The zymogens and PEDF–degrading activities in the BHK conditioned media and those in vitreal extracts shared identical MMP protease inhibition pattern. Increasing VEGF additions gradually promoted the increase of MMP–2 and PEDF–degrading activities in the BHK conditioned media. Limited proteolysis with MMP–2 and MMP–9 degraded PEDF in a Ca+2–dependent fashion to yield undetectable products. MMP–mediated proteolysis of PEDF abolished the retinal survival and antiangiogenic activities of PEDF. Conclusions: Hypoxia and VEGF can downregulate PEDF via proteolytic degradation. PEDF is a novel substrate for MMP–2/–9. These results reveal a novel post–translational mechanism for downregulating PEDF, and provide an explanation for hypoxia–provoked increases in VEGF/PEDF ratios, in angiogenesis and/or in neuronal death.
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