Hypoxia activates matrix metalloproteinase expression and theVEGF system in monkey choroid-retinal endothelial cells: Involvement ofcytosolic phospholipase A2 activity

[摘要] Purpose: To determine whether the gene expression of matrixmetalloproteinases (MMPs) as well as that of the pro-angiogenic cytokinevascular endothelial growth factor (VEGF) and its receptors change inresponse to hypoxic exposure in a primate choroid-retinal endothelialcell line, and furthermore, whether cytosolic phospholipase A2(cPLA2) plays a role in this process.Methods: Rhesus macaque choroid-retinal endothelial (RF/6A) cellswere incubated under hypoxic conditions for 1, 2, 4, or 8 h prior to RNAextraction. In some experiments cells were pretreated with thecPLA2 inhibitor AACOCF3 (10 μM) for 30 min prior to hypoxia.Changes in gene expression were determined by RT-PCR and quantified byreal-time PCR for urokinase plasminogen activator (uPA), collagenase-1(MMP-1), membrane type-1 metalloproteinase (MT1-MMP), gelatinases A andB (MMP-2, MMP-9), tissue inhibitor-2 (TIMP-2), VEGF and its receptors,Flt-1 (VEGFR-1), KDR (VEGFR-2), and neuropilin-1 (NP-1). MMP-2 secretedby the cells was evaluated by zymography. VEGF release was measured byELISA. In tube-formation studies, endothelial cells (EC) were seededinto collagen gel, exposed to hypoxia for 4 h, then incubated undernormoxic conditions for 72 h.Results: Hypoxia triggered a three fold increase in the geneexpression of MT1-MMP, MMP-2, and TIMP-2, and a ten fold increase inMMP-2 levels. Moreover it also induced tube formation in EC. Expressionof uPA, MMP-1, and MMP-9 mRNA was not detected. Pretreatment withAACOCF3 abolished hypoxia-induced tube formation and MT1-MMP, MMP-2, andTIMP-2 transcription. Furthermore, hypoxia produced a significant,sustained increase in the gene expression and release of VEGF-165, theonly VEGF-A isoform detected in these cells. AACOCF3 reduced thehypoxia-induced VEGF release at 8 h of hypoxia. VEGF receptors KDR andNP-1 were constitutively expressed in EC and up-regulated under hypoxicconditions.Conclusions: In monkey choroid-retinal EC, hypoxia selectivelyinduces MMP-2 activity. This induction is preceded by MT1-MMP, MMP-2,and TIMP-2 mRNA expression, as well as that of the VEGF-165 isoform andits receptors KDR and NP1. These increases possibly result fromhypoxia-induced activation of cPLA2 and subsequent release ofarachidonic acid and its conversion to prostaglandins. These molecularchanges in EC could, in part, contribute to the angiogenic response thatoccurs in the development of ischemic retinopathies and choroidalneovascularization.

[学科分类] 生物化学/生物物理