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GN Lambrou, P Ottino, A Ottlecz, J Finley, NG Bazan, HE P Bazan; Membrane Associated Matrix Metalloproteinase (mt1-mmp) and Mmp-2 Gene Expression Are Selectively Induced by Hypoxia in Monkey Choroid Retinal Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2752.
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Purpose: The extracellular matrix undergoes extensive changes during retinal pathoangiogenesis. MMPs may play an important role in ischemic retinal diseases in which neovascularization, migration and proliferation of vascular endothelial cells represent critical events in the progression of the disease. Here we investigated the expression of MMPs in monkey choroid retinal endothelial cells (RF/6A) exposed to hypoxia. Method: RF/6A cells (8 x 106, CRL-1780 from ATCC) were seeded onto Petri dishes (60 mm diameter) coated with collagen type-I and were grown in F12-K medium containing 5% fetal calf serum (FCS). When cells reached ∼90% confluency, the medium was replaced with hypoxic (degassed) medium containing 1% FCS and cells were placed in a cycling chamber and perfused with 95% N2 and 5% O2 at 37 0C for 2 hr. In post-hypoxia recovery experiments, cells were maintained in non-hypoxic media for an additional 2 hr. mRNA was extracted from endothelial cells using a SV total RNA isolation kit from Promega. Levels of gene expression for MMP-1, MT1-MMP, MMP-2, MMP-9 and the upstream stimulator of MMPs, urokinase plasminogen activator (uPA) were determined by real time PCR. All quantitations were normalized to the 18s rRNA endogenous control and changes in gene expression were assessed as relative to untreated controls. Results: Hypoxia triggered a 3-fold increase in the expression of the MT-MMP-1 and MMP-2 genes in monkey endothelial cells. Hypoxia produced no detectable changes in the expression of uPA, MMP-1 and MMP-9 mRNA in endothelial cells. In the post-hypoxia recovery period the induction of MT1-MMP and MMP-2 was decreased, whereas no changes were observed in the other MMPs and uPA. Conclusion: Hypoxia selectively induces MT1-MMP and MMP-2 transcription. Because MT1-MMP activates latent MMP-2, this increase in gene expression highlights a mechanism by which these proteases participate in the degradation of components of the extracellular matrix and promote retinal neovascularization.
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