Increased expression of activated matrix metalloproteinase-2 by human endothelial cells after sublethal H2O2 exposure

Abstract

Basement membranes form a boundary between intravascular and extravascular compartments that is remodeled by matrix metalloproteinases (MMP) expressed by endothelial cells. These cells are at risk of exposure to reactive oxygen intermediates generated as a consequence of interactions with drugs, x-radiation, activated neutrophils, or cancer cells. Herein we have investigated the hypothesis that endothelial cells alter their expression of MMP after sublethel exposure to H2O2 and that this leads to degradation of adjacent basement membranes. Cultured human umbilical vein endothelial cells were treated with concentrations of H2O2 ranging from 1.5 to 32 microM or with 2 x 10(-6)M phorbol myristate acetate (PMA). After 24 hours, the cells were placed into serum-free medium for an additional 24 hours. This conditioned medium or cell lysates were studied by matrix degradation assays, gelatin zymography, immunoblots, and Northern analysis. H2O2-treated or PMA-treated cells, or their serum-free conditioned medium, caused a 2-fold increase in degradation of [3H]-proline-labeled endothelial basement membranes or purified type IV collagen compared to untreated cells. Endothelial cells constitutively expressed gelatinases at Mr 96,000 and 72,000, consistent with MMP-9 and inactive MMP-2. H2O2 exposure caused increased expression of these MMP and appearance of Mr 64,000 to 66,000 gelatinases corresponding to activated MMP-2. In cell lysates, H2O2 or PMA treatment led to increased expression of membrane-type MMP-1, an activator of latent MMP-2. The results suggest that oxidants such as H2O2 may stimulate MMP expression and influence the remodeling of vascular basement membranes by endothelial cells.