INCREASED EXPRESSION OF ACTIVATED MATRIX METALLOPROTEINASE-2 BY HUMANENDOTHELIAL-CELLS AFTER SUBLETHAL H2O2 EXPOSURE

Basement membranes form a boundary between intravascular and extravasc ular compartments that is remodelled by matrix metalloproteinases (MMP ) expressed by endothelial cells. These cells are at risk of exposure to reactive oxygen intermediates generated as a consequence of interac tions with drugs, x-radiation, activated neutrophils, or cancer cells. Herein we have investigated the hypothesis that endothelial cells alt er their expression of MMP after sublethal exposure to H2O2 and that t his leads to degradation of adjacent basement membranes. Cultured huma n umbilical vein endothelial cells were treated with concentrations of H2O2 ranging from 1.5 to 32 mu M or with 2 x 10(-6) M phorbol myrista te acetate (PMA). After 24 hours, the cells were placed into serum-fre e medium for an additional 24 hours. This conditioned medium or cell l ysates 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 d egradation of [H-3]-proline-labeled endothelial basement membranes or purified type IV collagen compared to untreated cells. Endothelial cel ls constitutively expressed gelatinases at M, 96,000 and 72,000, consi stent with MMP-9 and inactive MMP-2. H2O2 exposure caused increased ex pression of these MMP and appearance of M-r 64,000 to 66,000 gelatinas es corresponding to activated MMP-2. In cell lysates, H2O2 or PMA trea tment led to increased expression of membrane-type MMP-1, an activator of latent MMP-2. The results suggest that oxidants such as H2O2 may s timulate MMP expression and influence the remodelling of vascular base ment membranes by endothelial cells.