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Increased
Expression of Activated Matrix Metalloproteinase-2 by Human Endothelial
Cells after Sublethal H2O2 Exposure |
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Abbes Belkhiri,
Carl Richards, Mark Whaley, Shelley A. McQueen, and F. William Orr
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Departments
of Pathology, University of Manitoba, Winnipeg, Manitoba (AB, SAM, FWO),
McMaster University, Hamilton, Ontario (CR), and the University of British
Columbia, Vancouver (MW), Canada |
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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 sublethal 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 [mu]m 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. |
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