Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration

Abstract

To explore the mechanisms by which NO elicits endothelial cell (EC) migration we used murine and bovine aortic ECs in an in vitro wound-healing model. We found that exogenous or endogenous NO stimulated EC migration. Moreover, migration was significantly delayed in ECs derived from endothelial NO synthase-deficient mice compared with WT murine aortic EC. To assess the contribution of matrix metalloproteinase (MMP)-13 to NO-mediated EC migration, we used RNA interference to silence MMP-13 expression in ECs. Migration was delayed in cells in which MMP-13 was silenced. In untreated cells MMP-13 was localized to caveolae, forming a complex with caveolin-1. Stimulation with NO disrupted this complex and significantly increased extracellular MMP-13 abundance, leading to collagen breakdown. Our findings show that MMP-13 is an important effector of NO-activated endothelial migration.angiogenesis ͉ endothelium ͉ vasular remodeling W ound healing is an orchestrated cascade of enzymatic activities that converge toward damage repair. Wound healing involves inflammation and angiogenesis and is tightly regulated by cytokines (1). Vascular endothelial growth factor (VEGF) is a critical cytokine involved in angiogenesis, and nitric oxide (NO) is a downstream effector (2, 3). VEGF increases NO levels in endothelial cells (ECs) by activating endothelial NO synthase (eNOS͞NOS3) (4-7). Recently, the role of eNOS in EC migration has been demonstrated in vivo and in vitro (8, 9) but the precise mechanism by which NO regulates migration is unknown.ECs migrate as a result of an injury and during angiogenesis from preexisting vessels. The process is tightly regulated by matrix turnover, in which matrix metalloproteinases (MMPs) play a pivotal role (10-12). We have previously reported that NO induces MMP-13 expression and activity in bovine aortic ECs (BAECs) (13,14).MMPs are extracellular matrix-degrading endopeptidases. MMP expression and activity can be found in physiological and pathological situations, such as tissue development, atherosclerosis, ovarian function, arthritis, osteoarthritis, cancer, angiogenesis, and wound healing (15). MMP-13 was initially discovered in mammalian cell carcinomas and is also expressed by several cell types, including endothelium (13,(16)(17)(18).Here, we present evidence that MMP-13 is a downstream effector of NO-activated EC movement. We have developed a wound-healing model in BAECs and aortic cells from eNOS WT and eNOS-deficient mice. We show that aortic ECs lacking MMP-13 experience delayed migration, and aortic ECs from eNOS null mice present delayed cell migration and a significant decrease in MMP-13 expression. In addition, we present data showing that MMP-13 exists in association with caveolin-1 in resting cells, and that this complex is disrupted in the presence of NO, leading to the secretion of MMP-13 to the extracellular media. We postulate that NO induces EC movement in part via the disruption of the MMP-13͞caveolin-1 complex, which in turn releases the secretion of active MMP-13 to the ...

Cyclic strain-mediated matrix metalloproteinase regulation within the vascular endothelium: a force to be reckoned with

“…These findings are consistent with related studies that show that NOS inhibition leads to increased MMP-2 activity in coronary vessels (90), whereas NOS overexpression (in SMCs) leads to reduction of MMP-2 activity and, consequently, migration (49). By contrast, another recent study has suggested that NO-mediated induction of MMP-13 stimulates BAEC migration (88).…”

“…The detailed molecular mechanism of how NO affects cellular physiology remains unclear, while studies of NO effects on cells have yielded controversial results. Reports have demonstrated both promigratory (72,88) and antimigratory (14,77,79,143) effects of NO on vascular ECs. As migration inevitably involves matrix remodeling, the relationship between NO and MMPs has received some attention.…”

Cbfa-1 mediates nitric oxide regulation of MMP-13 in osteoblasts

“…MMP-13 and Cbfa-1 from MC3T3-E1 were detected by confocal microscopy as previously described (Lopez-Rivera et al, 2005).…”

“…Cbfa-1 or GAPDH expression were silenced in MC3T3-E1 cells by transient transfection of cells with pre-annealed siRNA oligonucleotides, corresponding to specific regions of the murine Cbfa-1 and GAPDH genes (pre-validated siRNAs; Ambion), as described (Lopez-Rivera et al, 2005).…”

“…It was already known that NO is involved in bone metabolism, that Cbfa-1 is implicated in mediating MMP expression, and that NO regulates the expression and activity of MMP-13 in endothelium (Lopez-Rivera et al, 2005); this led us to investigate the mechanisms responsible for such actions. Here, we investigated the action of NO during differentiation of the osteoblastic cell line MC3T3-E1.…”