. Differential involvement of MMP-2 and VEGF during muscle stretch-versus shear stress-induced angiogenesis.

Microvascular endothelial cells embedded within three-dimensional (3D) type I collagen matrixes assemble into cellular networks, a process that requires the upregulation of membrane type 1 (MT1) matrix metalloproteinase (MMP) and MMP-2. The purpose of this study was to identify the signaling pathways responsible for the transcriptional activation of MT1-MMP and MMP-2 in endothelial cells in 3D collagen lattices. We hypothesized that the 3D type I collagen induction of MT1-MMP and MMP-2 is mediated by the mitogen-activated protein kinase family of enzymes. Here, we show that 3D type I collagen elicits a persistent increase in ERK1/2 and JNK activation and a decrease in p38 activation. Inhibition of ERK1/2 or JNK disrupted endothelial network formation in 3D type I collagen lattices, whereas inhibition of p38 promoted network formation. mRNA levels of both MT1-MMP and MMP-2 were attenuated by ERK1/2 inhibition but unaffected by either JNK or p38 inhibition. By contrast, expression of constitutively active MEK was sufficient to stimulate MMP-2 production in a monolayer of endothelial cells cultured on type I collagen. These results provide evidence that signaling through both ERK1/2 and JNK regulates endothelial assembly into cellular networks but that the ERK1/2 signaling cascade specifically regulates network formation and the production of both MT1-MMP and MMP-2 genes in response to 3D type I collagen.

Matrix metalloproteinase-2 (MMP-2) plays a critical role in endothelial cells during the processes of angiogenesis and vascular remodeling. Endothelial cell production of MMP-2 is greatly enhanced when cells are cultured within a three-dimensional type I collagen matrix coinciding with the increased invasive and migratory phenotype of the cells. To define the transcriptional regulation of MMP-2 in rat microvascular endothelial cells, we performed promoter-reporter assays with a series of promoter truncations. Activity of the full promoter was significantly greater in cells cultured within three-dimensional type I collagen compared with cells cultured as a monolayer (two-dimensional) on type I collagen. Truncation of the region encompassing base pairs ؊1562 to ؊1375 (relative to the start codon) of the MMP-2 promoter resulted in loss of this differential activity of the MMP-2 promoter. Analysis of this region indicated two putative GATA-2 binding domains between ؊1437 and ؊1387. Southwestern blot analysis and electrophoretic mobility shift assays confirmed the binding of GATA-2 to this region of the MMP-2 promoter. Overexpression of GATA-2 in COS-7 cells significantly increased the activity of the full-length MMP-2 promoter-luciferase construct. Endothelial cells expressed greater levels of GATA-2 protein in three-dimensional compared with two-dimensional cultures, and activity of the ؊1437/؊1387 region of the MMP-2 promoter was significantly greater in three-dimensional cultured endothelial cells. Together, these results indicate GATA-2 regulation of the MMP-2 promoter in endothelial cells and that the GATA-2 binding domain is sufficient to drive increased activity of the MMP-2 promoter in response to an extracellular matrix stimulus. The matrix metalloproteinases (MMPs)1 consist of a family of zinc-and calcium-dependent endopeptidases that cleave specific subsets of extracellular matrix proteins, growth factors, and cell surface receptors (1,2). Endothelial cell production and activation of MMPs, including MMP-2, are critical for the process of angiogenesis (3,4). Angiogenesis in skeletal muscle may be initiated by growth factors, by hemodynamic forces, or by mechanical stretching forces (5). The angiogenic process requires that endothelial cells proteolyze their basement membrane and then migrate through the interstitial matrix to form a new capillary. Inhibition of MMP activity prevents basement membrane degradation and the process of capillary sprouting in activity-stimulated skeletal muscle (6). When cultured within a three-dimensional type I collagen matrix, endothelial cells gain an invasive and migratory phenotype including increased production of matrix metalloproteinases and enhanced cell motility similar to what is seen in vivo during capillary sprouting (7,8). Regulated transcription of MMP-2 is important in determining this invasive phenotype in endothelial cells.Regulation of MMP-2 occurs at both transcriptional and post-translational levels. Although the rate of MMP-2 protein production is control...

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