Angiogenesis requires endothelial cell invasion and is crucial for wound healing and for tumor growth and metastasis. Invasion of native collagen is mediated by the alpha(5)beta(1) integrin fibronectin receptor. Thus, alpha(5)beta(1) up-regulation on the surfaces of endothelial cells may induce endothelial cell invasion to stimulate angiogenesis. We report that the interaction of alpha(5)beta(1) with its PHSRN peptide ligand induces human microvascular endothelial cell invasion and that PHSRN-induced endothelial cell invasion is regulated by alpha(4)beta(1) integrin and requires matrix metalloproteinase 1 (MMP-1). Moreover, our results show that exposure to PHSRN causes rapid, specific up-regulation of surface levels of alpha(5)beta(1) integrin and significantly increases alpha(5) integrin mRNA in microvascular endothelial cells. Consistent with these results, alpha(5) small interfering RNA abrogates PHSRN-induced surface alpha(5) and MMP-1 up-regulation, as well as blocking invasion induction. We also observed dose-dependent, PHSRN-induced alpha(5)beta(1) integrin up-regulation on endothelial cells in vivo in Matrigel plugs. We further report that the PHSCN peptide, an alpha(5)beta(1)-targeted invasion inhibitor, blocks PHSRN-induced invasion, alpha(5)beta(1) up-regulation, alpha(5) mRNA induction, and MMP-1 secretion in microvascular endothelial cells and that systemic PHSCN administration prevents PHSRN-induced alpha(5)beta(1) up-regulation and angiogenesis in Matrigel plugs. These results demonstrate a critical role for alpha(5)beta(1) integrin and MMP-1 in mediating the endothelial cell invasion and angiogenesis and suggest that PHSRN-induced alpha(5) transcription and alpha(5)beta(1) up-regulation may form an important feed-forward mechanism for stimulating angiogenesis.