Cleaved Kininogen Inhibits Endothelial Progenitor Cell Differentiation by Interfering with the avb3 Integrin-MMP-2 Pathway.

Circulating endothelial progenitor cells (EPCs) are involved in repairing damaged vasculature and in tumor angiogenesis, thus promoting wide interest in their therapeutic potential in ischemic diseases and cancer. Cleaved high molecular weight kininogen (HKa) potently inhibits endothelial cell (EC) functions including in vivo angiogenesis. We have shown that HKa inhibits differentiation of ECs. We now test a new hypothesis that HKa can inhibit EPC differentiation by blocking matrix metalloprotease-2 (MMP-2) activation. EPCs were isolated from human blood and cultured on collagen type I-coated surface. As detected by flow cytometry and Western-blot, they expressed MMP-2 and membrane-type 1-MMP (MT1-MMP), avb3 integrin, uPAR and caveolin-1, but did not express monocyte markers such as HLA-DR and CD14. A clone-forming assay using retroviral infection indicated that an early single EPC exhibited colony forming property, but mature ECs such as human umbilical vein endothelial cells (HUVECs) did not. Upon stimulation by VEGF, EPCs, but not HUVECs, formed vacuoles and differentiated into capillary networks. Gene silencing of either MT1-MMP or MMP-2 by siRNA completely blocked the vacuole and tube formation by EPCs, suggesting that MT1-MMP and MMP-2 are both essential for differentiation of EPCs. HKa inhibited tube formation by EPCs, as well as the conversion of pro-MMP-2 to MMP-2 as a functioning concentration in the conditioned medium. CS-1 cells expressing endogenous av subunit were transfected with human b3 cDNA. We found that HKa inhibition of MMP-2 activation in these cells is dependent on avb3 heterodimer. The binding of avb3 integrin to MMP-2 was via RGD-dependent and RGD-independent mechanisms, both of which, however, were completely blocked by HKa. Further, we found that HKa inhibited the association of MMP-2 with immunoprecipitated avb3 integrin, and prevented MMP-2 localization in caveolae in EPCs. Since HKa inhibited VEGF-stimulated proliferation of EPCs but not HUVECs on collagen-coated surface, we postulate that EPCs are more sensitive than mature ECs to HKa inhibition. Thus, HKa targets EPCs and inhibits their differentiation via blocking avb3 integrin and MMP-2 interaction. These observations provide novel insight into understanding the interactions between the kallikrein-kinin system and biologic functions of EPCs.