Abstract 3746: Human Tissue Kallikrein Overexpression Confers Upon Human Endothelial Progenitor Cells Enhanced Invasive and Pro-Angiogenic Activity
Under ischemic conditions endothelial progenitor cells (EPC) complement neovascularization afforded by local endothelial cells (EC). Efficient EPC homing is fundamental for their pro-angiogenic function. Although underlying mechanisms are incompletely understood, proteases are emerging modulators of stem cell invasive capacity. The serine-protease tissue kallikrein (hK1) stimulates neovascularization of ischemic tissues, by both kinin-dependent and independent mechanisms. Here we studied whether human EPC express hK1, and whether hK1 is necessary for EPC to invade the extracellular matrix and promote angiogenesis. We first demonstrated that both human EPC enriched from peripheral blood mononuclear cells by selective culture, and MACsorting-isolated circulating CD34+ and CD133+ cells, express hK1 mRNA (RT-PCR). Moreover, EPC secrete active hK1 as detected by both ELISA and amidolytic assay. Of note, kallikrein gene (KLK1) silencing by siRNA (four siRNAs pool, 100nM, 44% downregulation vs scrambled) resulted in decreased secreted MMP2 activity (gel zymography, n=3, p<0.05) and EPC invasion (transwell assay) (n=4, p<0.05 vs scrambled). Second, we overexpressed hK1 in EPC by adenoviral infection. Increased levels of secreted and cell-associated hK1 was confirmed by ELISA (2 fold) and Western Blot (18 fold) (n=3, p<0.05 vs null). HK1-EPC acquired increased migratory (1.6 fold) and invasive (1.8 fold) capacity, and stimulated HUVEC capillary-like structures formation on matrigel (1.4 fold) (n=5 replicates per assay, p<0.05 vs null). Migratory, invasive, and pro-angiogenic activities of hK1-EPC were contrasted by the serine protease inhibitor aprotinin, the specific hK1 inhibitor kallistatin, and the MMP inhibitor GM6001 (n=4, p<0.05 vs vehicle), but were preserved after B2 receptor blockade by icatibant (n=4, p>0.05). In summary, hK1 is expressed in human EPC, hK1-overexpressing EPC show enhanced invasive and pro-angiogenic capacities, and hK1 might cooperate with MMP2 for EPC invasion. These results could have important implications in post-ischemic vasculogenesis, since hK1 overexpression might confer upon EPC enhanced therapeutic activity for promotion of neovascularisation.