Abstract 5799: Overexpression of Integrin Beta5 Enhances the Paracrine Angiogenic Properties of Early Outgrowth Endothelial Progenitor Cells via Src Kinase-Mediated Activation of STAT3
Integrin alphavbeta5 is expressed on endothelial (progenitor) cells and has been implicated in angiogenesis. We have previously shown that stimulation of endothelial progenitor cells (EPC) with leptin upregulates alphavbeta5 integrin expression and enhances their angiogenic and reparative properties. The aim of the present study was to determine the intracellular mechanisms mediating the angiogenic effects of integrin alphavbeta5 in endothelial progenitor cells. For this purpose, EPC isolated from human venous blood and cultivated under endothelial conditions were transiently transfected with the full-length cDNA of human integrin beta 5 (EPC-ITGB5) or empty vector control (EPC-control), and analyzed 48 hours later (on day 7). Integrin beta5 overexpression was confirmed using flow cytometry (149±12%; P=0.0001 vs. EPC-control), Western blot (138±10%; P<0.01) and PCR analysis (135±13%; P<0.05). Moreover, it significantly enhanced the angiogenic capacities of EPC in vitro (spheroid and matrigel angiogenesis assay) and stimulated new vessel formation in vivo (chorioallantoic membrane assay and murine hindlimb ischemia model). Western blot and immunoprecipitation experiments revealed that overexpression of ITGB5 resulted in elevated integrin alphavbeta5 phosphorylation, followed by activation of Src kinase and signal transducer and activator of transcription 3 (STAT3). The specificity of ITGB5-dependent Src kinase and STAT3 phosphor-ylation was confirmed using function-blocking antibodies. Analysis of STAT3 target gene transcription revealed elevated mRNA and protein expression of the angiogenic chemokines IL8 and MCP1 in EPC-ITGB5, and significantly elevated IL8 and MCP1 protein levels in the conditioned medium (CdM) from EPC-ITGB5 were confirmed using quantitative ELISA. Moreover, CdM from EPC-ITGB5 enhanced the sprouting of co-incubated human umbilical vein endothelial cells (HUVEC) in a STAT3-dependent manner. Taken together, our findings suggest an important role for the cytoplasmic protein kinase Src in linking alphavbeta5 integrin to STAT3-dependent gene expression and angiogenesis. This pathway deserves further investigation as a possible means for (therapeutically) enhancing the paracrine potency of EPC.