Abstract 3661: Overexpression Of Integrin Beta 5 Enhances The Proangiogenic Properties Of Human Endothelial Progenitor Cells
Integrins alphav beta3 and alphav beta5 play an important role during angiogenesis. We have previously shown that stimulation of endothelial progenitor cells (EPC) with leptin significantly upregulated alphav beta5 integrin expression and enhanced their angiogenic properties. In order to further investigate how modulation of alphav beta5 expression may affect angiogenic processes, EPC were transiently transfected with full-length cDNA of human integrin beta5 (EPC-ITGB5) or control vector (EPC-control). Flow cytometry analysis confirmed significantly enhanced expression of alphav beta5 integrin (140±17%; P<0.05), and these findings were confirmed using RT-PCR, western blot and immunofluorescence. EPC adhesion to vitronectin (VN) was significantly enhanced after ITGB5 overexpression (P<0.005). Moreover, ITGB5 overexpression potentiated the VN-dependent EPC migration in the Boyden Chamber assay (422±61% of control; P<0.005). On the other hand, ITGB5 overexpression did not affect EPC proliferation. In the Matrigel angiogenesis assay, EPC incorporation into a network of co-cultured mature endothelial cells was significantly enhanced by ITGB5 overexpression (191±25% of control; P<0.005). In the 3-dimensional spheroid angiogenesis assay, an increased integration of EPC-ITGB5 into HUVEC sprouts (P<0.001), as well as an enhanced cumulative HUVEC sprout length (P<0.005 vs. control), could both be observed. Of note, the enhanced angiogenic properties of ITGB5-transfected EPC in all of the above assays were alphav beta5-dependent, as confirmed by preincubation of EPC with neutralizing alphav beta5 antibodies. In further studies, EPC-ITGB5 stimulated new avian vessel formation in the chorion allantoic membrane assay (128±5.3% vs. control; P<0.05), and increased the number of CD31-positive capillaries per muscle fiber in the in vivo murine hindlimb ischemia model (P<0.05 vs. EPC-control). Taken together, our findings suggest that alphav beta5 integrin overexpression enhances EPC adhesion and migration, promotes their incorporation into mature endothelial cell networks, and stimulates blood vessel formation in vivo. Our findings thus support a specific role for alphav beta5 integrin during angiogenic processes.