VEGF and inhibitors of TGFβ type-I receptor kinase synergistically promote blood-vessel formation by inducing α5-integrin expression

Vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF beta) are potent regulators of angiogenesis. How VEGF and TGF beta signaling pathways crosstalk is not well understood. Therefore, we analyzed the effects of the TGF beta type-I-receptor inhibitors (SB-431542 and LY-2157299) and VEGF on endothelial cell (EC) function and angiogenesis. We show that SB-431542 dramatically enhances VEGF-induced formation of EC sheets from fetal mouse metatarsals. Suboptimal doses of VEGF and SB-431542 synergistically induced EC migration and sprouting of EC spheroids, whereas overexpression of a constitutively active form of TGF beta type-I receptor had opposite effects. Using quantitative PCR, we demonstrated that VEGF and SB-431542 synergistically upregulated the mRNA expression of genes involved in angiogenesis, including the integrins alpha 5 and beta 3. Specific downregulation of alpha 5-integrin expression or functional blocking of alpha 5 integrin with a specific neutralizing antibody inhibited the cooperative effect of VEGF and SB-431542 on EC sprouting. In vivo, LY-2157299 induced angiogenesis and enhanced VEGF-and basic-fibroblast-growth-factor-induced angiogenesis in a Matrigel-plug assay, whereas adding an alpha 5-integrin-neutralizing antibody to the Matrigel selectively inhibited this enhanced response. Thus, induction of alpha 5-integrin expression is a key determinant by which inhibitors of TGF beta type-I receptor kinase and VEGF synergistically promote angiogenesis.