Abstract-Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF T he growth, repair, and regeneration of blood vessels are complex processes that involve coordinated regulation of endothelial cell proliferation, migration, and differentiation. 1 One of the most important vascular morphogens is vascular endothelial growth factor (VEGF). VEGF has been shown to play a major role in vasculogenesis and angiogenesis by gene deletion studies. 2,3 Targeted disruption of the VEGF receptor Flk-1 (VEGFR-2) in mice resulted in failure of blood-island formation and endothelial differentiation. 4 Flk-1 is also the first endothelial receptor tyrosine kinase to be expressed in the hemangioblast. 5 We and others recently demonstrated that the hematopoietic progenitor cell CD34 ϩ can differentiate into endothelial cells, and that VEGF was one of the critical factors promoting this differentiation. 6,7 Interactions between cells and their extracellular matrix (ECM) play an integral role in blood vessel development. The earliest ECM protein expressed in the embryo during vasculogenesis is fibronectin (FN). 8 Gene deletion studies have demonstrated that both FN and its major integrin receptor, ␣ 5  1 , are critical for vasculogenesis and angiogenesis in the developing embryo. 9 -11 Collectively, these observations suggest important roles for FN and its integrin receptor, ␣ 5  1 , in vasculogenesis and angiogenesis.In this study, we show that novel VEGF binding domains of FN are required for promoting the specific association of the FN receptor integrin ␣ 5  1 with the VEGF receptor, Flk-1. This association between VEGF and FN is required for the full effects of VEGF-induced endothelial cell migration and proliferation. This study demonstrates that FN can profoundly affect VEGF biological activity and consequently the behavior of endothelial cells through their coordinated effects on Flk-1 and ␣ 5  1 . Materials and Methods Solid-Phase VEGF Binding AssayECM proteins and FN peptides were purchased from Sigma and Gibco and were purified further by gel filtration and ion exchange chromatography. Microtiter plates were coated with the appropriate ECM proteins (50 L; 10 g/mL) in 100 mmol/L bicarbonate buffer (pH 9) overnight at 4°C.
Abstract-We describe extracellular interactions between fibronectin (Fn) and vascular endothelial growth factor (VEGF) that influence integrin-growth factor receptor crosstalk and cellular responses. In previous work, we found that VEGF bound specifically to fibronectin (Fn) but not vitronectin or collagens.
VEGF and FN together significantly promote the migration and differentiation of CD34(+) cells. This synergism is specific to FN and the alpha5beta1 integrin. Combinations of VEGF and FN may be useful in promoting differentiation of circulating endothelial progenitors into endothelial cells for tissue engineering. Clinical relevance Treatment of injured or diseased tissues with adult stem cells is a promising approach. In particular, bone marrow derived circulating endothelial progenitors (CEP's) have been shown to differentiate into endothelial cells in vitro and promote tissue revascularization of ischemic limbs and myocardium in vivo. Because of the relative ease of obtaining CEP's and as well as its high proliferative rate, CEP's may have clinical potential for endothelialization of prosthetic vascular grafts and revascularization of injured myocardium. However, there is a need to better understand the molecular pathways involved in the proliferation and differentiation of CEP's to take full advantage of its clinical potential.
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