Abstract 824: The Role of the Small GTPase Rap1 for Angiogenesis
The molecular mechanisms regulating angiogenesis are not completely understood. Integrins are critical mediators of angiogenesis. Recent evidence suggests that Rap1, a small GTPase of the Ras-family, is an important regulator of integrin activity and junctional adhesion in many cellular systems. However, the role of Rap1 genes in angiogenesis is unclear. In the present study, we investigated the role of Rap1a and Rap1b for angiogenesis. Human umbilical vein endothelial cells (HUVEC) express mRNA for Rap1a and Rap1b, as assessed by RT-PCR. In order to determine the relevance of Rap1 activity for angiogenesis, we overexpressed RapGAP1, a GTPase activating protein, which specifically inhibits the activity of both Rap1a and Rap1b. Overexpression of RapGAP1 led to a significant inhibition of angiogenic sprouting of HUVEC under basal conditions and after bFGF stimulation by 44 ± 5 % as assessed by a 3-dimensional spheroidal endothelial cell culture system. In addition, overexpression of RapGAP1 significantly blocked tube formation in a matrigel assay (by 62 ± 12 %) and the HUVEC migration and adhesion on fibronectin and collagen. In order to separately investigate the role of Rap1a and Rap1b genes for angiogenesis, we performed gene silencing with siRNA. Silencing of either Rap1a or Rap1b gene significantly and additively blocked the sprouting of HUVEC under basal and bFGF-stimulated conditions (Rap1a-siRNA: 55 ± 5 %, Rap1b-siRNA: 61 ± 9 % and Rap1a+Rap1b siRNA: 73 ± 5% inhibition). Moreover, silencing of either Rap1a or Rap1b or both significantly reduced HUVEC migration and adhesion, but had no effect on proliferation as assessed by BrdU-staining. These data reveal for the first time that Rap1a and Rap1b are essential mediators of angiogenesis by mediating endothelial cell migration and adhesion on extracellular matrix proteins. The in-vivo role of Rap1a is currently being investigated in Rap1a-deficient mice. Understanding of the molecular mechanisms of angiogenesis is crucial for the development of new therapeutic strategies in order to increase neovascularization in patients with ischemic disorders or to reduce angiogenesis in patients with tumor diseases.