Abstract 17622: Role of SHARPIN, an Integrin Inhibitor, in Angiogenesis
β1-integrins are mediating endothelial cell (EC) adhesion to extracellular matrix proteins and are critical mediators of neovascularization. In addition, intracellular signaling stimulating integrin affinity promotes angiogenesis. However, the role of intracellular integrin affinity inhibitors in angiogenesis remains elusive. SHARPIN is a negative regulator of integrin affinity in fibroblasts and leukocytes by binding to several α-integrin subunits. Nevertheless, the role of SHARPIN in EC biology and its contribution to angiogenesis is yet unknown. According to our findings, EC express SHARPIN. Silencing of SHARPIN with siRNA significantly reduced angiogenic sprouting of EC spheroids in collagen gels and inhibited tube formation in matrigel assays (by 41 ± 4 %) in comparison to scrambled siRNA-transfected EC. An essential angiogenic function of EC is chemotactic migration. In real-time video-microscopy chemotaxis assays, SHARPIN silencing almost abolished the ability of EC to follow chemotactic VEGF-gradients in comparison to scrambled siRNA-transfected EC. Furthermore, EC migration is dependent on integrin-mediated adhesion. Silencing of SHARPIN significantly increased β1-integrin-dependent adhesion of EC on fibronectin and collagen. In line with these results, silencing of SHARPIN reduced the abundance of the inactive conformation of β1-integrins on the EC surface as assessed by flow cytometry, while the total protein expression levels of β1-integrins remained unaltered. Interestingly, inhibition of β1-integrins with a neutralizing antibody (at low concentration) restored the ability of SHARPIN siRNA-transfected EC to follow chemotactic VEGF-gradients and significantly enhanced angiogenic sprouting in comparison to SHARPIN siRNA-transfected EC treated with the isotype control antibody. To address the in vivo relevance of our findings, we employed the murine model of post-natal retinal angiogenesis. SHARPIN-deficient mice displayed significantly lower vessel density and less vessel branching points in the retinal plexus when compared to the wild type mice. In conclusion, SHARPIN is a novel mediator of angiogenesis by promoting VEGF-induced EC chemotactic migration by downregulating β1-integrin-activity.
Author Disclosures: F. Bonini: None. P. Schuster: None. A. Gatsiou: None. K. Stellos: None. A.M. Zeiher: None. E. Chavakis: None.
- © 2016 by American Heart Association, Inc.