Abstract B25: R-Ras is critical for endothelial lumen formation, blood perfusion, and normalization of regenerating vasculature

Abstract

Abstract Formation of endothelial lumen is fundamental to angiogenesis and generation of functional vasculature. Tumors develop a large number of angiogenic blood vessels. However, a significant fraction of these vessels are poorly functional due to the impaired vessel maturation such as the improper formation of endothelial lumen. R-Ras is a small GTPase of the Ras family, which inhibits vessel sprouting and branching activities and redirects the vessel sprouts from these activities to the vessel maturation process. Through this effect, R-Ras normalizes pathologically regenerating blood vessels. Here we show that R-Ras regulates formation of endothelial lumen in the developing vessel sprouts. R-Ras deficient mice develop a large number of poorly perfused blood vessels in response to tissue ischemia. Notably, many of these vessels lack lumen assessed by the lack of PODXL staining of the endothelium. In 3D in vitro angiogenesis studies in fibrin gels, upregulation of R-Ras dramatically enhances formation of lumens in the endothelial sprouts. On the other hand, knockdown of the endogenous R-Ras abrogates lumen formation and endothelial cell differentiation into tubular structures resulting in abnormal extension of endothelial cells with numerous membrane protrusions. The stabilization of microtubules in endothelial cells is critical to the formation of endothelial lumen. Upregulation of R-Ras signaling in endothelial cells results in the formation of prominent microtubule cytoskeleton network that extends to the membrane periphery in 2D culture. This phenomenon is accompanied by the significant spreading of endothelial cells. We found that R-Ras stabilizes microtubules by increasing acetylation of alpha-tubulin. This effect of R-Ras is mediated by the PI3-kinase-Akt1/2 pathway, which phosphorylates GSK3-beta and inhibits the inhibitory activity of GSK3-beta on tubulin acetylation. GSK3-beta inhibition partially rescues tubulogenesis of R-Ras-silenced endothelial cells. These findings demonstrate a novel Akt-dependent mechanism of endothelial lumen formation and blood vessel maturation. The endothelial-specific upregulation of R-Ras in vivo leads to improved blood perfusion and normalization of VEGF-induced angiogenic vessels developing in Matrigel implants. The enhanced endothelial lumen formation and resulting improvement of vessel perfusion would significantly increase the delivery of chemotherapeutic drugs to tumors, which is otherwise hampered by the abnormal, poorly perfused vasculature. This R-Ras-dependent mechanism of endothelial lumen formation should be further exploited to potentially develop a strategy for improving drug delivery and enhancing treatment efficacy via normalization of the tumor vasculature. Citation Format: Fangfei Li, Junko Sawada, Masanobu Komatsu. R-Ras is critical for endothelial lumen formation, blood perfusion, and normalization of regenerating vasculature. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B25.