Temporal and Spatial Modulation of Rho GTPases during in Vitro Formation of Capillary Vascular Network

Abstract

Endothelial cells (ECs) self-organize into capillary networks when plated on extracellular matrix. In this process, Rho GTPases-mediated cytoskeletal dynamics control cell movement and organization of cell-to-matrix and cell-to-cell contacts. Time course analysis of RhoA and Rac1 activation matches specific morphological aspects of nascent pattern. RhoA-GTP increases early during EC adhesion and accumulates at sites of membrane ruffling. Rac1 is activated later and localizes in lamellipodia and at cell-to-cell contacts of organized cell chains. When ECs stretch and remodel to form capillary structures, RhoA-GTP increases again and associates with stress fibers running along the major cell axis. N17Rac1 and N19RhoA mutants impair pattern formation. Cell-to-cell contacts and myosin light chains (MLC) are targets of Rac1 and RhoA, respectively. N17Rac1 reduces the shift of ␤-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs ␤-catenin distribution at adherens junctions, suggesting that Rac1 controls the dynamics of cadherin-catenin complex with F-actin. During the remodeling phase of network formation, ECs show an intense staining for phosphorylated MLC along the plasma membrane; in contrast, MLC is less phosphorylated and widely diffused in N19RhoA ECs. Both N17Rac1 and N19RhoA have been used to investigate the role of wild type molecules in the main steps characterizing in vitro angiogenesis: (i) cell adhesion to the substrate, (ii) cell movement, and (iii) mechanical remodeling of matrix. N17Rac1 has a striking inhibitory effect on haptotaxis, whereas N19RhoA slightly inhibits EC adhesion and motility but more markedly Matrigel contraction. We conclude that different Rho GTPases control distinct morphogenetic aspects of vascular morphogenesis.

Rho activity critically and selectively regulates endothelial cell organization during angiogenesis

“…Finally, consistent with our experiments implicating Rho and Rho kinase critically in EC contraction of collagen I gels (Fig. 3B), others have reported that both N19RhoA and Y-27632 (5 M) suppressed EC contraction of three-dimensional Matrigel (31).…”

“…We observed no significant differences in EC density between the N19RhoA and V14RhoA groups; however, differences in cross-sectional area of blood vessel lumens were highly significant (P Ͻ 0.001). In addition, others have reported that transduction with N19RhoA suppressed EC motility (31), but it appears that they achieved considerably greater inhibition of Rho activity (Ϸ75%) in comparison with that achieved in our experiments (Ϸ20%). Thus, the consequences of modulating Rho activity for EC migration may depend critically on percent change in Rho activity.…”

FAK Potentiates Rac1 Activation and Localization to Matrix Adhesion Sites: A Role for βPIX

“…These include fibroblast motility during wound repair and endothelial metamorphosis during angiogenesis (Cascone et al, 2003;Harms et al, 2005). The FAK-␤PIX-Rac1 pathway that is defined in this work may be a critical determinant of success in these endeavors by providing the targeting and assembly of Rac1-containing molecular networks at focal complex and focal adhesion sites.…”

Gene Therapy for Gastric Diseases

“…Rac1, a major member of the Rho GTPase family, is related to tumorigenesis [153], invasion and metastasis [154], and angiogenesis [81,155]. Xue et al showed that VEGF and HIF-1α were down-regulated in gastric cancer cells which were transfected with a Rac1 siRNA expression vector [156].…”