Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity

Endothelial cell–cell junctions regulate vascular permeability, vasculogenesis, and angiogenesis. Familial cerebral cavernous malformations (CCMs) in humans result from mutations of CCM2 (malcavernin, OSM, MGC4607), PDCD10 (CCM3), or KRIT1 (CCM1), a Rap1 effector which stabilizes endothelial cell–cell junctions. Homozygous loss of KRIT1 or CCM2 produces lethal vascular phenotypes in mice and zebrafish. We report that the physical interaction of KRIT1 and CCM2 proteins is required for endothelial cell–cell junctional localization, and lack of either protein destabilizes barrier function by sustaining activity of RhoA and its effector Rho kinase (ROCK). Protein haploinsufficient Krit1+/− or Ccm2+/− mouse endothelial cells manifested increased monolayer permeability in vitro, and both Krit1+/− and Ccm2+/− mice exhibited increased vascular leak in vivo, reversible by fasudil, a ROCK inhibitor. Furthermore, we show that ROCK hyperactivity occurs in sporadic and familial human CCM endothelium as judged by increased phosphorylation of myosin light chain. These data establish that KRIT1–CCM2 interaction regulates vascular barrier function by suppressing Rho/ROCK signaling and that this pathway is dysregulated in human CCM endothelium, and they suggest that fasudil could ameliorate both CCM disease and vascular leak

Monocyte Adhesion and Spreading on Human Endothelial Cells Is Dependent on Rho-regulated Receptor Clustering

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation

Isoform-specific differences between Rap1A and Rap1B GTPases in the formation of endothelial cell junctions

Rap1 is a Ras-like GTPase that has been studied with respect to its role in cadherin-based cell adhesion. Rap1 exists as two separate isoforms, Rap1A and Rap1B, which are 95% identical and yet the phenotype of the isoform-specific knockout mice is different. We and others have previously identified a role for Rap1 in regulating endothelial adhesion, junctional integrity and barrier function; however, these early studies did not distinguish a relative role for each isoform. To dissect the individual contribution of each isoform in regulating the endothelial barrier, we utilized an engineered microRNA-based approach to silence Rap1A, Rap1B or both, then analyzed barrier properties of the endothelium. Electrical impedance sensing experiments show that Rap1A is the predominant isoform involved in endothelial cell junction formation. Quantification of monolayer integrity by VE-cadherin staining revealed that knockdown of Rap1A, but not Rap1B, increased the number of gaps in the confluent monolayer. This loss of monolayer integrity could be rescued by re-expression of exogenous Rap1A protein. Expression of GFP-tagged Rap1A or 1B revealed quantifiable differences in localization of each isoform, with the junctional pool of Rap1A being greater. The junctional protein AF-6 also co-immunoprecipitates more strongly with expressed GFP-Rap1A. Our results show that Rap1A is the more critical isoform in the context of endothelial barrier function, indicating that some cellular processes differentially utilize Rap1A and 1B isoforms. Studying how Rap1 isoforms differentially regulate EC junctions may thus reveal new targets for developing therapeutic strategies during pathological situations where endothelial barrier disruption leads to disease