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

Between Rho(k) and a hard place: the relation between vessel wall stiffness, endothelial contractility, and cardiovascular disease

Vascular stiffness is a mechanical property of the vessel wall that affects blood pressure, permeability, and inflammation. As a result, vascular stiffness is a key driver of (chronic) human disorders, including pulmonary arterial hypertension, kidney disease, and atherosclerosis. Responses of the endothelium to stiffening involve integration of mechanical cues from various sources, including the extracellular matrix, smooth muscle cells, and the forces that derive from shear stress of blood. This response in turn affects endothelial cell contractility, which is an important property that regulates endothelial stiffness, permeability, and leukocyte-vessel wall interactions. Moreover, endothelial stiffening reduces nitric oxide production, which promotes smooth muscle cell contraction and vasoconstriction. In fact, vessel wall stiffening, and microcirculatory endothelial dysfunction, precedes hypertension and thus underlies the development of vascular disease. Here, we review the cross talk among vessel wall stiffening, endothelial contractility, and vascular disease, which is controlled by Rho-driven actomyosin contractility and cellular mechanotransduction. In addition to discussing the various inputs and relevant molecular events in the endothelium, we address which actomyosin-regulated changes at cell adhesion complexes are genetically associated with human cardiovascular disease. Finally, we discuss recent findings that broaden therapeutic options for targeting this important mechanical signaling pathway in vascular pathogenesis