ROCK2 primes the endothelium for vascular hyperpermeability responses by raising baseline junctional tension

Abstract

Rho kinase mediates the effects of inflammatory permeability factors by increasing actomyosin-generated traction forces on endothelial adherens junctions, resulting in disassembly of intercellular junctions and increased vascular leakage. In vitro, this is accompanied by the Rho kinase-driven formation of prominent radial F-actin fibers, but the in vivo relevance of those F-actin fibers has been debated, suggesting other Rho kinase-mediated events to occur in vascular leak. Here, we delineated the contributions of the highly homologous isoforms of Rho kinase (ROCK1 and ROCK2) to vascular hyperpermeability responses. We show that ROCK2, rather than ROCK1 is the critical Rho kinase for regulation of thrombin receptor-mediated vascular permeability. Novel traction force mapping in endothelial monolayers, however, shows that ROCK2 is not required for the thrombin-induced force enhancements. Rather, ROCK2 is pivotal to baseline junctional tension as a novel mechanism by which Rho kinase primes the endothelium for hyperpermeability responses, independent from subsequent ROCK1-mediated contractile stress-fiber formation during the late phase of the permeability response.

Selective inhibition of brain endothelial Rho-kinase-2 provides optimal protection of an in vitro blood-brain barrier from tissue-type plasminogen activator and plasmin

“…Indeed, siRNA-induced silencing of ROCK-2, but not ROCK-1, significantly reduced thrombin-mediated permeability increases both in vitro and in vivo [31]. ROCK-2 was similarly involved in rapid permeability rises post thrombin treatment (within 1 h) while ROCK-1 became more prominent at a later stage [31].…”

Protein Interactions at Endothelial Junctions and Signaling Mechanisms Regulating Endothelial Permeability

“…Despite their structural similarity, the two proteins have distinct functions in endothelial cells. ROCKII but not ROCKI regulates basal tension across AJs and shifts the endothelium towards hyper-permeability 326 . The deletion of either ROCKI or ROCKII genes in mice has no apparent phenotype except for a defective placenta–embryo interaction 366, 367 .…”

“…mDia promotes the assembly of actin stress fibers, which are re-enforced by ROCK-mediated activation of myosin II 324 . ROCKI and ROCKII are differentially regulated in endothelial cells 325, 326 . ROCKI is basally active 325 and contributes to early responses of endothelial cells to pro-inflammatory mediators such as TNFα and Lipopolysaccharide (LPS) 194, 327 .…”

“…In contrast, activation of ROCKII in response to pro-inflammatory stimuli is required for the long-term effects of LPS and TNFα in disrupting endothelial barrier integrity 325, 328 . Evidence also indicates that ROCKII maintains baseline junctional tension and primes the endothelium for hyperpermeability responses such as during thrombin challenge, independent from subsequent ROCKI-mediated contractile stress-fiber formation 326 . Both ROCKs maintain MLC in a phosphorylated state through interaction with the PI3K/AKT pathway 329 .…”

Lung Circulation

“…Within this family, RhoA diminishes barrier function through its effects on myosin light chains (146) while Rac1 improves barrier function by stabilizing adherens junctions (61,147,327). Along these lines, silencing or pharmacological inhibition of the downstream effector molecule, ROCK1, prevented F-actin organization induced by in vivo agonism of the thrombin receptor, but had no effect on hyperpermeability (45). Rather, ROCK2 activation was established as the main mediator of pulmonary edema, via regulation of basal tension and cell-cell adhesion.…”