Matvey Gorovoy

Tiam1 and Rac1 Are Required for Platelet-activating Factor-induced Endothelial Junctional Disassembly and Increase in Vascular Permeability

It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases.

RhoGDI-1 Modulation of the Activity of Monomeric RhoGTPase RhoA Regulates Endothelial Barrier Function in Mouse Lungs

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1−/− mice. We observed that basal endothelial permeability in lungs of RhoGDI-1−/− mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1−/− lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1–mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

A Novel Mechanism of G Protein-dependent Phosphorylation of Vasodilator-stimulated Phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) α-thrombin induced phosphorylation of VASP. Specific inhibition of Gα13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Gα13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-κB signaling pathway. α-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Gα13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Gα13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IκB and pretreatment with the proteasome inhibitor MG-132 abolished Gα13- and α-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Gα13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IκB, release of PKA catalytic subunit from the complex with IκB and NF-κB, and subsequent phosphorylation of VASP.

LIM Kinase 1 Promotes Endothelial Barrier Disruption and Neutrophil Infiltration in Mouse Lungs

Rationale: Disruption of endothelial barrier function and neutrophil-mediated injury are two major mechanisms underlying the pathophysiology of sepsis-induced acute lung injury (ALI). Recently we reported that endotoxin induced activation of RhoA in mice lungs that led to the disruption of endothelial barrier and lung edema formation; however, the molecular mechanism of this phenomenon remained unknown. Objective: We reasoned that LIMK1, which participates in the regulation of endothelial cell contractility and is activated by RhoA/Rho kinase pathway, could mediate RhoA-dependent disruption of endothelial barrier function in mouse lungs during ALI. And if that is the case, then attenuation of endothelial cell contractility by downregulating LIMK1 may lead to the enhancement of endothelial barrier function, which could protect mice from endotoxin-induced ALI. Methods and Results: Here we report that LIMK1 deficiency in mice significantly reduced mortality induced by endotoxin. Data showed that lung edema formation, lung microvascular permeability, and neutrophil infiltration into the lungs were suppressed in limk1−/− mice. Conclusions: We identified that improvement of endothelial barrier function along with impaired neutrophil chemotaxis were the underlying mechanisms that reduced severity of ALI in limk1−/− mice, pointing to a new therapeutic target for diseases associated with acute inflammation of the lungs.

61 THE LOSS OF LIMK1 PROTECTS ENDOTHELIAL BARRIER FUNCTION.

Introduction Acute lung injury (ALI) is a syndrome of acute respiratory failure that results from acute pulmonary edema and inflammation. The development of ALI is associated with direct pulmonary injury from pneumonia and aspiration as well as indirect pulmonary injury from trauma and sepsis. LIMK1 is a serine/threonine kinase that is involved in cytoskeleton dynamics. Methods The role of LIMK1 in the regulation of endothelial permeability was evaluated using in vivo lung perfusion studies, transendothelial resistance of cell culture measurements, Western blotting, and electron and confocal microscopy. Results As enhanced pulmonary vascular permeability is a hallmark of acute lung injury, we examined the lung microcirculation in LIMK1 knockout mice. We found that endothelial permeability in the lungs of LIMK1 -/- mice was lower than that of wild-type mice. Notably, the endothelial permeability of the lungs of LIMK1 -/- mice after PAR1 peptide perfusion was significantly lower than that of wild type. Down-regulation of endogenous LIMK1 with siRNA in HUVECs resulted in increased transendothelial resistance. The overexpression of w.t. LIMK1 in HUVECs led to the decreased transendothelial resistance and opening of tight junctions as was revealed by confocal microscopy with the staining for ZO-1 and VE-cadherin. To study endotoxin-induced acute lung injury, anesthetized mice received LPS (ip) and the wet to dry ratio of the lungs of wild-type mice was compared to that of LIMK1 -/- mice. We found a decreased edema formation in LIMK1 -/- mice upon LPS treatment. Conclusions We suggest that the loss of LIMK1 protein leads to less permeable pulmonary blood vessels. These results favor the possibility that the inhibition of LIMK1 function may attenuate acute lung injury. This study was supported by NIH grants GM56159 and GM65160 and an American Heart Association (AHA) grant to T.V.Y. and an AHA predoctoral fellowship 0510133Z to M.G.

4 INCREASED ENDOTHELIAL PERMEABILITY IN RHOGDI ALPHA NULL MICE DUE TO THE ACTIVATION OF RHOA SIGNALING CASCADE

Changes in the endothelial permeability that are essential for vascular repair or inflammatory responses may also contribute to pathological conditions such as vascular leakage, septic shock, edema, atherosclerosis, hypertension, or cancer. Rho family GTPases have been implicated in the regulation of endothelial permeability via action on both actin cytoskeletal organization and the integrity of intercellular junctions. Regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs) prevent activation of RhoA, Rac1, and Cdc42. We studied the lung microcirculation to address the in-vivo role of RhoGDIα signaling in endothelial barrier regulation. We found that endothelial permeability in the lungs of RhoGDIα null mice was two-fold higher than that in the lungs of wild type mice. Perfusion of the mice lungs with the ROCK kinase inhibitor Y-27632 restored endothelial permeability to control level. The activity of RhoA and Rac1, but not Cdc42 was increased in RhoGDIα knock-out mice. Interestingly, myosin light chain 2 and cofilin were hyperphosphorylated, indicating the involvement of the RhoA signaling cascade. Thus, we concluded that regulation of RhoA and Rac1 activity via RhoGDIα has barrier protective effect in endothelium.