Radu Neamu

Cdc42 Regulates Adherens Junction Stability and Endothelial Permeability by Inducing α-Catenin Interaction With the Vascular Endothelial Cadherin Complex

The endothelial adherens junctions (AJs) consist of trans-oligomers of membrane spanning vascular endothelial (VE)-cadherin proteins, which bind β-catenin through their cytoplasmic domain. β-Catenin in turn binds α-catenin and connects the AJ complex with the actin cytoskeleton. We addressed the in vivo effects of loss of VE-cadherin interactions on lung vascular endothelial permeability and the role of specific Rho GTPase effectors in regulating the increase in permeability induced by AJ destabilization. We used cationic liposomes encapsulating the mutant of VE-cadherin lacking the extracellular domain (&Dgr;EXD) to interfere with AJ assembly in mouse lung endothelial cells. We observed that lung vascular permeability (quantified as microvessel filtration coefficient [Kf,c]) was increased 5-fold in lungs expressing &Dgr;EXD. This did not occur to the same degree on expression of the VE-cadherin mutant, &Dgr;EXD&Dgr;β, lacking the β-catenin–binding site. The increased vascular permeability was the result of destabilization of VE-cadherin homotypic interaction induced by a shift in the binding of β-catenin from wild-type VE-cadherin to the expressed &Dgr;EXD mutant. Because &Dgr;EXD expression in endothelial cells activated the Rho GTPase Cdc42, we addressed its role in the mechanism of increased endothelial permeability induced by AJ destabilization. Coexpression of dominant-negative Cdc42 (N17Cdc42) prevented the increase in Kf,c induced by &Dgr;EXD. This was attributed to inhibition of the association of α-catenin with the &Dgr;EXD–β-catenin complex. The results demonstrate that Cdc42 regulates AJ permeability by controlling the binding of α-catenin with β-catenin and the consequent interaction of the VE-cadherin/catenin complex with the actin cytoskeleton.

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.

MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.

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.

Impaired Caveolae Function and Upregulation of Alternative Endocytic Pathways Induced by Experimental Modulation of Intersectin-1s Expression in Mouse Lung Endothelium

Intersectin-1s (ITSN-1s), a protein containing five SH3 (A-E) domains, regulates via the SH3A the function of dynamin-2 (dyn2) at the endocytic site. ITSN-1s expression was modulated in mouse lung endothelium by liposome delivery of either a plasmid cDNA encoding myc-SH3A or a specific siRNA targeting ITSN-1 gene. The lung vasculature of SH3A-transduced and ITSN-1s- deficient mice was perfused with gold albumin (Au-BSA) to analyze by electron microscopy the morphological intermediates and pathways involved in transendothelial transport or with dinitrophenylated (DNP)-BSA to quantify by ELISA its transport. Acute modulation of ITSN-1s expression decreased the number of caveolae, impaired their transport, and opened the interendothelial junctions, while upregulating compensatory nonconventional endocytic/transcytotic structures. Chronic inhibition of ITSN-1s further increased the occurrence of nonconventional intermediates and partially restored the junctional integrity. These findings indicate that ITSN-1s expression is required for caveolae function and efficient transendothelial transport. Moreover, our results demonstrate that ECs are highly adapted to perform their transport function while maintaining lung homeostasis.

Regulation of dynamin‐2 assembly–disassembly and function through the SH3A domain of intersectin‐1s

Intersectin‐1s (ITSN‐1s), a five Src homology 3 (SH3) domain‐containing protein, is critically required for caveolae and clathrin‐mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A‐E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN‐1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high‐resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining‐dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full‐length ITSN‐1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN‐1s were used as controls. Strikingly, in the presence of SH3A, dyn2–dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free‐transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN‐1s, via its SH3A has the unique ability to regulate dyn2 assembly–disassembly and function during endocytosis.

Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles.

Vasodilator‐stimulated phosphoprotein deficiency potentiates PAR‐1‐induced increase in endothelial permeability in mouse lungs

Vasodilator‐stimulated phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. The function of VASP in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA‐mediated VASP downregulation in HUVECs leads to a potentiation of thrombin‐ and protease‐activated receptor 1 (PAR‐1) agonist‐induced increase in endothelial permeability. Compared to control cells, VASP‐deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE‐cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP−/− mice have increased microvascular permeability in response to infusion with PAR‐1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP‐deficient HUVECs after thrombin stimulation and VASP−/− mouse lungs after PAR‐1 agonist infusion, indicating that VASP effects on thrombin signaling may be correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin‐dependent disruption of the endothelial barrier function. J. Cell. Physiol. 226: 1255–1264, 2011.

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.