Noureddine Zebda

Focal adhesion kinase regulation of mechanotransduction and its impact on endothelial cell functions.

Vascular endothelial cells lining the blood vessels form the interface between the bloodstream and the vessel wall and as such they are continuously subjected to shear and cyclic stress from the flowing blood in the lumen. Additional mechanical stimuli are also imposed on these cells in the form of substrate stiffness transmitted from the extracellular matrix components in the basement membrane, and additional mechanical loads imposed on the lung endothelium as the result of respiration or mechanical ventilation in clinical settings. Focal adhesions (FAs) are complex structures assembled at the abluminal endothelial plasma membrane which connect the extracellular filamentous meshwork to the intracellular cytoskeleton and hence constitute the ideal checkpoint capable of controlling or mediating transduction of bidirectional mechanical signals. In this review we focus on focal adhesion kinase (FAK), a component of FAs, which has been studied for a number of years with regards to its involvement in mechanotransduction. We analyzed the recent advances in the understanding of the role of FAK in the signaling cascade(s) initiated by various mechanical stimuli with particular emphasis on potential implications on endothelial cell functions.

Interaction of p190RhoGAP with C-terminal domain of p120-catenin modulates endothelial cytoskeleton and permeability

Background: p120-catenin protein interactions regulate vascular permeability. Results: We identified p190RhoGAP-binding domain of p120-catenin and evaluated its functional significance. Conclusion: Binding of p190RhoGAP occurs at the amino acid 820–843 domain of p120-catenin and promotes activation of Rac and down-regulation of Rho signaling, leading to increased endothelial barrier. Significance: These data demonstrate functional significance of uncoupling the p120-catenin-p190RhoGAP interaction in the context of agonist-induced endothelial permeability. p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820–843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation.

Association between adherens junctions and tight junctions via Rap1 promotes barrier protective effects of oxidized phospholipids.

Previous studies showed that cyclopenthenone‐containing products resulting from oxidation of a natural phospholipid, 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphorylcholine (OxPAPC) exhibit potent barrier‐protective effects in the in vitro and in vivo models of lung endothelial cell (EC) barrier dysfunction, and these effects are associated with enhancement of peripheral actin cytoskeleton, cell–cell and cell–substrate contacts driven by activation of Rac and Cdc42 GTPases. Rap1 GTPase is another member of small GTPase family involved in control of cell–cell interactions; however, its involvement in EC barrier‐protective effects by OxPAPC remains unknown. This study examined a role of Rap1 in regulation of OxPAPC‐induced interactions in adherens junctions (AJ) and tight junctions (TJ) as a novel mechanism of EC barrier preservation in vitro and in vivo. Immunofluorescence analysis, subcellular fractionation, and co‐immunoprecipitation assays indicate that OxPAPC promoted accumulation of AJ proteins: VE‐cadherin, p120‐catenin, and β‐catenin; and TJ proteins: ZO‐1, occludin, and JAM‐A in the cell membrane, and induced novel cross‐interactions between AJ and TJ protein complexes, that were dependent on OxPAPC‐induced Rap1 activation. Inhibition of Rap1 function suppressed OxPAPC‐mediated pulmonary EC barrier enhancement and AJ and TJ interactions in vitro, as well as inhibited protective effects of OxPAPC against ventilator‐induced lung injury in vivo. These results show for the first time a role of Rap1‐mediated association between adherens junctions and tight junction complexes in the OxPAPC‐induced pulmonary vascular EC barrier protection. J. Cell. Physiol. 226: 2052–2062, 2011.

p190RhoGAP mediates protective effects of oxidized phospholipids in the models of ventilator-induced lung injury.

Products resulting from oxidation of cell membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent protective effects against lung endothelial cell (EC) barrier dysfunction caused by pathologically relevant mechanical forces and inflammatory agents. These effects were linked to enhancement of peripheral cytoskeleton and cell adhesion interactions mediated by small GTPase Rac and inhibition of Rho-mediated barrier-disruptive signaling. However, the mechanism of OxPAPC-induced, Rac-dependent Rho downregulation critical for vascular barrier protection remains unclear. This study tested the hypothesis that Rho negative regulator p190RhoGAP is essential for OxPAPC-induced lung barrier protection against ventilator-induced lung injury (VILI), and investigated potential mechanism of p190RhoGAP targeting to adherens junctions (AJ) via p120-catenin. OxPAPC induced peripheral translocation of p190RhoGAP, which was abolished by knockdown of Rac-specific guanine nucleotide exchange factors Tiam1 and Vav2. OxPAPC also induced Rac-dependent tyrosine phosphorylation and association of p190RhoGAP with AJ protein p120-catenin. siRNA-induced knockdown of p190RhoGAP attenuated protective effects of OxPAPC against EC barrier compromise induced by thrombin and pathologically relevant cyclic stretch (18% CS). In vivo, p190RhoGAP knockdown significantly attenuated protective effects of OxPAPC against ventilator-induced lung vascular leak, as detected by increased cell count and protein content in the bronchoalveolar lavage fluid, and tissue neutrophil accumulation in the lung. These results demonstrate for the first time a key role of p190RhoGAP for the vascular endothelial barrier protection in VILI.

VE-cadherin trans-interactions modulate Rac activation and enhancement of lung endothelial barrier by iloprost.

Small GTPase Rac is important regulator of endothelial cell (EC) barrier enhancement by prostacyclin characterized by increased peripheral actin cytoskeleton and increased interactions between VE‐cadherin and other adherens junction (AJ) proteins. This study utilized complementary approaches including siRNA knockdown, culturing in Ca2+‐free medium, and VE‐cadherin blocking antibody to alter VE‐cadherin extracellular interactions to investigate the role of VE‐cadherin outside‐in signaling in modulation of Rac activation and EC barrier regulation by prostacyclin analog iloprost. Spatial analysis of Rac activation in pulmonary EC by FRET revealed additional spike in iloprost‐induced Rac activity at the sites of newly formed cell–cell junctions. In contrast, disruption of VE‐cadherin extracellular trans‐interactions suppressed iloprost‐activated Rac signaling and attenuated EC barrier enhancement and cytoskeletal remodeling. These inhibitory effects were associated with decreased membrane accumulation and activation of Rac‐specific guanine nucleotide exchange factors (GEFs) Tiam1 and Vav2. Conversely, plating of pulmonary EC on surfaces coated with extracellular VE‐cadherin domain further promoted iloprost‐induced Rac signaling. In the model of thrombin‐induced EC barrier recovery, blocking of VE‐cadherin trans‐interactions attenuated activation of Rac pathway during recovery phase and delayed suppression of Rho signaling and restoration of EC barrier properties. These results suggest that VE‐cadherin outside‐in signaling controls locally Rac activity stimulated by barrier protective agonists. This control is essential for maximal EC barrier enhancement and accelerated barrier recovery. J. Cell. Physiol. 227: 3405–3416, 2012.

Barrier Enhancing Signals in Pulmonary Edema

Increased endothelial permeability and reduction of alveolar liquid clearance capacity are two leading pathogenic mechanisms of pulmonary edema, which is a major complication of acute lung injury, severe pneumonia, and acute respiratory distress syndrome, the pathologies characterized by unacceptably high rates of morbidity and mortality. Besides the success in protective ventilation strategies, no efficient pharmacological approaches exist to treat this devastating condition. Understanding of fundamental mechanisms involved in regulation of endothelial permeability is essential for development of barrier protective therapeutic strategies. Ongoing studies characterized specific barrier protective mechanisms and identified intracellular targets directly involved in regulation of endothelial permeability. Growing evidence suggests that, although each protective agonist triggers a unique pattern of signaling pathways, selected common mechanisms contributing to endothelial barrier protection may be shared by different barrier protective agents. Therefore, understanding of basic barrier protective mechanisms in pulmonary endothelium is essential for selection of optimal treatment of pulmonary edema of different etiology. This article focuses on mechanisms of lung vascular permeability, reviews major intracellular signaling cascades involved in endothelial monolayer barrier preservation and summarizes a current knowledge regarding recently identified compounds which either reduce pulmonary endothelial barrier disruption and hyperpermeability, or reverse preexisting lung vascular barrier compromise induced by pathologic insults.

Anti-Inflammatory Effects of OxPAPC Involve Endothelial Cell Mediated Generation of LXA4

Rationale: Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) generates a group of bioactive oxidized phospholipid products with a broad range of biological activities. Barrier-enhancing and anti-inflammatory effects of OxPAPC on pulmonary endothelial cells are critical for prevention of acute lung injury caused by bacterial pathogens or excessive mechanical ventilation. Anti-inflammatory properties of OxPAPC are associated with its antagonistic effects on Toll-like receptors and suppression of RhoA GTPase signaling. Objective: Because OxPAPC exhibits long-lasting anti-inflammatory and lung-protective effects even after single administration in vivo, we tested the hypothesis that these effects may be mediated by additional mechanisms, such as OxPAPC-dependent production of anti-inflammatory and proresolving lipid mediator, lipoxin A4 (LXA4). Methods and Results: Mass spectrometry and ELISA assays detected significant accumulation of LXA4 in the lungs of OxPAPC-treated mice and in conditioned medium of OxPAPC-exposed pulmonary endothelial cells. Administration of LXA4 reproduced anti-inflammatory effect of OxPAPC against tumor necrosis factor-&agr; in vitro and in the animal model of lipopolysaccharide-induced lung injury. The potent barrier-protective and anti-inflammatory effects of OxPAPC against tumor necrosis factor-&agr; and lipopolysaccharide challenge were suppressed in human pulmonary endothelial cells with small interfering RNA–induced knockdown of LXA4 formyl peptide receptor-2 (FPR2/ALX) and in mFPR2−/− (mouse formyl peptide receptor 2) mice lacking the mouse homolog of human FPR2/ALX. Conclusions: This is the first demonstration that inflammation- and injury-associated phospholipid oxidation triggers production of anti-inflammatory and proresolution molecules, such as LXA4. This lipid mediator switch represents a novel mechanism of OxPAPC-assisted recovery of inflamed lung endothelium.

Understanding dynamic changes in live cell adhesion with neutron reflectometry.

Neutron reflectometry (NR) was used to examine various live cells adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutron reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell - surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies.

Tuning endothelial monolayer adhesion: a neutron reflectivity study

Endothelial cells, master gatekeepers of the cardiovascular system, line its inner boundary from the heart to distant capillaries constantly exposed to blood flow. Interendothelial signaling and the monolayers adhesion to the underlying collagen-rich basal lamina are key in physiology and disease. Using neutron scattering, we report the first ever interfacial structure of endothelial monolayers under dynamic flow conditions mimicking the cardiovascular system. Endothelial adhesion (defined as the separation distance ℓ between the basal cell membrane and solid boundary) is explained using developed interfacial potentials and intramembrane segregation of specific adhesion proteins. Our method provides a powerful tool for the biophysical study of cellular layer adhesion strength in living tissues.