Hepatocyte Growth Factor-induced Asef-IQGAP1 Complex Controls Cytoskeletal Remodeling and Endothelial Barrier

Hepatocyte growth factor (HGF) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via Rac-dependent enhancement of the peripheral actin cytoskeleton. However, the precise mechanisms of HGF effects on the peripheral cytoskeleton are not well understood. This study evaluated a role for Rac/Cdc42-specific guanine nucleotide exchange factor Asef and the multifunctional Rac effector, IQGAP1, in the mechanism of HGF-induced EC barrier enhancement. HGF induced Asef and IQGAP1 co-localization at the cell cortical area and stimulated formation of an Asef-IQGAP1 functional protein complex. siRNA-induced knockdown of Asef or IQGAP1 attenuated HGF-induced EC barrier enhancement. Asef knockdown attenuated HGF-induced Rac activation and Rac association with IQGAP1, and it abolished both IQGAP1 accumulation at the cell cortical layer and IQGAP1 interaction with actin cytoskeletal regulators cortactin and Arp3. Asef activation state was essential for Asef interaction with IQGAP1 and protein complex accumulation at the cell periphery. In addition to the previously reported role of the IQGAP1 RasGAP-related domain in the Rac-dependent IQGAP1 activation and interaction with its targets, we show that the IQGAP1 C-terminal domain is essential for HGF-induced IQGAP1/Asef interaction and Asef-Rac-dependent activation leading to IQGAP1 interaction with Arp3 and cortactin as a positive feedback mechanism of IQGAP1 activation. These results demonstrate a novel feedback mechanism of HGF-induced endothelial barrier enhancement via Asef/IQGAP1 interactions, which regulate the level of HGF-induced Rac activation and promote cortical cytoskeletal remodeling via IQGAP1-Arp3/cortactin interactions.     

Hepatocyte growth factor triggers distinct mechanisms of Asef and Tiam1 activation to induce endothelial barrier enhancement

Previous reports described an important role of hepatocyte growth factor (HGF) in mitigation of pulmonary endothelial barrier dysfunction and cell injury induced by pathologic agonists and mechanical forces. HGF protective effects have been associated with Rac-GTPase signaling pathway activated by Rac-specific guanine nucleotide exchange factor Tiam1 and leading to enhancement of intercellular adherens junctions. This study tested involvement of a novel Rac-specific activator, Asef, in endothelial barrier enhancement by HGF and investigated a mechanism of HGF-induced Asef activation. Si-RNA-based knockdown of Tiam1 and Asef had an additive effect on attenuation of HGF-induced Rac activation and endothelial cell (EC) barrier enhancement. Tiam1 and Asef activation was abolished by pharmacologic inhibitors of HGF receptor and PI3-kinase. In contrast to Tiam1, Asef interacted with APC and associated with microtubule fraction upon HGF stimulation. EC treatment by low dose nocodazole to inhibit peripheral microtubule dynamics partially attenuated HGF-induced Asef peripheral translocation, but had negligible effect on Tiam1 translocation. These effects were associated with attenuation of HGF-induced barrier enhancement in EC pretreated with low ND dose and activation of Rac and its cytoskeletal effectors PAK1 and cortactin. These data demonstrate, that in addition to microtubule-independent Tiam1 activation, HGF engages additional microtubule- and APC-dependent pathway of Asef activation. These mechanisms may complement each other to provide the fine tuning of Rac signaling and endothelial barrier enhancement in response to various agonists.     

Microtubule Dynamics Control HGF-Induced Lung Endothelial Barrier Enhancement

Microtubules (MT) play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC) barrier by hepatocyte growth factor (HGF) which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics.     

Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met (1). We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.     

CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin

The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.     

IQGAP1 Regulates Endothelial Barrier Function via EB1-Cortactin Cross Talk

Cross talk between the actin cytoskeleton and microtubules (MT) has been implicated in the amplification of agonist-induced Rho signaling, leading to increased vascular endothelial permeability. This study tested the involvement of actin-MT cross talk in the mechanisms of barrier enhancement induced by hepatocyte growth factor (HGF) and evaluated the role of the adaptor protein IQGAP1 in integrating the MT- and actin-dependent pathways of barrier enhancement. IQGAP1 knockdown by small interfering RNA attenuated the HGF-induced increase in endothelial barrier properties and abolished HGF-activated cortical actin dynamics. IQGAP1 reduction abolished HGF-induced peripheral accumulation of Rac cytoskeletal effector cortactin and cortical actin remodeling. In addition, HGF stimulated peripheral MT growth in an IQGAP1-dependent fashion. HGF also induced Rac1-dependent IQGAP1 association with the MT fraction and the formation of a protein complex containing end-binding protein 1 (EB1), IQGAP1, and cortactin. Decreasing endogenous IQGAP1 abolished HGF-induced EB1-cortactin colocalization at the cell periphery. In turn, expression of IQGAP1ΔC (IQGAP1 lacking the C-terminal domain) attenuated the cortactin association with EB1 and suppressed HGF-induced endothelial cell peripheral actin cytoskeleton enhancement. These results demonstrate for the first time the MT-actin cross talk mechanism of HGF-induced endothelial barrier enhancement and suggest that IQGAP1 functions as a hub linking HGF-induced signaling to MT and actin remodeling via EB1-IQGAP1-cortactin interactions.     

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 Ca(2+) -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.     

Control of Vascular Permeability by Atrial Natriuretic Peptide via a GEF-H1-dependent Mechanism

Microtubule (MT) dynamics is involved in a variety of cell functions, including control of the endothelial cell (EC) barrier. Release of Rho-specific nucleotide exchange factor GEF-H1 from microtubules activates the Rho pathway of EC permeability. In turn, pathologic vascular leak can be prevented by treatment with atrial natriuretic peptide (ANP). This study investigated a novel mechanism of vascular barrier protection by ANP via modulation of GEF-H1 function. In pulmonary ECs, ANP suppressed thrombin-induced disassembly of peripheral MT and attenuated Rho signaling and cell retraction. ANP effects were mediated by the Rac1 GTPase effector PAK1. Activation of Rac1-PAK1 promoted PAK1 interaction with the Rho activator GEF-H1, inducing phosphorylation of total and MT-bound GEF-H1 and leading to attenuation of Rho-dependent actin remodeling. In vivo, ANP attenuated lung injury caused by excessive mechanical ventilation and TRAP peptide (TRAP/HTV), which was further exacerbated in ANP^−/− mice. The protective effects of ANP against TRAP/HTV-induced lung injury were linked to the increased pool of stabilized MT and inactivation of Rho signaling via ANP-induced, PAK1-dependent inhibitory phosphorylation of GEF-H1. This study demonstrates a novel protective mechanism of ANP against pathologic hyperpermeability and suggests a novel pharmacological intervention for the prevention of increased vascular leak via PAK1-dependent modulation of GEF-H1 activity.     

Prostaglandins PGE(2) and PGI(2) promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation

Prostaglandin E(2) (PGE(2)) and prostacyclin are lipid mediators produced by cyclooxygenase and implicated in the regulation of vascular function, wound repair, inflammatory processes, and acute lung injury. Although protective effects of these prostaglandins (PGs) are associated with stimulation of intracellular cAMP production, the crosstalk between cAMP-activated signal pathways in the regulation of endothelial cell (EC) permeability is not well understood. We studied involvement of cAMP-dependent kinase (PKA), cAMP-Epac-Rap1 pathway, and small GTPase Rac in the PGs-induced EC barrier protective effects and cytoskeletal remodeling. PGE(2) and PGI(2) synthetic analog beraprost increased transendothelial electrical resistance and decreased dextran permeability, enhanced peripheral F-actin rim and increased intercellular adherens junction areas reflecting EC barrier-protective response. Furthermore, beraprost dramatically attenuated thrombin-induced Rho activation, MLC phosphorylation and EC barrier dysfunction. In vivo, beraprost attenuated lung barrier dysfunction induced by high tidal volume mechanical ventilation. Both PGs caused cAMP-mediated activation of PKA-, Epac/Rap1- and Tiam1/Vav2-dependent pathways of Rac1 activation and EC barrier regulation. Knockdown of Epac, Rap1, Rac-specific exchange factors Tiam1 and Vav2 using siRNA approach, or inhibition of PKA activity decreased Rac1 activation and PG-induced EC barrier enhancement. Thus, our results show that barrier-protective effects of PGE(2) and prostacyclin on pulmonary EC are mediated by PKA and Epac/Rap pathways, which converge on Rac activation and lead to enhancement of peripheral actin cytoskeleton and adherens junctions. These mechanisms may mediate protective effects of PGs against agonist-induced lung vascular barrier dysfunction in vitro and against mechanical stress-induced lung injury in vivo.     

Magnitude-dependent effects of cyclic stretch on HGF- and VEGF-induced pulmonary endothelial remodeling and barrier regulation

Mechanical ventilation at high tidal volumes compromises the blood-gas barrier and increases lung vascular permeability, which may lead to ventilator-induced lung injury and pulmonary edema. Using pulmonary endothelial cell (ECs) exposed to physiologically [5% cyclic stretch (CS)] and pathologically (18% CS) relevant magnitudes of CS, we evaluated the potential protective effects of hepatocyte growth factor (HGF) on EC barrier dysfunction induced by CS and vascular endothelial growth factor (VEGF). In static culture, HGF enhanced EC barrier function in a Rac-dependent manner and attenuated VEGF-induced EC permeability and paracellular gap formation. The protective effects of HGF were associated with the suppression of Rho-dependent signaling triggered by VEGF. Five percent CS promoted HGF-induced enhancement of the cortical F-actin rim and activation of Rac-dependent signaling, suggesting synergistic barrier-protective effects of physiological CS and HGF. In contrast, 18% CS further enhanced VEGF-induced EC permeability, activation of Rho signaling, and formation of actin stress fibers and paracellular gaps. These effects were attenuated by HGF pretreatment. EC preconditioning at 5% CS before HGF and VEGF further promoted EC barrier maintenance. Our data suggest synergistic effects of HGF and physiological CS in the Rac-mediated mechanisms of EC barrier protection. In turn, HGF reduced the barrier-disruptive effects of VEGF and pathological CS via downregulation of the Rho pathway. These results support the importance of HGF-VEGF balance in control of acute lung injury/acute respiratory distress syndrome severity via small GTPase-dependent regulation of lung endothelial permeability.     

SRC kinase activator CDCP1 promotes hepatocyte growth factor–induced cell migration/invasion of a subset of breast cancer cells

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain–containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.     

Epac/Rap and PKA are novel mechanisms of ANP-induced Rac-mediated pulmonary endothelial barrier protection

Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in the modulation of lung injury. Besides well recognized physiological effects on vascular tone, plasma volume, and renal function, ANP may exhibit protective effects in models of lung vascular endothelial cell (EC) barrier dysfunction. However, the molecular mechanisms of ANP protective effects are not well understood. The recently described cAMP-dependent guanine nucleotide exchange factor (GEF) Epac activates small GTPase Rap1, which results in activation of small GTPase Rac-specific GEFs Tiam1 and Vav2 and Rac-mediated EC barrier protective responses. Our results show that ANP stimulated protein kinase A and the Epac/Rap1/Tiam/Vav/Rac cascade dramatically attenuated thrombin-induced pulmonary EC permeability and the disruption of EC monolayer integrity. Using pharmacological and molecular activation and inhibition of cAMP-and cGMP-dependent protein kinases (PKA and PKG), Epac, Rap1, Tiam1, Vav2, and Rac we linked ANP-mediated protective effects to the activation of Epac/Rap and PKA signaling cascades, which dramatically inhibited the Rho pathway of thrombin-induced EC hyper-permeability. These results suggest a novel mechanism of ANP protective effects against agonist-induced pulmonary EC barrier dysfunction via inhibition of Rho signaling by Epac/Rap1-Rac and PKA signaling cascades.     

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.     

Endothelial cell barrier enhancement by ATP is mediated by the small GTPase Rac and cortactin

ATP is a physiologically relevant agonist released by various sources, including activated platelets, with complex effects mediated via activation of P(2) purinergic receptors. ATP-induced endothelial cell (EC) production of prostacyclin and nitric oxide is recognized, and EC barrier enhancement evoked by ATP has been described. ATP effects on EC barrier function and vascular permeability, however, remain poorly characterized. Although the mechanisms involved are unclear, we previously identified activation of the small GTPase Rac and translocation of cortactin, an actin-binding protein, as key to EC barrier augmentation induced by simvastatin and sphingosine 1-phosphate and therefore examined the role of these molecules in ATP-induced EC barrier enhancement. ATP induced rapid, dose-dependent barrier enhancement in human pulmonary artery EC as measured by transendothelial electrical resistance, with a peak effect appreciable at 25 min (39% increase, 10 microM) and persisting at 2 h. These effects were associated with rearrangement of the EC actin cytoskeleton, early myosin light chain phosphorylation, and spatially defined (cell periphery) translocation of both Rac and cortactin. ATP (10 microM)-treated EC demonstrated a significant increase in Rac activation relative to controls, with a maximal effect (approximately 4-fold increase) at 10 min. Finally, ATP-induced barrier enhancement was markedly attenuated by reductions of either Rac or cortactin (small interfering RNA) relative to controls. Our results suggest for the first time that ATP-mediated barrier protection is associated with cytoskeletal activation and is dependent on both Rac activation and cortactin.     

Asef is a Cdc42-specific guanine nucleotide exchange factor

Asef is a member of the Dbl-family of guanine nucleotide exchange factors (GEFs) with a proposed specificity for the small GTPase Rac1. Here we investigated the specificity and regulation of Asef by measuring its GEF activity in vitro and observed hardly any activity towards Rac1, Rac2 and Rac3, or RhoA and TC10. In contrast, various purified Asef protein fragments catalyzed the nucleotide exchange reaction of Cdc42. The Cdc42GEF activity of the Dbl homology (DH) domain of Asef was significantly higher in the presence of the pleckstrin homology (PH) domain. Our data strongly suggest that Asef is a canonical Cdc42GEF, which employs its PH domain to efficiently stabilize its autoinhibited state, but also to facilitate nucleotide exchange activity of the DH domain after its activation by upstream signals.     

Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.     

Lipopolysaccharide-induced Lung Injury Involves the Nitration-mediated Activation of RhoA

Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr³⁴ in RhoA. Tyr³⁴ is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr³⁴ against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr³⁴ nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of “shielding” peptides to prevent RhoA nitration in the management of ALI.     

Rap-afadin axis in control of Rho signaling and endothelial barrier recovery

Activation of the Rho GTPase pathway determines endothelial cell (EC) hyperpermeability after injurious stimuli. To date, feedback mechanisms of Rho down-regulation critical for barrier restoration remain poorly understood. We tested a hypothesis that Rho down-regulation and barrier recovery of agonist-stimulated ECs is mediated by the Ras family GTPase Rap1. Thrombin-induced EC permeability driven by rapid activation of the Rho GTPase pathway was followed by Src kinase–dependent phosphorylation of the Rap1-specific guanine nucleotide exchange factor (GEF) C3G, activation of Rap1, and initiation of EC barrier recovery. Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution. Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin. This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps. These data demonstrate that activation of the Rap1-afadin axis is a physiological mechanism driving restoration of barrier integrity in agonist-stimulated EC monolayers via negative-feedback regulation of Rho signaling, stimulation of actin peripheral dynamics, and reestablishment of cell–cell adhesive complexes.     

Stiffness-Activated GEF-H1 Expression Exacerbates LPS-Induced Lung Inflammation

Acute lung injury (ALI) is accompanied by decreased lung compliance. However, a role of tissue mechanics in modulation of inflammation remains unclear. We hypothesized that bacterial lipopolysacharide (LPS) stimulates extracellular matrix (ECM) production and vascular stiffening leading to stiffness-dependent exacerbation of endothelial cell (EC) inflammatory activation and lung barrier dysfunction. Expression of GEF-H1, ICAM-1, VCAM-1, ECM proteins fibronectin and collagen, lysyl oxidase (LOX) activity, interleukin-8 and activation of Rho signaling were analyzed in lung samples and pulmonary EC grown on soft (1.5 or 2.8 kPa) and stiff (40 kPa) substrates. LPS induced EC inflammatory activation accompanied by expression of ECM proteins, increase in LOX activity, and activation of Rho signaling. These effects were augmented in EC grown on stiff substrate. Stiffness-dependent enhancement of inflammation was associated with increased expression of Rho activator, GEF-H1. Inhibition of ECM crosslinking and stiffening by LOX suppression reduced EC inflammatory activation and GEF-H1 expression in response to LPS. In vivo, LOX inhibition attenuated LPS-induced expression of GEF-H1 and lung dysfunction. These findings present a novel mechanism of stiffness-dependent exacerbation of vascular inflammation and escalation of ALI via stimulation of GEF-H1 - Rho pathway. This pathway represents a fundamental mechanism of positive feedback regulation of inflammation.     

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.