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Biomedical subjects

Konstantin G Birukov

Publications and source records attributed to Konstantin G Birukov.

At least 19 recordsLinked to original sources
PubMed2007

Tiam1 and betaPIX mediate Rac-dependent endothelial barrier protective response to oxidized phospholipids.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibits potent barrier protective effects on pulmonary endothelium, which are mediated by small GTPases Rac and Cdc42. However, upstream mechanisms of OxPAPC-induced small GTPase activation are not known. We studied involvement of Rac/Cdc42-specific guanine nucleotide exchange factors (GEFs) Tiam1 and betaPIX in OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective responses in the human pulmonary endothelial cells (EC). OxPAPC induced membrane translocation of Tiam1, betaPIX, Cdc42, and Rac, but did not affect intracellular distribution of Rho and Rho-specific GEF p115-RhoGEF. Protein depletion of Tiam1 and betaPIX using siRNA approach abolished OxPAPC-induced activation of Rac and its effector PAK1. EC transfection with Tiam1-, betaPIX-, or PAK1-specific siRNA dramatically attenuated OxPAPC-induced barrier enhancement, peripheral actin cytoskeletal enhancement, and translocation of actin-binding proteins cortactin and Arp3. These results show for the first time that Tiam1 and betaPIX mediate OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective response in pulmonary endothelium.

Capillary Permeability↗
PubMed2006

Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium.

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.

Actins↗
PubMed2006

EGFR-activated signaling and actin remodeling regulate cyclic stretch-induced NRF2-ARE activation.

Cyclic stretch (CS) associated with mechanical ventilation (MV) can cause excessive alveolar and endothelial distention, resulting in lung injury and inflammation. Antioxidant enzymes (AOEs) play a major role in suppressing these effects. The transcription factor Nrf2, via the antioxidant response element (ARE), alleviates pulmonary toxicant- and oxidant-induced oxidative stress by up-regulating the expression of several AOEs. Although gene expression profiling has revealed the induction of AOEs in the lungs of rodents exposed to MV, the mechanisms by which mechanical forces, such as CS, regulate the activation of Nrf2-dependent ARE-transcriptional responses are poorly understood. To mimic mechanical stress associated with MV, we have cultured pulmonary alveolar epithelial and endothelial cells on collagen I-coated BioFlex plates and subjected them to CS. CS exposure stimulated ARE-driven transcriptional responses and subsequent AOE expression. Ectopic expression of a dominant-negative Nrf2 suppressed the CS-stimulated ARE-driven responses. Our findings suggest that actin remodeling is necessary but not sufficient for high-level CS-induced ARE activation in both epithelial and endothelial cells. We also found that inhibition of EGFR activity by a pharmacologic agent ablated the CS-induced ARE transcriptional response in both cell types. Additional studies revealed that amphiregulin, an EGFR ligand, regulates this process. We further demonstrated that the PI3K-Akt pathway acts as the downstream effector of EGFR and regulates CS-induced ARE-activation in an oxidative stress-dependent manner. Collectively, these novel findings suggest that EGFR-activated signaling and actin remodeling act in concert to regulate the CS-induced Nrf2-ARE transcriptional response and subsequent AOE expression.

Actins↗
PubMed2006

Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways.

Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.

Animals↗
PubMed2006

Focal adhesions: paradigm for a signaling nexus.

The vascular wall contains intimal endothelium and medial smooth muscle that act as contiguous tissues with tight spatial and functional coordination in response to tonic and episodic input from the bloodstream and the surrounding parenchyma. Focal adhesions are molecular bridges between the intracellular and extracellular spaces that integrate a variety of environmental stimuli and mediate 2-way crosstalk between the extracellular matrix and the cytoskeleton. Focal adhesion components are targets for biochemical and mechanical stimuli that evoke crucial developmental and injury response mechanisms including cell growth, movement, and differentiation, and tailoring of the extracellular microenvironment. Focal adhesions provide the vascular wall constituents with flexible and specific tools for exchanging cues in a complex system. The molecular mechanisms that underlie these vital communications are detailed in this review with the goal of defining future targets for vascular tissue engineering and for the therapeutic modulation of disordered vascular growth, inflammation, thrombosis, and angiogenesis.

Animals↗
PubMed2006

Oxidized phospholipids reduce vascular leak and inflammation in rat model of acute lung injury.

RATIONALE: Acute inflammation and vascular leak are cardinal features of acute lung injury and the acute respiratory distress syndrome. Nonspecific tissue inflammation and injury in response to infectious and noninfectious insults lead to oxidative stress and the generation of lipid oxidation products, which may inhibit the acute inflammatory response to bacterial components. OBJECTIVE: To test the hypothesis that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) may attenuate the acute lung inflammatory response to lipopolysaccharide (LPS) and enhance lung vascular barrier recovery, we used in vivo and in vitro models of LPS-induced lung injury. METHODS: Rats received intratracheal aerosolized LPS (5 mg/kg) or sterile water with concurrent intravenous injection of OxPAPC (0.5-6.0 mg/kg) or saline alone. Nonoxidized PAPC was used as a control. At 18 h, bronchoalveolar lavage was performed and the lungs were removed for histologic analysis. Measurements of endothelial transmonolayer electrical resistance and immunofluorescent analysis of monolayer integrity were used in an in vitro model of LPS-induced lung vascular barrier dysfunction. MEASUREMENTS AND MAIN RESULTS: In vivo, aerosolized intratracheal LPS induced lung injury with profound increases in bronchoalveolar lavage neutrophils, protein content, and the inflammatory cytokines interleukin 6 and interleukin 1beta, as well as tissue neutrophils. OxPAPC, but not nonoxidized PAPC, markedly attenuated the LPS-induced tissue inflammation, barrier disruption, and cytokine production over a range of doses. In vitro, oxidized phospholipids attenuated LPS-induced endothelial barrier disruption and reversed LPS-induced cytoskeletal remodeling and disruption of monolayer integrity. CONCLUSIONS: These studies demonstrate in vivo and in vitro protective effects of oxidized phospholipids on LPS-induced lung dysfunction.

Animals↗
PubMed2006

Oxidized lipids: the two faces of vascular inflammation.

Elevated plasma levels of low-density lipoprotein and generation of oxidized low-density lipoprotein have been directly associated with the pathogenesis of atherosclerosis, and lipid oxidation products have been directly linked with induction and propagation of monocytic subendothelial accumulation and other inflammatory reactions associated with chronic vascular inflammation. However, accumulating data suggest that oxidized lipids may also exhibit anti-inflammatory potential and serve as potent inhibitors of nuclear factor-kappaB-dependent proinflammatory cascade. In addition, we have characterized a group of bioactive components of oxidized phospholipids with barrier-protective effects towards endothelial cells in the models of agonist-induced endothelial permeability and lipopolysaccharide-induced lung dysfunction. This review discusses the role of oxidized lipids in the progression of atherosclerosis as well as the important anti-inflammatory effects of oxidized phospholipids and their potential role in the modulation of vascular barrier integrity.

Anti-Inflammatory Agents↗
PubMed2006

Differential regulation of pulmonary endothelial monolayer integrity by varying degrees of cyclic stretch.

Ventilator-induced lung injury is a life-threatening complication of mechanical ventilation at high-tidal volumes. Besides activation of proinflammatory cytokine production, excessive lung distension directly affects blood-gas barrier and lung vascular permeability. To investigate whether restoration of pulmonary endothelial cell (EC) monolayer integrity after agonist challenge is dependent on the magnitude of applied cyclic stretch (CS) and how these effects are linked to differential activation of small GTPases Rac and Rho, pulmonary ECs were subjected to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of CS. Pathological CS enhanced thrombin-induced gap formation and delayed monolayer recovery, whereas physiological CS induced nearly complete EC recovery accompanied by peripheral redistribution of focal adhesions and cortactin after 50 minutes of thrombin. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation during the EC recovery phase. Rac inhibition dramatically attenuated restoration of monolayer integrity after thrombin challenge. Physiological CS preconditioning (5% CS, 24 hours) enhanced EC paracellular gap resolution after step-wise increase to 18% CS (30 minutes) and thrombin challenge. These results suggest a critical role for the CS amplitude and the balance between Rac and Rho in mechanochemical regulation of lung EC barrier.

Cell Culture Techniques↗
PubMed2005

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction.

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.

Actin Cytoskeleton↗
PubMed2005

ALK5 and Smad4 are involved in TGF-beta1-induced pulmonary endothelial permeability.

The ability of inflammatory cytokine TGF-beta1 to alter endothelial cell phenotype suggests its role in the regulation of vascular endothelial cell permeability. We demonstrate that depletion of TGF-beta1 receptor ALK5 and regulatory protein Smad4, but not ALK1 receptor attenuates TGF-beta1-induced permeability increase and significantly inhibits TGF-beta1-induced EC contraction manifested by actin stress fiber formation and increased MLC and MYPT1 phosphorylation. Consistent with these results, EC treatment with SB 431542, an inhibitor of ALK5 but not ALK1 receptor, significantly attenuates TGF-beta1-induced permeability. Thus, our data demonstrate for the first time direct link between TGF-beta1-mediated activation of ALK5/Smad and EC barrier dysfunction.

Actins↗
PubMed2005

MAP kinases in lung endothelial permeability induced by microtubule disassembly.

Lung endothelial barrier function is regulated by multiple signaling pathways, including mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK) 1/2 and p38. We have recently shown involvement of microtubule (MT) disassembly in endothelial cell (EC) barrier failure. In this study, we examined potential involvement of ERK1/2 and p38 MAPK in lung EC barrier dysfunction associated with MT disassembly. MT inhibitors nocodazole (0.2 microM) and vinblastine (0.1 microM) induced sustained activation of Ras-Raf-MEK1/2-ERK1/2 and MKK3/6-p38-MAPKAPK2 MAPK cascades in human and bovine pulmonary EC, as detected by phosphospecific antibodies and in MAPK activation assays. These effects were linked to increased permeability assessed by measurements of transendothelial electrical resistance and cytoskeletal remodeling analyzed by morphometric analysis of EC monolayers. MT stabilization by taxol (5 microM, 1 h) attenuated nocodazole-induced ERK1/2 and p38 MAPK activation and phosphorylation of p38 MAPK substrate 27-kDa heat shock protein and regulatory myosin light chains, the proteins involved in actin polymerization and actomyosin contraction. Importantly, only pharmacological inhibition of p38 MAPK by SB-203580 (20 microM, 1 h) attenuated nocodazole-induced MT depolymerization, actin remodeling, and EC barrier dysfunction, whereas the MEK/ERK1/2 inhibitor U0126 (5 microM, 1 h) exhibited no effect. These data suggest a direct link between p38 MAPK activation, remodeling of MT network, and EC barrier regulation.

Actins↗
PubMed2005

Involvement of microtubules and Rho pathway in TGF-beta1-induced lung vascular barrier dysfunction.

Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling.

Actins↗
PubMed2004

Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells.

Regulation of endothelial cell (EC) permeability by bioactive molecules is associated with specific patterns of cytoskeletal and cell contact remodeling. A role for mechanical factors such as shear stress (SS) and cyclic stretch (CS) in cytoskeletal rearrangements and regulation of EC permeability becomes increasingly recognized. This paper examined redistribution of focal adhesion (FA) proteins, site-specific focal adhesion kinase (FAK) phosphorylation, small GTPase activation and barrier regulation in human pulmonary EC exposed to laminar shear stress (15 dyn/cm2) or cyclic stretch (18% elongation) in vitro. SS caused peripheral accumulation of FAs, whereas CS induced randomly distributed FAs attached to the ends of newly formed stress fibers. SS activated small GTPase Rac without effects on Rho, whereas 18% CS activated without effect on Rac. SS increased transendothelial electrical resistance (TER) in EC monolayers, which was further elevated by barrier-protective phospholipid sphingosine 1-phosphate. Finally, SS induced FAK phosphorylation at Y576, whereas CS induced FAK phosphorylation at Y397 and Y576. These results demonstrate for the first time differential effects of SS and CS on Rho and Rac activation, FA redistribution, site-specific FAK phosphorylation, and link them with SS-mediated barrier enhancement. Thus, our results suggest common signaling and cytoskeletal mechanisms shared by mechanical and chemical factors involved in EC barrier regulation.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac.

After an acute phase of inflammation or injury, restoration of the endothelial barrier is important to regain vascular integrity and to prevent edema formation. However, little is known about mediators that control restoration of endothelial barrier function. We show here that oxidized phospholipids that accumulate at sites of inflammation and tissue damage are potent regulators of endothelial barrier function. Oxygenated epoxyisoprostane-containing phospholipids, but not fragmented oxidized phospholipids, exhibited barrier-protective effects mediated by small GTPases Cdc42 and Rac and their cytoskeletal, focal adhesion, and adherens junction effector proteins. Oxidized phospholipid-induced cytoskeletal rearrangements resulted in a unique peripheral actin rim formation, which was mimicked by coexpression of constitutively active Cdc42 and Rac, and abolished by coexpression of dominant-negative Rac and Cdc42. Thus, oxidative modification of phospholipids during inflammation leads to the formation of novel regulators that may be critically involved in restoration of vascular barrier function.

Butylated Hydroxytoluene↗
PubMed2004

Differential regulation of human lung epithelial and endothelial barrier function by thrombin.

Lung epithelial and endothelial barrier dysfunction is critical to the physiologic derangement observed in acute lung injury, but remains poorly understood. We utilized human alveolar epithelial (A549) and endothelial cells (EC) to study cytoskeletal remodeling, myosin light chain (MLC) phosphorylation and barrier regulation evoked by the edemagenic agent, thrombin. Thrombin-challenged human EC monolayers demonstrated increased MLC phosphorylation, actin stress fiber formation and loss of barrier integrity reflected by decreased transmonolayer electrical resistance (TER). In contrast, thrombin produced prominent circumferential localization of actin fibers, increased MLC phosphorylation and increased TER across epithelial monolayers, consistent with barrier protection. Reductions in MLC phosphorylation induced by cell pretreatment with pharmacological inhibitors of MLC kinase (ML-7) and Rho kinase (Y-27632) significantly attenuated thrombin-mediated TER changes and MLC phosphorylation in both lung cell types. Thrombin-produced, time-dependent activation of Rho GTPase in both epithelial and EC, whereas Rac GTPase activation was observed only in A549 cells. Molecular inhibition of Rac activity by adenoviral transfer of dominant-negative Rac mutant abolished thrombin-induced TER increases in alveolar epithelial cells. Finally, A549 cells, but not endothelium, demonstrated increased levels of tight junction proteins (ZO-1 and occludin) after thrombin at the cell-cell interface areas linked to thrombin-elicited barrier protection. These results demonstrate differential pulmonary endothelial and alveolar epithelial barrier regulation via unique actomyosin remodeling and cytoskeletal interactions with tight junction complexes, which confer selective barrier responses to edemagenic stimuli.

Actins↗
PubMed2004

Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase.

We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P.

Actins↗
PubMed2004

Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms.

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.

Actin Cytoskeleton↗