Konstantin G. Birukov

Stretch affects phenotype and proliferation of vascular smooth muscle cells

The exertion of periodic dynamic strain on the arterial wall is hypothesized to be relevant to smooth muscle cell morphology and function. This study has investigated the effect of cyclic mechanical stretching on rabbit aortic smooth muscle cell proliferation and expression of contractile phenotype protein markers. Cells were cultured on flexible-bottomed dishes and cyclic stretch was applied (frequency 30 cycles/min, 15% elongation) using a Flexercell Strain unit. Cyclic stretch potentiated smooth muscle cell proliferation in serum-activated cultures but not in cultures maintained in 0.5% fetal calf serum. Stretching induced a serum-independent increase of h-caldesmon expression and this effect was reversible following termination of mechanical stimulation. Strain was without effect on smooth muscle myosin or calponin expression. In cells grown on laminin stretch-induced h-caldesmon expression was more prominent than in cells cultured on collagen types I and IV, poly-L-lysine and gelatin. These data suggest that cyclic mechanical stimulation possesses dual effect on vascular smooth muscle cell phenotype characteristics since it: 1) potentiates proliferation, an attribute of a dedifferentiated phenotype; and 2) increases expression of h-caldesmon considered a marker of a differentiated smooth muscle cell state.

Novel role of microtubules in thrombin-induced endothelial barrier dysfunction

Disturbances in endothelial cell (EC) barrier regulation are critically dependent upon rearrangements of EC actin cytoskeleton. However, the role of microtubule (MT) network in the regulation of EC permeability is not well understood. We examined involvement of MT remodeling in thrombin‐induced EC permeability and explored MT regulation by heterotrimeric G12/13 proteins and by small GTPase Rho. Thrombin induced phosphorylation of MT regulatory protein tau at Ser409 and Ser262 and peripheral MT disassembly, which was linked to increased EC permeability. MT stabilization by taxol attenuated thrombin‐ induced permeability, actin remodeling, and paracellular gap formation and diminished thrombin‐induced activation of Rho and Rho‐kinase. Expression of activated Gα12/13 subunits involved in thrombin‐mediated signaling or their effector p115RhoGEF involved in Rho activation caused MT disassembly, whereas p115RhoGEF‐specific negative regulator RGS preserved MT from thrombin‐induced disassembly. Consistent with these results, expression of activated RhoA and Rho‐kinase induced MT disassembly. Conversely, thrombin‐induced disassembly of peripheral MT network was attenuated by expression of dominant negative RhoA and Rho‐kinase mutants or by pharmacological inhibition of Rho‐kinase. Collectively, our data demonstrate for the first time a critical involvement of MT disassembly in thrombin‐induced EC barrier dysfunction and indicate G‐protein‐dependent mechanisms of thrombin‐induced MT alteration.—Birukova, A. A., Birukov, K G., Smurova, K., Adyshev, D., Kaibuchi, K., Alieva, I., Garcia, J. G. N., Verin, A. D. Novel role of microtubules in thrombin‐induced endothelial barrier dysfunction. FASEB J. 18, 1879‐1890 (2004)

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 Thr696 and Thr850 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. J. Cell. Physiol. 201: 55–70, 2004.

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

The Ca2+/calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210–214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436–505) that contains potentially important consensus sites for phosphorylation by p60Src kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256–267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction ofV max, and a 2-fold increase inK 0.5 CaM when compared with the SM MLCK isoform, whereas K m was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60 Src in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK). This increased activity of phospho-MLCK-1 was observed over a broad range of submaximal [Ca2+] levels with comparable EC50 [Ca2+] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60Src are Tyr464 and Tyr471 within the 69-residue stretch deleted in the MLCK-2 splice variant. These results demonstrate for the first time that p60Src-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of nonmuscle MLCK and vascular cell function.

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.

Involvement of site-specific FAK phosphorylation in sphingosine-1 phosphate- and thrombin-induced focal adhesion remodeling: role of Src and GIT

Sphingosine‐1 phosphate (S1P) and thrombin are agents with profound but divergent ef‐ fects on vascular endothelial cell (EC) barrier proper‐ ties. We have previously reported that S1P‐induced focal adhesion (FA) remodeling involves interactions between focal adhesion kinase (FAK), paxillin, and G‐protein‐coupled receptor kinase‐interacting proteins GIT1 and GIT2 and suggested a critical involvement of focal adhesions in the EC barrier regulation. In this study, we examined redistribution of FA proteins (FAK, paxillin, GIT1, and GIT2) and site‐specific FAK ty‐ rosine phosphorylation in human pulmonary artery endothelial cells stimulated with thrombin. In contrast to S1P, which we have shown to induce peripheral translocation of FA proteins associated with cortical actin ring formation, thrombin caused the redistribution of FA proteins to the ends of the newly formed massive stress fibers. S1P and thrombin induced dis‐ tinct patterns of FAK site‐specific phosphorylation with the FAK Y576 phosphorylation site targeted by SIP challenge and phosphorylation of three FAK sites (Y397, Y576, and Y925) in response to thrombin stimulation. Pharmacological inhibition of Src with Src‐specific in‐ hibitor PP2 abolished S1P‐induced translocation of FA proteins, cortical actin ring formation, and FAK [Y576] phosphorylation. However, PP2 failed to alter throm‐ bin‐induced morphological changes and exhibited only partial inhibition of FAK site‐specific tyrosine phos‐ phorylation. These observations highlight the differen‐ tial mechanisms of focal adhesion protein complex remodeling and FAK activation by S1P and thrombin and link differential FA remodeling to EC barrier regulation.—Shikata, Y., Birukov, K. G., Birukova, A. A., Verin, A., Garcia, J. G. N. Involvement of site‐specific FAK phosphorylation in sphingosine‐1 phosphate‐ and thrombin‐induced focal adhesion re‐ modeling: role of Src and GIT. FASEB J. 17, 2240‐2249 (2003)

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. J. Cell. Physiol. 215: 715–724, 2008.

Role of Phospholipid Oxidation Products in Atherosclerosis

There is increasing clinical evidence that phospholipid oxidation products (Ox-PL) play a role in atherosclerosis. This review focuses on the mechanisms by which Ox-PL interact with endothelial cells, monocyte/macrophages, platelets, smooth muscle cells, and HDL to promote atherogenesis. In the past few years major progress has been made in identifying these mechanisms. It has been recognized that Ox-PL promote phenotypic changes in these cell types that have long-term consequences for the vessel wall. Individual Ox-PL responsible for specific cellular effects have been identified. A model of the configuration of bioactive truncated Ox-PL within membranes has been developed that demonstrates that the oxidized fatty acid moiety protrudes into the aqueous phase, rendering it accessible for receptor recognition. Receptors and signaling pathways for individual Ox-PL species are now determined and receptor independent signaling pathways identified. The effects of Ox-PL are mediated both by gene regulation and transcription independent processes. It has now become apparent that Ox-PL affects multiple genes and pathways, some of which are proatherogenic and some are protective. However, at concentrations that are likely present in the vessel wall in atherosclerotic lesions, the effects promote atherogenesis. There have also been new insights on enzymes that metabolize Ox-PL and the significance of these enzymes for atherosclerosis. With the knowledge we now have of the regulation and effects of Ox-PL in different vascular cell types, it should be possible to design experiments to test the role of specific Ox-PL on the development of atherosclerosis.

HGF attenuates thrombin-induced endothelial permeability by Tiam1-mediated activation of the Rac pathway and by Tiam1/Rac-dependent inhibition of the Rho pathway

Reorganization of the endothelial cell (EC) cytoskeleton and cell adhesive complexes provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases, including asthma, sepsis, and acute respiratory distress syndrome (ARDS). We have recently described the barrier‐protective effects of hepatocyte growth factor (HGF) on the human pulmonary EC. In the present study, we explored the involvement of Rac‐GTPase and Rac‐specific nucleotide exchange factor Tiam1 in the mechanisms of EC barrier protection by HGF. HGF protected EC monolayers from thrombin‐induced hy‐perpermeability, disruption of intercellular junctions, and formation of stress fibers and paracellular gaps by inhibiting thrombin‐induced activation of Rho GTPase, Rho association with nucleotide exchange factor p115‐RhoGEF, and myosin light chain phosphorylation, which was opposed by stimulation of Rac‐dependent signaling. The pharmacological Rac inhibitor or silencing RNA (siRNA) based depletion of either Rac or Tiam1 significantly attenuated HGF‐induced peripheral translocation of Rac effector cortactin, cortical actin ring formation, and EC barrier enhancement. Moreover, Tiam1 knockdown using the siRNA approach, attenuated the protective effect of HGF against throm‐bin‐induced activation of Rho signaling, monolayer disruption, and EC hyperpermeability. This study demonstrates the Tiam1/Rac‐dependent mechanism of HGF‐induced EC barrier protection and provides novel mechanistic insights into regulation of EC permeabilityvia dynamic interactions between Rho‐ and Tiam1/Rac‐medi‐ated pathways.—Birukova, A., Alekseeva, E., Mikaelyan, A., and Birukov, K. G. HGF attenuates thrombin‐induced endothelial permeability by Tiam1‐mediated activation of the Rac pathway and by Tiam1/Rac‐dependent inhibition of the Rho pathway. FASEB J. 21, 2776–2786 (2007)

Involvement of microtubules and Rho pathway in TGF‐β1‐induced lung vascular barrier dysfunction

Transforming growth factor‐β1 (TGF‐β1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF‐β1‐mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF‐β1‐induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF‐β1 (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‐β1 receptor kinase RI (5 μM) abolished TGF‐β1‐induced TER decline, whereas inhibitor of caspase‐3 zVAD (10 μM) was without effect. TGF‐β1‐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 μM) attenuated TGF‐β1‐induced barrier dysfunction and actin remodeling. TGF‐β1 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 μM) attenuated TGF‐β1‐induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF‐β1‐mediated Rho regulation, EC barrier dysfunction, and actin remodeling.