scholarly journals Mechanisms of sodium fluoride-induced endothelial cell barrier dysfunction: role of MLC phosphorylation

2001 ◽  
Vol 281 (6) ◽  
pp. L1472-L1483 ◽  
Author(s):  
Peiyi Wang ◽  
Alexander D. Verin ◽  
Anna Birukova ◽  
Lydia I. Gilbert-McClain ◽  
Keri Jacobs ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Phosphatase Activity ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Actin Stress Fiber ◽  
Gap Formation ◽  
Barrier Dysfunction ◽  
Transient Activation ◽  
Mlc Phosphorylation

NaF, a potent G protein activator and Ser/Thr phosphatase inhibitor, significantly increased albumin permeability and decreased transcellular electrical resistance (TER), indicating endothelial cell (EC) barrier impairment. EC barrier dysfunction induced by NaF was accompanied by the development of actin stress fibers, intercellular gap formation, and significant time-dependent increases in myosin light chain (MLC) phosphorylation. However, despite rapid, albeit transient, activation of Ca2+/calmodulin-dependent MLC kinase (MLCK), the specific MLCK inhibitor ML-7 failed to affect NaF-induced MLC phosphorylation, actin cytoskeletal rearrangement, and reductions in TER, suggesting a limited role of MLCK in NaF-induced EC activation. In contrast, strategies to reduce Rho (C3 exoenzyme or toxin B) or to inhibit Rho-associated kinase (Y-27632 or dominant/negative RhoK) dramatically reduced MLC phosphorylation and actin stress fiber formation and significantly attenuated NaF-induced EC barrier dysfunction. Consistent with this role for RhoK activity, NaF selectively inhibited myosin-specific phosphatase activity, whereas the total Ser/Thr phosphatase activity remained unchanged. These data strongly suggest that MLC phosphorylation, mediated primarily by RhoK, and not MLCK, participates in NaF-induced EC actin cytoskeletal changes and barrier dysfunction.

Differential effect of MLC kinase in TNF-α-induced endothelial cell apoptosis and barrier dysfunction

2001 ◽  
Vol 280 (6) ◽  
pp. L1168-L1178 ◽  
Author(s):  
Irina Petrache ◽  
Alexander D. Verin ◽  
Michael T. Crow ◽  
Anna Birukova ◽  
Feng Liu ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Apoptosis ◽  
Electrical Resistance ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Endothelial Cell Apoptosis ◽  
Gap Formation ◽  
Barrier Dysfunction ◽  
Tnf Α ◽  
Mlc Phosphorylation

Tumor necrosis factor (TNF)-α is released in acute inflammatory lung syndromes linked to the extensive vascular dysfunction associated with increased permeability and endothelial cell apoptosis. TNF-α induced significant decreases in transcellular electrical resistance across pulmonary endothelial cell monolayers, reflecting vascular barrier dysfunction (beginning at 4 h and persisting for 48 h). TNF-α also triggered endothelial cell apoptosis beginning at 4 h, which was attenuated by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Exploring the involvement of the actomyosin cytoskeleton in these important endothelial cell responses, we determined that TNF-α significantly increased myosin light chain (MLC) phosphorylation, with prominent stress fiber and paracellular gap formation, which paralleled the onset of decreases in transcellular electrical resistance and enhanced apoptosis. Reductions in MLC phosphorylation by the inhibition of either MLC kinase (ML-7, cholera toxin) or Rho kinase (Y-27632) dramatically attenuated TNF-α-induced stress fiber formation, indexes of apoptosis, and caspase-8 activity but not TNF-α-induced barrier dysfunction. These studies indicate a central role for the endothelial cell cytoskeleton in TNF-α-mediated apoptosis, whereas TNF-α-induced vascular permeability appears to evolve independently of contractile tension generation.

scholarly journals Microtubule disassembly increases endothelial cell barrier dysfunction: role of MLC phosphorylation

2001 ◽  
Vol 281 (3) ◽  
pp. L565-L574 ◽  
Author(s):  
Alexander D. Verin ◽  
Anna Birukova ◽  
Peiyi Wang ◽  
Feng Liu ◽  
Patrice Becker ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Electrical Resistance ◽  
Rho Gtpase ◽  
Dependent Manner ◽  
Transendothelial Electrical Resistance ◽  
Barrier Dysfunction ◽  
Microtubule Disruption ◽  
Barrier Regulation ◽  
Mlc Phosphorylation

Endothelial cell (EC) barrier regulation is critically dependent on cytoskeletal components (microfilaments and microtubules). Because several edemagenic agents induce actomyosin-driven EC contraction tightly linked to myosin light chain (MLC) phosphorylation and microfilament reorganization, we examined the role of microtubule components in bovine EC barrier regulation. Nocodazole or vinblastine, inhibitors of microtubule polymerization, significantly decreased transendothelial electrical resistance in a dose-dependent manner, whereas pretreatment with the microtubule stabilizer paclitaxel significantly attenuated this effect. Decreases in transendothelial electrical resistance induced by microtubule disruption correlated with increases in lung permeability in isolated ferret lung preparations as well as with increases in EC stress fiber content and MLC phosphorylation. The increases in MLC phosphorylation were attributed to decreases in myosin-specific phosphatase activity without significant increases in MLC kinase activity and were attenuated by paclitaxel or by several strategies (C3 exotoxin, toxin B, Rho kinase inhibition) to inhibit Rho GTPase. Together, these results suggest that microtubule disruption initiates specific signaling pathways that cross talk with microfilament networks, resulting in Rho-mediated EC contractility and barrier dysfunction.

scholarly journals Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction

2009 ◽  
Vol 297 (1) ◽  
pp. L73-L83 ◽  
Author(s):  
Nutan Prasain ◽  
Mikhail Alexeyev ◽  
Ron Balczon ◽  
Troy Stevens
Keyword(s):  
Endothelial Cell ◽  
Adenylyl Cyclase ◽  
Fluorescent Protein ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Adenylyl Cyclases ◽  
Gap Formation ◽  
Soluble Adenylyl Cyclase ◽  
Cell Retraction

Soluble adenylyl cyclase toxins, such as Pseudomonas aeruginosa exoY, generate a cAMP pool that retracts cell borders. However, the cytoskeletal basis by which this cAMP signal retracts cell borders is not known. We sought to determine whether activation of chimeric, soluble adenylyl cyclase I/II (sACI/II) reorganizes either microtubules or peripheral actin. Endothelial cells were stably transfected with either green fluorescent protein-labeled α-tubulin or β-actin, and then infected with adenovirus to express sACI/II. Forskolin, which stimulates both the endogenously expressed transmembrane adenylyl cyclases and sACI/II, induced cell retraction accompanied by the reorganization of peripheral microtubules. However, cortical filamentous-actin (f-actin) did not reorganize into stress fibers, and myosin light-chain-20 phosphorylation was decreased. Isoproterenol, which activates endogenous adenylyl cyclases but does not activate sACI/II, did not induce endothelial cell gaps and did not influence microtubule or f-actin architecture. Thus, sACI/II generates a cAMP signal that reorganizes microtubules and induces cell retraction, without inducing f-actin stress fibers. These findings illustrate that endothelial cell gap formation can proceed without f-actin stress fiber formation, and provide mechanistic insight how bacterial adenylyl cyclase toxins reorganize the cytoskeleton to induce cell rounding.

Lung endothelial heparan sulfates mediate cationic peptide-induced barrier dysfunction: a new role for the glycocalyx

2003 ◽  
Vol 285 (5) ◽  
pp. L986-L995 ◽  
Author(s):  
Randal O. Dull ◽  
Ramani Dinavahi ◽  
Lawrence Schwartz ◽  
Donald E. Humphries ◽  
David Berry ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
P38 Mapk ◽  
Barrier Function ◽  
Fiber Formation ◽  
Cell Permeability ◽  
Endothelial Glycocalyx ◽  
Microvascular Endothelial Cell ◽  
Actin Stress Fiber ◽  
Cationic Peptide ◽  
Barrier Dysfunction

The endothelial glycocalyx is believed to play a major role in microvascular permeability. We tested the hypothesis that specific components of the glycocalyx, via cytoskeletal-mediated signaling, actively participate in barrier regulation. With the use of polymers of arginine and lysine as a model of neutrophil-derived inflammatory cationic proteins, we determined size- and dose-dependent responses of cultured bovine lung microvascular endothelial cell permeability as assessed by transendothelial electrical resistance (TER). Polymers of arginine and lysine >11 kDa produced maximal barrier dysfunction as demonstrated by a 70% decrease in TER. Monomers of l-arginine and l-lysine did not alter barrier function, suggesting a cross-linking requirement of cell surface “receptors”. To test the hypothesis that glycosaminoglycans (GAGs) are candidate receptors for this response, we used highly selective enzymes to remove specific GAGs before polyarginine (PA) treatment and examined the effect on TER. Heparinase III attenuated PA-induced barrier dysfunction by 50%, whereas heparinase I had no effect. To link changes in barrier function with structural alterations, we examined actin organization and syndecan localization after PA. PA induced actin stress fiber formation and clustering of syndecan-1 and syndecan-4, which were significantly attenuated by heparinase III. PA-induced cytoskeletal rearrangement and barrier function did not involve myosin light chain kinase (MLCK) or p38 MAPK, as ML-7, a specific MLCK inhibitor, or SB-20358, a p38 MAPK inhibitor, did not alter PA-induced barrier dysfunction. In summary, lung endothelial cell heparan sulfate proteoglycans are key participants in inflammatory cationic peptide-induced signaling that links cytoskeletal reorganization with subsequent barrier dysfunction.

Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction

2000 ◽  
Vol 279 (3) ◽  
pp. L441-L451 ◽  
Author(s):  
Shu Shi ◽  
Joe G. N. Garcia ◽  
Shukla Roy ◽  
Narasimham L. Parinandi ◽  
Viswanathan Natarajan
Keyword(s):  
Endothelial Cell ◽  
Barrier Function ◽  
Kinase Inhibitors ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Barrier Dysfunction ◽  
Transient Overexpression ◽  
Dose Dependent

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.

Role of protein kinase G in barrier-protective effects of cGMP in human pulmonary artery endothelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. L919-L930 ◽  
Author(s):  
Aigul Moldobaeva ◽  
Laura E. Welsh-Servinsky ◽  
Larissa A. Shimoda ◽  
R. Scott Stephens ◽  
Alexander D. Verin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Pulmonary Artery ◽  
Recombinant Adenovirus ◽  
Fiber Formation ◽  
Independent Effect ◽  
Actin Stress Fiber ◽  
Barrier Dysfunction ◽  
Human Pulmonary Artery

Increases in endothelial cGMP prevent oxidant-mediated endothelial barrier dysfunction, but the downstream mechanisms remain unclear. To determine the role of cGMP-dependent protein kinase (PKG)I, human pulmonary artery endothelial cells (HPAEC) lacking PKGI expression were infected with a recombinant adenovirus encoding PKGIβ (Ad.PKG) and compared with uninfected and control-infected (Ad.βgal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H2O2 (250 μM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.βgal, expressed PKGI protein and demonstrated Ser239 and Ser157 phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.βgal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 μM) caused a PKGI-independent decrease in permeability (8.2 ± 0.6%). In all three groups, H2O2 (250 μM) caused a similar ∼35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca2+ concentration ([Ca2+]i). In uninfected and Ad.βgal-infected HPAEC, pretreatment with CPT-cGMP (100 μM) partially blocked the increased permeability induced by H2O2. In Ad.PKG-infected HPAEC, CPT-cGMP (50 μM) prevented the H2O2-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca2+]i caused by H2O2 similarly (23%) in Ad.βgal- and Ad.PKG-infected HPAEC, indicating a PKGI-independent effect. These data suggest that cGMP decreased HPAEC basal permeability by a PKGI-independent process, whereas the ability of cGMP to prevent H2O2-induced barrier dysfunction was predominantly mediated by PKGI through a Ca2+-independent mechanism.

scholarly journals Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin

2013 ◽  
Vol 305 (3) ◽  
pp. L240-L255 ◽  
Author(s):  
Djanybek M. Adyshev ◽  
Steven M. Dudek ◽  
Nurgul Moldobaeva ◽  
Kyung-mi Kim ◽  
Shwu-Fan Ma ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fiber Formation ◽  
Actin Binding ◽  
Actin Stress Fiber ◽  
Gap Formation ◽  
Barrier Dysfunction ◽  
Protease Activated Receptors ◽  
Erm Proteins ◽  
Threonine Residue ◽  
Sphingosine 1 Phosphate

Endothelial cell (EC) barrier disruption induced by inflammatory agonists such as thrombin leads to potentially lethal physiological dysfunction such as alveolar flooding, hypoxemia, and pulmonary edema. Thrombin stimulates paracellular gap and F-actin stress fiber formation, triggers actomyosin contraction, and alters EC permeability through multiple mechanisms that include protein kinase C (PKC) activation. We previously have shown that the ezrin, radixin, and moesin (ERM) actin-binding proteins differentially participate in sphingosine-1 phosphate-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the COOH-terminus of ERM proteins causes conformational changes in ERM to unmask binding sites and is considered a hallmark of ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (ezrin Thr567, radixin Thr564, moesin Thr558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone or of all three ERM proteins significantly attenuates thrombin-induced increase in EC barrier permeability (transendothelial electrical resistance), cytoskeletal rearrangements, paracellular gap formation, and accumulation of phospho-myosin light chain. In contrast, radixin depletion exerts opposing effects on these indexes. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it.

scholarly journals Protective role of FKBP51 in calcium entry-induced endothelial barrier disruption

2017 ◽  
Vol 8 (1) ◽  
pp. 204589321774998 ◽  
Author(s):  
Caleb L. Hamilton ◽  
Pierre I. Kadeba ◽  
Audrey A. Vasauskas ◽  
Viktoriya Solodushko ◽  
Anna K. McClinton ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Endothelial Permeability ◽  
Protective Role ◽  
Calcium Entry ◽  
Endothelial Barrier ◽  
Protective Mechanism ◽  
Actin Stress Fiber ◽  
Gap Formation ◽  
Microtubule Network

Pulmonary artery endothelial cells (PAECs) express a cation current, ISOC (store-operated calcium entry current), which when activated permits calcium entry leading to inter-endothelial cell gap formation. The large molecular weight immunophilin FKBP51 inhibits ISOC but not other calcium entry pathways in PAECs. However, it is unknown whether FKBP51-mediated inhibition of ISOC is sufficient to protect the endothelial barrier from calcium entry-induced disruption. The major objective of this study was to determine whether FKBP51-mediated inhibition of ISOC leads to decreased calcium entry-induced inter-endothelial gap formation and thus preservation of the endothelial barrier. Here, we measured the effects of thapsigargin-induced ISOC on the endothelial barrier in control and FKBP51 overexpressing PAECs. FKBP51 overexpression decreased actin stress fiber and inter-endothelial cell gap formation in addition to attenuating the decrease in resistance observed with control cells using electric cell-substrate impedance sensing. Finally, the thapsigargin-induced increase in dextran flux was abolished in FKBP51 overexpressing PAECs. We then measured endothelial permeability in perfused lungs of FKBP51 knockout (FKBP51–/–) mice and observed increased calcium entry-induced permeability compared to wild-type mice. To begin to dissect the mechanism underlying the FKBP51-mediated inhibition of ISOC, a second goal of this study was to determine the role of the microtubule network. We observed that FKBP51 overexpressing PAECs exhibited increased microtubule polymerization that is critical for inhibition of ISOC by FKBP51. Overall, we have identified FKBP51 as a novel regulator of endothelial barrier integrity, and these findings are significant as they reveal a protective mechanism for endothelium against calcium entry-induced disruption.

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

2007 ◽  
Vol 293 (1) ◽  
pp. H366-H375 ◽  
Author(s):  
MaryEllen Carlile-Klusacek ◽  
Victor Rizzo
Keyword(s):  
Endothelial Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Signaling Cascade ◽  
Membrane Rafts ◽  
Actin Stress Fiber ◽  
Caveolin 1 ◽  
Stress Fiber Formation ◽  
Mlc Phosphorylation ◽  
Actin Stress Fiber Formation

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH2 terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 μg/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.

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