Phorbol ester-mediated pulmonary artery endothelial barrier dysfunction through regulation of actin cytoskeletal mechanics

The mechanisms of phorbol ester- and thrombin-mediated pulmonary artery endothelial barrier dysfunction were compared. Phorbol ester dibutyrate (PDBU) mediated slow force velocity and less force than thrombin. Taxol did not attenuate PDBU-mediated tension, while it reversed nocodazole-mediated tension. PDBU-mediated tension was not affected by acrylamide; PDBU increased cell stiffness and produced greater declines in transendothelial resistance (TER) than acrylamide. Thus PDBU caused a net increase in tension and did not unload microtubule or intermediate filaments. Microfilament remodeling, determined on the basis of immunocytochemistry and actin solubility, lacked the sensitivity and specificity to predict actin-dependent mechanical properties. Thrombin increased myosin light chain (MLC) kinase site-specific MLC phosphorylation, according to peptide map analysis, whereas PDBU did not increase PKC-specific MLC phosphorylation. The initial PDBU-mediated tension development temporally correlated with PDBU-mediated decline in TER and increased low-molecular-weight caldesmon ( l-CaD) phosphorylation. PDBU-mediated tension development and decreases in TER were associated with a temporal loss of endothelial cell-matrix adhesion, based on a numerical model of TER. Although, on the basis of immunocytochemistry, thrombin-mediated tension was associated with actin insolubility, actin reorganization, and gap formation, these changes did not predict thrombin-mediated gap formation, based on TER and time-lapse differential interference contrast microscopy. These data suggest that PDBU may disrupt endothelial barrier function through loss of cell-matrix adhesion through l-CaD-dependent actin contraction.

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Barrier dysfunction and RhoA activation are blunted by homocysteine and adenosine in pulmonary endothelium

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00421.2003 ◽

2004 ◽

Vol 287 (6) ◽

pp. L1091-L1097 ◽

Cited By ~ 22

Author(s):

Elizabeth O. Harrington ◽

Julie Newton ◽

Nicole Morin ◽

Sharon Rounds

Keyword(s):

Endothelial Cells ◽

Pulmonary Artery ◽

Barrier Function ◽

Main Pulmonary Artery ◽

Endothelial Barrier ◽

Gap Formation ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Rhoa Activation

RhoA GTPases modulate endothelial permeability. We have previously shown that adenosine and homocysteine enhance basal barrier function in pulmonary artery endothelial cells by a mechanism involving diminution of RhoA carboxyl methylation and activity. In the current study, we investigated the effects of adenosine and homocysteine on endothelial monolayer permeability in cultured monolayers. Adenosine and homocysteine significantly attenuated thrombin-induced endothelial barrier dysfunction and intercellular gap formation. We found significantly diminished RhoA associated with the membrane subcellular fraction in endothelial cells pretreated with adenosine and homocysteine, compared with vehicle-treated endothelial cells. Additionally, adenosine and homocysteine significantly blunted RhoA activation following thrombin exposure. Incubation with adenosine and homocysteine also enhanced in vitro interactions between RhoA and RhoGDI, as well as subcellular translocation of p190RhoGAP to the cytosol. These data demonstrate that elevated intracellular concentrations of homocysteine and adenosine enhance endothelial barrier function in cultured endothelial cells isolated from the main pulmonary artery and lung microvasculature, suggesting a potentially protective effect against pulmonary edema in response to lung injury. We speculate that homocysteine and adenosine modulate the level of endothelial barrier dysfunction through modulation of RhoA posttranslational processing resulting in diminished GTPase activity through altered interactions with modulators of RhoA activation.

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Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: Concurrent F-actin depolymerization and new actin synthesis

Journal of Cellular Physiology ◽

10.1002/jcp.1041570103 ◽

1993 ◽

Vol 157 (1) ◽

pp. 13-23 ◽

Cited By ~ 75

Author(s):

Simeon E. Goldblum ◽

Xueda Ding ◽

Terrance W. Brann ◽

Judith Campbell-Washington

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Bacterial Lipopolysaccharide ◽

Endothelial Barrier ◽

Vascular Endothelial ◽

Gap Formation ◽

Barrier Dysfunction ◽

Actin Reorganization ◽

Actin Depolymerization ◽

Endothelial Barrier Dysfunction

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Phytoestrogen Genistein Protects Against Endothelial Barrier Dysfunction in Vascular Endothelial Cells Through PKA-Mediated Suppression of RhoA Signaling

Endocrinology ◽

10.1210/en.2012-1774 ◽

2012 ◽

Vol 154 (2) ◽

pp. 727-737 ◽

Cited By ~ 5

Author(s):

Zhenquan Jia ◽

Wei Zhen ◽

Pon Velayutham Anandh Babu ◽

Dongmin Liu

Keyword(s):

Endothelial Cells ◽

Protective Effect ◽

Vascular Function ◽

Vascular Endothelial Cells ◽

Small Gtpase ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Mlc Phosphorylation ◽

Rhoa Signaling

The soy-derived phytoestrogen genistein has received attention for its potential to improve vascular function, but its mechanism remains unclear. Here, we report that genistein at physiologically relevant concentrations (0.1–10 μM) significantly inhibited thrombin-induced increase in endothelial monolayer permeability. Genistein also reduced the formation of stress fibers by thrombin and suppressed thrombin-induced phosphorylation of myosin light chain (MLC) on Ser19/Thr18 in endothelial cells (ECs). Genistein had no effect on resting intracellular [Ca2+] or thrombin-induced increase in Ca2+ mobilization. Addition of the inhibitors of endothelial nitric oxide synthase or estrogen receptor did not alter the protective effect of genistein. RhoA is a small GTPase that plays an important role in actin-myosin contraction and endothelial barrier dysfunction. RhoA inhibitor blocked the protective effect of genistein on endothelial permeability and also ablated thrombin-induced MLC-phosphorylation in ECs. Inhibition of PKA significantly attenuated the effect of genistein on thrombin-induced EC permeability, MLC phosphorylation, and RhoA membrane translocation in ECs. Furthermore, thrombin diminished cAMP production in ECs, which were prevented by treatment with genistein. These findings demonstrated that genistein improves thrombin-induced endothelial barrier dysfunction in ECs through PKA-mediated suppression of RhoA signaling.

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Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00417.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. L686-L697 ◽

Cited By ~ 24

Author(s):

Otgonchimeg Rentsendorj ◽

Tamara Mirzapoiazova ◽

Djanybek Adyshev ◽

Laura E. Servinsky ◽

Thomas Renné ◽

...

Keyword(s):

Protein Kinase ◽

Pulmonary Artery ◽

Actin Polymerization ◽

Cell Junctions ◽

Endothelial Barrier ◽

Protective Effects ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Human Pulmonary Artery

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKGI). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKGI barrier protection because VASP is a cytoskeletal phosphorylation target of PKGI expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKGI-mediated phosphorylation of Ser157 and Ser239. To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H2O2 in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKGI expression introduced by adenoviral infection (Ad.PKG). In the absence of PKGI expression, H2O2 (100–250 μM) caused a transient increased permeability and pSer157-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser157 phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H2O2. With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H2O2-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated PKGI. Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKGI to attenuate H2O2-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKGI on H2O2-induced endothelial barrier dysfunction in HPAEC.

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Endotoxin induces endothelial barrier dysfunction through protein tyrosine phosphorylation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.1.l217 ◽

1997 ◽

Vol 273 (1) ◽

pp. L217-L226 ◽

Cited By ~ 10

Author(s):

D. D. Bannerman ◽

S. E. Goldblum

Keyword(s):

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Endothelial Barrier ◽

Protein Tyrosine Phosphorylation ◽

Gap Formation ◽

Barrier Dysfunction ◽

Protein Tyrosine ◽

Actin Depolymerization ◽

Endothelial Barrier Dysfunction

Bacterial lipopolysaccharide (LPS) induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in vitro. We studied whether LPS-induced increments in 14C-labeled bovine serum albumin (BSA) flux across bovine pulmonary artery endothelial cell (EC) monolayers and actin depolymerization are mediated through protein tyrosine phosphorylation. Lysates from EC exposed to LPS derived from Escherichia coli 0111:B4 (100 ng/ml, 1 h) demonstrated increased tyrosine phosphorylation of the cytoskeletal protein paxillin. Protein tyrosine kinase inhibition, with either herbimycin A (1 microM) or genistein (50 micrograms/ml), protected against LPS-induced actin depolymerization, intercellular gap formation, and increments in [14C]BSA flux. In contrast, inhibition of tyrosine phosphatases with sodium orthovanadate (2.5 microM) or phenylarsine oxide (0.1 microM) enhanced the LPS-induced increments in the G-actin pool and the transendothelial flux of [14C]BSA compared with that seen after exposure to LPS alone. Our data indicate that the influence of LPS on EC actin organization and barrier function is mediated, in part, through a signaling pathway that is dependent on tyrosine phosphorylation.

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