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

2008 ◽  
Vol 294 (4) ◽  
pp. L686-L697 ◽  
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.

scholarly journals Role of IQGAP1 in endothelial barrier enhancement caused by OxPAPC

2016 ◽  
Vol 311 (4) ◽  
pp. L800-L809 ◽  
Author(s):  
Yufeng Tian ◽  
Xinyong Tian ◽  
Grzegorz Gawlak ◽  
Nicolene Sarich ◽  
David B. Sacks ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Cell Junction ◽  
Protective Effects ◽  
P120 Catenin

Oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphatidylcholine (OxPAPC) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via enhancement of both the peripheral actin cytoskeleton and cell junctions mediated by Rac1 and Cdc42 GTPases. This study evaluated the role for the multifunctional Rac1/Cdc42 effector and regulator, IQ domain containing GTPase-activating protein (IQGAP1), as a molecular transducer of the OxPAPC-mediated EC barrier-enhancing signal. IQGAP1 knockdown in endothelial cells by gene-specific small-interfering RNA abolished OxPAPC-induced enlargement of VE-cadherin-positive adherens junctions, suppressed peripheral accumulation of actin polymerization regulators, namely cortactin, neural Wiskott-Aldrich syndrome protein (N-WASP), and actin-related protein 3, and attenuated remodeling of the peripheral actin cytoskeleton. Inhibition of OxPAPC-induced barrier enhancement by IQGAP1 knockdown was due to suppressed Rac1 and Cdc42 activation. Expression of an IQGAP1 truncated mutant showed that the GTPase regulatory domain of IQGAP1 was essential for the OxPAPC-induced membrane localization of cortactin, adherens junction proteins VE-cadherin and p120-catenin, as well as for EC permeability response. IQGAP1 knockdown attenuated the protective effect of OxPAPC against thrombin-induced cell contraction, cell junction disruption, and EC permeability. These results demonstrate for the first time the role of IQGAP1 as a critical transducer of OxPAPC-induced Rac1/Cdc42 signaling to the actin cytoskeleton and adherens junctions, which promotes cortical cytoskeletal remodeling and EC barrier-protective effects of oxidized phospholipids.

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.

PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction

2003 ◽  
Vol 284 (6) ◽  
pp. L972-L980 ◽  
Author(s):  
Jing Qiao ◽  
Fei Huang ◽  
Hazel Lum
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Rho Gtpases ◽  
Endothelial Barrier ◽  
Intracellular Camp ◽  
Protective Effects ◽  
Barrier Dysfunction ◽  
Similar Time ◽  
Endothelial Barrier Dysfunction ◽  
Rhoa Activation

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 μM forskolin plus 1 μM IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of32P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of ∼23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.

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

2004 ◽  
Vol 287 (1) ◽  
pp. L153-L167 ◽  
Author(s):  
Alan B. Moy ◽  
Ken Blackwell ◽  
Ning Wang ◽  
Kari Haxhinasto ◽  
Mary K. Kasiske ◽  
...  
Keyword(s):  
Pulmonary Artery ◽  
Phorbol Ester ◽  
Endothelial Barrier ◽  
Gap Formation ◽  
Barrier Dysfunction ◽  
Tension Development ◽  
Endothelial Barrier Dysfunction ◽  
Cell Matrix ◽  
Mlc Phosphorylation ◽  
Cell Matrix Adhesion

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.

scholarly journals Protein Kinase G and p38 MAPK in H 2 O 2 ‐induced pulmonary endothelial barrier dysfunction

2006 ◽  
Vol 20 (4) ◽  
Author(s):  
Otgonchimeg Rentsendorj ◽  
Laura E. Servinsky ◽  
Larissa A. Shimoda ◽  
Aigul Moldobaeva ◽  
Tamara Mirzapoiazova ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Endothelial Barrier ◽  
Protein Kinase G ◽  
Barrier Dysfunction ◽  
Endothelial Barrier Dysfunction

scholarly journals Atrial natriuretic peptide protects against Staphylococcus aureus-induced lung injury and endothelial barrier dysfunction

2011 ◽  
Vol 110 (1) ◽  
pp. 213-224 ◽  
Author(s):  
Junjie Xing ◽  
Nurgul Moldobaeva ◽  
Anna A. Birukova
Keyword(s):  
Staphylococcus Aureus ◽  
Lung Inflammation ◽  
Endothelial Barrier ◽  
Protective Effects ◽  
Barrier Dysfunction ◽  
Gram Positive ◽  
Gram Negative ◽  
Endothelial Barrier Dysfunction

Lung inflammation and alterations in endothelial cell (EC) permeability are key events to development of acute lung injury (ALI). Protective effects of atrial natriuretic peptide (ANP) have been shown against inflammatory signaling and endothelial barrier dysfunction induced by gram-negative bacterial wall liposaccharide. We hypothesized that ANP may possess more general protective effects and attenuate lung inflammation and EC barrier dysfunction by suppressing inflammatory cascades and barrier-disruptive mechanisms shared by gram-negative and gram-positive pathogens. C57BL/6J wild-type or ANP knockout mice (Nppa−/−) were treated with gram-positive bacterial cell wall compounds, Staphylococcus aureus-derived peptidoglycan (PepG) and/or lipoteichoic acid (LTA) (intratracheal, 2.5 mg/kg each), with or without ANP (intravenous, 2 μg/kg). In vitro, human pulmonary EC barrier properties were assessed by morphological analysis of gap formation and measurements of transendothelial electrical resistance. LTA and PepG markedly increased pulmonary EC permeability and activated p38 and ERK1/2 MAP kinases, NF-κB, and Rho/Rho kinase signaling. EC barrier dysfunction was further elevated upon combined LTA and PepG treatment, but abolished by ANP pretreatment. In vivo, LTA and PepG-induced accumulation of protein and cells in the bronchoalveolar lavage fluid, tissue neutrophil infiltration, and increased Evans blue extravasation in the lungs was significantly attenuated by intravenous injection of ANP. Accumulation of bronchoalveolar lavage markers of LTA/PepG-induced lung inflammation and barrier dysfunction was further augmented in ANP−/− mice and attenuated by exogenous ANP injection. These results strongly suggest a protective role of ANP in the in vitro and in vivo models of ALI associated with gram-positive infection. Thus ANP may have important implications in therapeutic strategies aimed at the treatment of sepsis and ALI-induced gram-positive bacterial pathogens.

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