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.

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 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.

Role of the Na/H antiport in pH-dependent cell death in pulmonary artery endothelial cells

2000 ◽  
Vol 278 (3) ◽  
pp. L536-L544 ◽  
Author(s):  
M. Cutaia ◽  
K. Tollefson ◽  
J. Kroczynski ◽  
N. Parks ◽  
S. Rounds
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Pulmonary Artery ◽  
Phorbol Ester ◽  
Atp Depletion ◽  
Live Cells ◽  
Cell Death Pathway ◽  
Ph Dependent ◽  
Human Pulmonary Artery

We investigated the role of intracellular pH (pHi) and Na/H exchange in cell death in human pulmonary artery endothelial cells (HPAEC) following a metabolic insult (inhibition-oxidative phosphorylation, glycolysis). Metabolic inhibition in medium at pH 7.4 decreased viability (0–15% live cells) over 6 h. Cell death was attenuated by maneuvers that decreased pHi and inhibited Na/H exchange (acidosis, Na/H antiport inhibitors). In contrast, cell death was potentiated by maneuvers that elevated pHi or increased Na/H exchange (monensin, phorbol ester treatment) before the insult. HPAEC demonstrated a biphasic pHi response following a metabolic insult. An initial decrease in pHi was followed by a return to baseline over 60 min. Maneuvers that protected HPAEC and inhibited Na/H exchange (acidosis, Na+-free medium, antiport inhibitors) altered this pattern. pHi decreased, but no recovery was observed, suggesting that the return of pHi to normal was mediated by antiport activation. Although Na/H antiport activity was reduced (55–60% of control) following a metabolic insult, the cells still demonstrated active Na/H exchange despite significant ATP depletion. Phorbol ester pretreatment, which potentiated cell death, increased Na/H antiport activity above the level observed in monolayers subjected to a metabolic insult alone. These results demonstrate that HPAEC undergo a pH-dependent loss of viability linked to active Na/H exchange following a metabolic insult. Potentiation of cell death with phorbol ester treatment suggests that this cell death pathway involves protein kinase C-mediated phosphorylation events.

scholarly journals Inhibition of Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinase 2 (MK2) is Protective in Pulmonary Hypertension

Hypertension ◽  
2021 ◽  
Author(s):  
Mohammad Shafiq ◽  
Kumaravelu Jagavelu ◽  
Hina Iqbal ◽  
Pankaj Yadav ◽  
Debabrata Chanda ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Pulmonary Artery ◽  
Right Ventricle ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Pulmonary Artery Smooth Muscle ◽  
Human Pulmonary Artery

Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinase 2 (MK2), downstream to p38MAPK (p38mitogen-activated protein kinase), regulates cellular inflammation and proliferation. So far, the role of MK2 has been studied in many cardiovascular diseases, but it remains unexplored in pulmonary hypertension (PH). Therefore, to investigate the role of MK2 in the PH pathogenesis, human pulmonary artery smooth muscle cells were exposed to hypoxia (1% O 2 ) for 72 hours, and MK2 was inhibited by siRNA. We observed significantly increased MK2 expression, inflammatory cytokines, proliferation, mitochondrial dysfunction, and apoptosis resistance in hypoxic human pulmonary artery smooth muscle cells, which were reversed by treatment with MK2 siRNA. For in vivo studies, male Sprague Dawley rats were treated with monocrotaline (60 mg/kg, SC, once) to induce PH. To inhibit MK2, a peptide MMI-0100 (40 μg/kg, IP daily, 5 weeks for preventive and 3 weeks for curative study) was administered. MMI-0100 treatment decreased right ventricle pressure and hypertrophy, hallmarks of PH, in both preventive and curative study. MMI-0100-treated rats showed better cardiac functions as revealed by 2-dimensional echocardiography study. Furthermore, MMI-0100 reversed pulmonary vascular remodeling and improved pulmonary vascular relaxation in monocrotaline-treated rats. Finally, the above results were confirmed in MK2 knockout mice. MK2 knockout mice, received 600 mg/kg monocrotaline, subcutaneous weekly for 5 weeks, failed to develop PH and showed no increase in right ventricle pressure and hypertrophy. This study, therefore, proved that MK2 is involved in PH, and its inhibition may be a novel target for PH treatment.

Thrombin enhances the barrier function of rat microvascular endothelium in a PAR-1-dependent manner

2008 ◽  
Vol 294 (2) ◽  
pp. L266-L275 ◽  
Author(s):  
B. Troyanovsky ◽  
D. F. Alvarez ◽  
J. A. King ◽  
K. L. Schaphorst
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Barrier Function ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Gap Formation ◽  
Vascular Effects ◽  
Microvascular Endothelium

Thrombin is a multifunctional coagulation protease with pro- and anti-inflammatory vascular effects. We questioned whether thrombin may have segmentally differentiated effects on pulmonary endothelium. In cultured rat endothelial cells, rat thrombin (10 U/ml) recapitulated the previously reported decrease in transmonolayer electrical resistance (TER), F-actin stress fiber formation, paracellular gap formation, and increased permeability. In contrast, in rat pulmonary microvascular endothelial cells (PMVEC), isolated on the basis of Griffonia simplicifolia lectin recognition, thrombin increased TER, induced fewer stress fibers, and decreased permeability. To assess for differential proteinase-activated receptor (PAR) expression as a basis for the different responses, PAR family expression was analyzed. Both pulmonary artery endothelial cells and PMVEC expressed PAR-1 and PAR-2; however, only PMVEC expressed PAR-3, as shown by both RT-PCR and Western analysis. PAR-1 activating peptides (PAR-APs: SFLLRN-NH2and TFLLRN-NH2) were used to confirm a role for the PAR-1 receptor. PAR-APs (25–250 μM) also increased TER, formed fewer stress fibers, and did not induce paracellular gaps in PMVEC in contrast to that shown in pulmonary artery endothelial cells. These results were confirmed in isolated perfused rat lung preparations. PAR-APs (100 μg/ml) induced a 60% increase in the filtration coefficient over baseline. However, by transmission electron microscopy, perivascular fluid cuffs were seen only along conduit veins and arteries without evidence of intra-alveolar edema. We conclude that thrombin exerts a segmentally differentiated effect on endothelial barrier function in vitro, which corresponds to a pattern of predominant perivascular fluid cuff formation in situ. This may indicate a distinct role for thrombin in the microcirculation.

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