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Protein kinase C-? and arginase I mediate pneumolysin-induced pulmonary endothelial hyperpermeability.

ABSTRACT: Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-? activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-? inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-? activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-? and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.

SUBMITTER: Lucas R

PROVIDER: S-EPMC3488628 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Protein kinase C-α and arginase I mediate pneumolysin-induced pulmonary endothelial hyperpermeability.

Lucas Rudolf R Yang Guang G Gorshkov Boris A BA Zemskov Evgeny A EA Sridhar Supriya S Umapathy Nagavedi S NS Jezierska-Drutel Agnieszka A Alieva Irina B IB Leustik Martin M Hossain Hamid H Fischer Bernhard B Catravas John D JD Verin Alexander D AD Pittet Jean-François JF Caldwell Ruth B RB Mitchell Timothy J TJ Cederbaum Stephen D SD Fulton David J DJ Matthay Michael A MA Caldwell Robert W RW Romero Maritza J MJ Chakraborty Trinad T

American journal of respiratory cell and molecular biology 20120510 4

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial ce ...[more]

PMID: 22582175

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Project description:ObjectivesWe previously reported microvascular leakage resulting from fibrinogen-? chain C-terminal products (?C) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which ?C induces endothelial hyperpermeability. Since it is known that ?C binds and activates endothelial ?v?3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that ?C alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation.Methods and resultsUsing intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from ?C administration. In addition, capillary fluid filtration coefficient (Kfc) indicated ?C-induced elevated lung vascular permeability. Furthermore, ?C decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced ?C-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved ?C-induced endothelial barrier dysfunction, indicating the involvement of FAK in ?C mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in ?C-mediated hyperpermeability. In addition, ?C-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition.ConclusionThe FAK-Src pathway contributes to ?C-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.

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Protein kinase C-? and arginase I mediate pneumolysin-induced pulmonary endothelial hyperpermeability.

Publications

Protein kinase C-α and arginase I mediate pneumolysin-induced pulmonary endothelial hyperpermeability.

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