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EETs reduces LPS-induced hyperpermeability by targeting GRP78 mediated Src activation and subsequent Rho/ROCK signaling pathway.

ABSTRACT: Integrity of endothelial barrier is a determinant of the prognosis in the acute lung injury caused by sepsis. The epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid, exhibit protective effects in various pathogenic states, however, whether EETs play a role in endothelial barrier enhancement and the involved mechanisms remain to be investigated. Here, we show that increased EETs level by endothelial specific cytochrome P450 epoxygenase 2J2 over-expression and soluble epoxide hydrolase (sEH) inhibitor TPPU reduced lipopolysaccharide-induced endothelial hyper-permeability in vivo, accompanied by improved survival of septic mice. In addition, sEH inhibitor AUDA and 11,12-EET also decreased endothelial hyper-permeability in the in-vitro study. Importantly, the relative mechanisms were associated with reduced GRP78-Src interaction and ROS production, and subsequently reduced RhoA/ROCK activation, and eventually decreased VE-cadherin and myosin light chain (MLC) phosphorylation. Thus CYP2J2-EETs is crucial for RhoA-dependent regulation of cytoskeletal architecture leading to reversible changes in vascular permeability, which may contribute to the development of new therapeutic approaches for pulmonary edema and other diseases caused by abnormal vascular permeability.

SUBMITTER: Dong R

PROVIDER: S-EPMC5584221 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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EETs reduces LPS-induced hyperpermeability by targeting GRP78 mediated Src activation and subsequent Rho/ROCK signaling pathway.

Dong Ruolan R Hu Danli D Yang Yan Y Chen Zhihui Z Fu Menglu M Wang Dao Wen DW Xu Xizhen X Tu Ling L

Oncotarget 20170421 31

Integrity of endothelial barrier is a determinant of the prognosis in the acute lung injury caused by sepsis. The epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid, exhibit protective effects in various pathogenic states, however, whether EETs play a role in endothelial barrier enhancement and the involved mechanisms remain to be investigated. Here, we show that increased EETs level by endothelial specific cytochrome P450 epoxygenase 2J2 over-expression and soluble epoxide hydrol ...[more]

PMID: 28881620

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Project description:Neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, consist of highly phosphorylated tau proteins. Tau protein binds to microtubules and is best known for its role in regulating microtubule dynamics. However, if tau protein is phosphorylated by activated major tau kinases, including glycogen synthase kinase 3β or cyclin-dependent kinase 5, or inactivated tau phosphatase, including protein phosphatase 2A, its affinity for microtubules is reduced, and the free tau is believed to aggregate, thereby forming neurofibrillary tangles. We previously reported that pitavastatin decreases the total and phosphorylated tau protein using a cellular model of tauopathy. The reduction of tau was considered to be due to Rho-associated coiled-coil protein kinase (ROCK) inhibition by pitavastatin. ROCK plays important roles to organize the actin cytoskeleton, an expected therapeutic target of human disorders. Several ROCK inhibitors are clinically applied to prevent vasospasm postsubarachnoid hemorrhage (fasudil) and for the treatment of glaucoma (ripasudil). We have examined the effects of ROCK inhibitors (H1152, Y-27632, and fasudil [HA-1077]) on tau protein phosphorylation in detail. A human neuroblastoma cell line (M1C cells) that expresses wild-type tau protein (4R0N) by tetracycline-off (TetOff) induction, primary cultured mouse neurons, and a mouse model of tauopathy (rTG4510 line) were used. The levels of phosphorylated tau and caspase-cleaved tau were reduced by the ROCK inhibitors. Oligomeric tau levels were also reduced by ROCK inhibitors. After ROCK inhibitor treatment, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and caspase were inactivated, protein phosphatase 2A was activated, and the levels of IFN-γ were reduced. ROCK inhibitors activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Collectively, these results suggest that ROCK inhibitors represent a viable therapeutic route to reduce the pathogenic forms of tau protein in tauopathies, including Alzheimer's disease.

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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EETs reduces LPS-induced hyperpermeability by targeting GRP78 mediated Src activation and subsequent Rho/ROCK signaling pathway.

Publications

EETs reduces LPS-induced hyperpermeability by targeting GRP78 mediated Src activation and subsequent Rho/ROCK signaling pathway.

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