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Fibrinogen-gamma C-terminal fragments induce endothelial barrier dysfunction and microvascular leak via integrin-mediated and RhoA-dependent mechanism.

ABSTRACT: The purposes of this study were to characterize the direct effect of the C-terminal fragment of fibrinogen gamma chain (gammaC) on microvascular endothelial permeability and to examine its molecular mechanism of action.Intravital microscopy was performed to measure albumin extravasation in intact mesenteric microvasculature, followed by quantification of hydraulic conductivity in single perfused microvessels. Transendothelial electric resistance was measured in microvascular endothelial cells in combination with immunoblotting and immunocytochemistry. The results show that gammaC induced time- and concentration-dependent increases in protein transvascular flux and water permeability and decreases in endothelial barrier function, coupled with Rho GTPase activation, myosin light chain phosphorylation, and stress fiber formation. Depletion of RhoA via siRNA knockdown or pharmacological inhibition of RhoA signaling attenuated gammaC-induced barrier dysfunction. Imaging analyses demonstrated binding of gammaC to endothelial cells; the interaction was inhibited during blockage of the alphavbeta3 integrin. Furthermore, in vivo experiments showed that the microvascular leak response to gammaC was attenuated in integrin beta3(-/-) animals.Fibrinogen-gamma C terminus directly interacts with the microvascular endothelium causing fluid and protein leak. The endothelial response to gammaC involves an integrin receptor-mediated RhoA-dependent signaling pathway that leads to paracellular hyperpermeability.

SUBMITTER: Guo M

PROVIDER: S-EPMC2737386 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Fibrinogen-gamma C-terminal fragments induce endothelial barrier dysfunction and microvascular leak via integrin-mediated and RhoA-dependent mechanism.

Guo Mingzhang M Daines Dayle D Tang Jing J Shen Qiang Q Perrin Rachel M RM Takada Yoshikazu Y Yuan Sarah Y SY Wu Mack H MH

Arteriosclerosis, thrombosis, and vascular biology 20090102 3

<h4>Objectives</h4>The purposes of this study were to characterize the direct effect of the C-terminal fragment of fibrinogen gamma chain (gammaC) on microvascular endothelial permeability and to examine its molecular mechanism of action.<h4>Methods and results</h4>Intravital microscopy was performed to measure albumin extravasation in intact mesenteric microvasculature, followed by quantification of hydraulic conductivity in single perfused microvessels. Transendothelial electric resistance was ...[more]

PMID: 19122172

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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.

Project description:p.R375W (Fibrinogen Aguadilla) is one out of seven identified mutations (Brescia, Aguadilla, Angers, Al du Pont, Pisa, Beograd, and Ankara) causing hepatic storage of the mutant fibrinogen ?. The Aguadilla mutation has been reported in children from the Caribbean, Europe, Japan, Saudi Arabia, Turkey, and China. All reported children presented with a variable degree of histologically proven chronic liver disease and low plasma fibrinogen levels. In addition, one Japanese and one Turkish child had concomitant hypo-APOB-lipoproteinemia of unknown origin. We report here on an additional child from Turkey with hypofibrinogenemia due to the Aguadilla mutation, massive hepatic storage of the mutant protein, and severe hypo-APOB-lipoproteinemia. The liver biopsy of the patient was studied by light microscopy, electron microscopy (EM), and immunohistochemistry. The investigation included the DNA sequencing of the three fibrinogen and APOB-lipoprotein regulatory genes and the analysis of the encoded protein structures. Six additional Fibrinogen Storage Disease (FSD) patients with either the Aguadilla, Ankara, or Brescia mutations were investigated with the same methodology. A molecular analysis revealed the fibrinogen gamma p.R375W mutation (Aguadilla) but no changes in the APOB and MTTP genes. APOB and MTTP genes showed no abnormalities in the other study cases. Light microscopy and EM studies of liver tissue samples from the child led to the demonstration of the simultaneous accumulation of both fibrinogen and APOB in the same inclusions. Interestingly enough, APOB-containing lipid droplets were entrapped within the fibrinogen inclusions in the hepatocytic Endoplasmic Reticulum (ER). Similar histological, immunohistochemical, EM, and molecular genetics findings were found in the other six FSD cases associated with the Aguadilla, as well as with the Ankara and Brescia mutations. The simultaneous retention of fibrinogen and APOB-lipoproteins in FSD can be detected in routinely stained histological sections. The analysis of protein structures unraveled the pathomorphogenesis of this unexpected phenomenon. Fibrinogen gamma chain mutations provoke conformational changes in the region of the globular domain involved in the "end-to-end" interaction, thus impairing the D-dimer formation. Each monomeric fibrinogen gamma chain is left with an abnormal exposure of hydrophobic patches that become available for interactions with APOB and lipids, causing their intracellular retention and impairment of export as a secondary unavoidable phenomenon.

Dataset Information

Fibrinogen-gamma C-terminal fragments induce endothelial barrier dysfunction and microvascular leak via integrin-mediated and RhoA-dependent mechanism.

Publications

Fibrinogen-gamma C-terminal fragments induce endothelial barrier dysfunction and microvascular leak via integrin-mediated and RhoA-dependent mechanism.

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