Abstract
ObjectivesWe previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvβ3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC 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 γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC 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 γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC 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 γC-mediated hyperpermeability. In addition, γC-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 γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.
Highlights
When severe injury results in bleeding, fibrinogen, a soluble protein consisting of α, β and γ polypeptide pairs, is converted at the wound into fibrin by thrombin [1]
FITC-albumin flux was observed after perfusion ofC or vehicle control into mesenteric microcirculation. ̊C treatment increased microvascular leakage of albumin throughout the time intervals of 5, 10 and 30 minutes (Fig 1A)
Given that focal adhesion kinase (FAK) acts as an upstream regulator of Src [7, 34, 35], we investigated whether FAK is an upstream regulator of Src in γ chain C-terminal products (γC)-induced barrier min was compared with control and γC only treatment values obtained from Fig 1A, (n = 4)
Summary
When severe injury results in bleeding, fibrinogen, a soluble protein consisting of α, β and γ polypeptide pairs, is converted at the wound into fibrin by thrombin [1]. The proteolysis of fibrin is coupled with its breakdown into fibrin degradation products (FDPs), which includes a D-dimer and soluble C-termini of the α, β and γ chains [1]. Of all the soluble fibrinogen monomers, the C-terminus of the γ chain is of specific interest due to its reactivity imparted by a calcium binding site, polymerization pocket and cross-binding site. This reactive region allows for surface receptor binding and stimulates fibrin cross-linking [5]. The mechanism behind fibrinogen γC microvascular hyperpermeability is not fully understood
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