Abstract
The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.
Highlights
Endothelial cells (EC) constitute the inner lining of all blood vessels and regulate the interface between the circulating blood and the vessel wall including vascular barrier regulation, passive diffusion and active transport of blood-borne substances, regulation of vascular smooth muscle tone and blood coagulation properties [1,2]
CD44, a major hyaluronan (HA) receptor localized in caveolin-enriched microdomains (CEM), has been implicated in regulating hepatocyte growth factor (HGF)/c-Met signaling [10,11,12,13]
Considering our results indicating that CD44 regulates HGF-induced EC barrier enhancement (Figure 4), we examined whether Tiam1, cortactin and/or dynamin 2 are involved in HGF-induced increases in EC barrier integrity
Summary
Vessel wall including vascular barrier regulation, passive diffusion and active transport of blood-borne substances, regulation of vascular smooth muscle tone and blood coagulation properties [1,2] Disruption of this semi-selective cellular barrier is a critical feature of inflammation as well as an important contributing factor to atherosclerosis and tumor angiogenesis [3,4]. Our prior studies indicated that hepatocyte growth factor (HGF) binding to its receptor tyrosine kinase, c-Met, promotes EC barrier function [5], the exact mechanism by this occurs is incompletely defined. Cortactin is a ~80-85 kDa actin-binding protein we have shown to be critically involved in EC cortical cytoskeletal rearrangements [26,27,28] and agonist-mediated EC barrier enhancement [29,30]. HGF promotes increased EC barrier function may lead to novel treatments for diseases involving vascular barrier disruption including inflammation, tumor angiogenesis, atherosclerosis and acute lung injury
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