Role of Claudin-5 in the Attenuation of Murine Acute Lung Injury by Simvastatin

Abstract

The statins are now recognized to have pleiotropic properties, including augmentation of endothelial barrier function. To explore the mechanisms involved, we investigated the effect of simvastatin on endothelial cell (EC) tight junctions. Western blotting of human pulmonary artery ECs treated with simvastatin (5 mM) confirmed a significant time-dependent increase (16-48 h) in claudin-5 protein expression compared with controls, without detectable alterations in zonula occludens-1 or occludin. These effects were associated with membrane translocation of VE-cadherin, whereas translocation of vascular endothelial cadherin (VE-cadherin; silencing RNA) inhibited simvastatin-induced claudin-5 up-regulation. Moreover, simvastatin treatment of ECs induced increased phosphorylation of both FoxO1 and b-catenin, transcriptional regulators of claudin-5 expression mediated by VE-cadherin. Subsequently, we found no effect of claudin-5 silencing on EC barrier protection by simvastatin in response to thrombin stimulation, as measured by either transendothelial electrical resistance or by EC monolayer flux of FITC-dextran (2,000 kD). However, silencing of claudin-5 did significantly attenuate simvastatin-mediated EC barrier protection in response to thrombin, as measured by monolayer flux of sodium fluorescein (376 Da). Finally, employing a murine model of LPSinduced acute lung injury, there was no effect of claudin-5 silencing in vivo (intratracheal injection) on bronchoalveolar lavage fluid protein or cell counts, but LPS-induced lung tissue extravasation of the small molecular weight markers, sodium fluorescein and Hochst stain (562 Da), were significantly increased in claudin-5-silenced animals compared with simvastatin-treated control animals. These findings implicate a distinct mechanism underlying size-selective endothelial barrier-protective properties of statins, and may ultimately lead to new novel therapeutic targets for patients with acute lung injury.

Cytoskeletal mechanisms regulating vascular endothelial barrier function in response to acute lung injury

“…138 Up-regulation of ECspecific claudin-5 isoform is involved in EC barrier enhancement in some, but not all models. 139,140 Conversely, edemagenic agonists decreased claudin-5 and ZO-1 expression accompanied by translocation of ZO-1 from the cytoskeleton to the membrane/ nuclear fractions. 141,142 Recent study implicated the involvement of PKCe/Erk1/2 MAPK axis in phosphorylation of ZO-1 at Thr 770,772 .…”

Claudins: Gatekeepers of lung epithelial function

“…Because of the dual nature of this barrier, the close apposition of alveolar endothelial and epithelial tight junctions and the fact that both tissues express claudin-5 has led to some apparently contradictory data related to roles for claudin-5 in regulating alveolar barrier function. In brief, claudin-5 is likely to have a protective effect by increasing the barrier function of the endothelial part of the barrier [128–133]. However, increased claudin-5 expression decreases alveolar epithelial barrier function [100, 122, 125].…”

“…Related to the endothelial barrier, there was no effect on barrier function at baseline in studies using pulmonary artery endothelial cells in vitro where claudin-5 was silenced using siRNA [128]. However, cells treated with claudin-5 siRNA had increased permeability to fluorescein (0.4 kDa) after thrombin treatment as compared to controls expressing normal levels of claudin-5.…”

Roles of the Mevalonate Pathway and Cholesterol Trafficking in Pulmonary Host Defense

“…Statins inhibit LPS-induced acute lung injury in murine models through reducing neutrophil trafficking [133] and vascular leak [134]. Protection from LPS-induced lung injury has been linked to upregulation of claudin-5 and integrin-β4 on lung endothelial cells [135], and also to inhibition of LPS signaling by macrophages due to upregulation of the tetraspanin CD9 [136]. Suggesting relevance to humans, simvastatin is reported to attenuate lung inflammation induced by LPS inhalation in human volunteers [137], and plasma cytokines induced by intravenous LPS in human volunteers [138].…”