Differential Regulation of Pulmonary Endothelial Monolayer Integrity by Varying Degrees of Cyclic Stretch

Abstract

Ventilator-induced lung injury is a life-threatening complication of mechanical ventilation at high-tidal volumes. Besides activation of proinflammatory cytokine production, excessive lung distension directly affects blood-gas barrier and lung vascular permeability. To investigate whether restoration of pulmonary endothelial cell (EC) monolayer integrity after agonist challenge is dependent on the magnitude of applied cyclic stretch (CS) and how these effects are linked to differential activation of small GTPases Rac and Rho, pulmonary ECs were subjected to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of CS. Pathological CS enhanced thrombin-induced gap formation and delayed monolayer recovery, whereas physiological CS induced nearly complete EC recovery accompanied by peripheral redistribution of focal adhesions and cortactin after 50 minutes of thrombin. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation during the EC recovery phase. Rac inhibition dramatically attenuated restoration of monolayer integrity after thrombin challenge. Physiological CS preconditioning (5% CS, 24 hours) enhanced EC paracellular gap resolution after step-wise increase to 18% CS (30 minutes) and thrombin challenge. These results suggest a critical role for the CS amplitude and the balance between Rac and Rho in mechanochemical regulation of lung EC barrier.

Prostaglandins PGE2 and PGI2 promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation

“…Endothelial cells plated on glass coverslips were grown to confluence, stimulated with PGs or left untreated, and immunofluorescent staining of proteins of interest using corresponding antibodies was performed as described elsewhere [12,13,43]. Images were processed with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA) software.…”

“…Immunoblotting detection of proteins of interest was performed as described previously [12,43]. In brief, protein extracts were subjected to 12.5% or 7.5% SDS-PAGE, transferred to nitrocellulose membrane in semidry transfer apparatus (BioRad, Hercules, CA) 10 V for 1 hour, and probed with antibody of interest.…”

Paxillin Is Involved in the Differential Regulation of Endothelial Barrier by HGF and VEGF

“…Specific patterns of FA protein interactions and site-specific phosphorylation have been associated with barrier-protective and barrier-disruptive EC responses to mechanical and chemical stimuli (21)(22)(23)(24)(25)(26)(27). Several FA-associated proteins play a key role in FA assembly, interaction with cytoskeleton, and FA-dependent signal transduction.…”

Cell cultures as models of cardiac mechanoelectric feedback

“…Thus, it may be essential to examine the behavior of tens or hundreds of thousands of cells, which is possible in cultured cell monolayers. Yet another advantage of cell culture models is the potential to study cumulative changes in cellular physiology with prolonged exposure to a given mechanical perturbation, as has been the case with vascular endothelial cells (Sato and Ohashi, 2005), vascular smooth muscle cells (Stegemann et al, 2005), chondrocytes (Smith et al, 2004), pulmonary epithelial cells (Trepat et al, 2006), pulmonary endothelial cells (Birukova et al, 2006), cortical neurons (Cohen et al, 2007) and other cell types.…”