Acute lung injury/acute respiratory distress syndrome (ALI/ARDS), an illness characterized by life-threatening vascular leak, is a significant cause of morbidity and mortality in critically ill patients. Recent preclinical studies and clinical observations have suggested a potential role for the chemotherapeutic agent imatinib in restoring vascular integrity. Our prior work demonstrates differential effects of imatinib in mouse models of ALI, namely attenuation of LPS-induced lung injury but exacerbation of ventilator-induced lung injury (VILI). Because of the critical role of mechanical ventilation in the care of patients with ARDS, in the present study we pursued an assessment of the effectiveness of imatinib in a “two-hit” model of ALI caused by combined LPS and VILI. Imatinib significantly decreased bronchoalveolar lavage protein, total cells, neutrophils, and TNF-α levels in mice exposed to LPS plus VILI, indicating that it attenuates ALI in this clinically relevant model. In subsequent experiments focusing on its protective role in LPS-induced lung injury, imatinib attenuated ALI when given 4 h after LPS, suggesting potential therapeutic effectiveness when given after the onset of injury. Mechanistic studies in mouse lung tissue and human lung endothelial cells revealed that imatinib inhibits LPS-induced NF-κB expression and activation. Overall, these results further characterize the therapeutic potential of imatinib against inflammatory vascular leak.

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure yet has few pharmacologic therapies, reflecting the mechanistic heterogeneity of lung injury. We hypothesized that damage to the alveolar epithelial glycocalyx, a layer of glycosaminoglycans interposed between the epithelium and surfactant, contributes to lung injury in patients with ARDS. Using mass spectrometry of airspace fluid noninvasively collected from mechanically ventilated patients, we found that airspace glycosaminoglycan shedding (an index of glycocalyx degradation) occurred predominantly in patients with direct lung injury and was associated with duration of mechanical ventilation. Male patients had increased shedding, which correlated with airspace concentrations of matrix metalloproteinases. Selective epithelial glycocalyx degradation in mice was sufficient to induce surfactant dysfunction, a key characteristic of ARDS, leading to microatelectasis and decreased lung compliance. Rapid colorimetric quantification of airspace glycosaminoglycans was feasible and could provide point-of-care prognostic information to clinicians and/or be used for predictive enrichment in clinical trials.

The vascular endothelium separates circulating fluid and inflammatory cells from the surrounding tissues. Vascular leak occurs in response to wide-spread inflammatory processes, such as sepsis and acute respiratory distress syndrome, because of the formation of gaps between endothelial cells. Although these disorders are leading causes of mortality in the intensive care unit, no medical therapies exist to restore endothelial cell barrier function. Recent evidence highlights a key role for the Abl family of nonreceptor tyrosine kinases in regulating vascular barrier integrity. These kinases have well-described roles in cancer progression and neuronal morphogenesis, but their functions in the vasculature have remained enigmatic until recently. The Abl family kinases, c-Abl (Abl1) and A bl related gene (Arg, Abl2), phosphorylate several cytoskeletal effectors that mediate vascular permeability, including nonmuscle myosin light chain kinase, cortactin, vinculin, and β-catenin. They also regulate cell–cell and cell–matrix junction dynamics, and the formation of actin-based cellular protrusions in multiple cell types. In addition, both c-Abl and Arg are activated by hyperoxia and contribute to oxidant-induced endothelial cell injury. These numerous roles of Abl kinases in endothelial cells and the current clinical usage of imatinib and other Abl kinase inhibitors have spurred recent interest in repurposing these drugs for the treatment of vascular barrier dysfunction. This review will describe the structure and function of Abl kinases with an emphasis on their roles in mediating vascular barrier integrity. We will also provide a critical evaluation of the potential for exploiting Abl kinase inhibition as a novel therapy for inflammatory vascular leak syndromes.

Endothelial dysfunction underlies the pathophysiology of vascular disorders such as acute lung injury (ALI) syndromes. Recent work has identified the Abl family kinases (c-Abl and Arg) as important regulators of endothelial cell (EC) barrier function and suggests that their inhibition by currently available pharmaceutical agents such as imatinib may be EC protective. Here we describe novel and differential effects of imatinib in regulating lung pathophysiology in two clinically relevant experimental models of ALI. Imatinib attenuates endotoxin (LPS)-induced vascular leak and lung inflammation in mice but exacerbates these features in a mouse model of ventilator-induced lung injury (VILI). We next explored these discrepant observations in vitro through investigation of the roles for Abl kinases in cultured lung EC. Imatinib attenuates LPS-induced lung EC permeability, restores VE-cadherin junctions, and reduces inflammation by suppressing VCAM-1 expression and inflammatory cytokine (IL-8 and IL-6) secretion. Conversely, in EC exposed to pathological 18% cyclic stretch (CS) (in vitro model of VILI), imatinib decreases VE-cadherin expression, disrupts cell-cell junctions, and increases IL-8 levels. Downregulation of c-Abl expression with siRNA attenuates LPS-induced VCAM-1 expression, whereas specific reduction of Arg reduces VE-cadherin expression in 18% CS-challenged ECs to mimic the imatinib effects. In summary, imatinib exhibits pulmonary barrier-protective and anti-inflammatory effects in LPS-injured mice and lung EC; however, imatinib exacerbates VILI as well as dysfunction in 18% CS-EC. These findings identify the Abl family kinases as important modulators of EC function and potential therapeutic targets in lung injury syndromes.

Endothelial nitric oxide synthase (eNOS) is a multifunctional enzyme with roles in diverse cellular processes including angiogenesis, tissue remodeling, and the maintenance of vascular tone. Monomeric and dimeric forms of eNOS exist in various tissues. The dimeric form of eNOS is considered the active form and the monomeric form is considered inactive. The activity of eNOS is also regulated by many other mechanisms, including amino acid phosphorylation and interactions with other proteins. However, the precise mechanisms regulating eNOS dimerization, phosphorylation, and activity remain incompletely characterized. We utilized purified eNOS and bovine aorta endothelial cells (BAECs) to investigate the mechanisms regulating eNOS degradation. Both eNOS monomer and dimer existed in purified bovine eNOS. Incubation of purified bovine eNOS with protein phosphatase 2A (PP2A) resulted in dephosphorylation at Serine 1179 (Ser1179) in both dimer and monomer and decrease in eNOS activity. However, the eNOS dimer∶monomer ratio was unchanged. Similarly, protein phosphatase 1 (PP1) induced dephosphorylation of eNOS at Threonine 497 (Thr497), without altering the eNOS dimer∶monomer ratio. Different from purified eNOS, in cultured BAECs eNOS existed predominantly as dimers. However, eNOS monomers accumulated following treatment with the proteasome inhibitor lactacystin. Additionally, treatment of BAECs with vascular endothelial growth factor (VEGF) resulted in phosphorylation of Ser1179 in eNOS dimers without altering the phosphorylation status of Thr497 in either form. Inhibition of heat shock protein 90 (Hsp90) or Hsp90 silencing destabilized eNOS dimers and was accompanied by dephosphorylation both of Ser1179 and Thr497. In conclusion, our study demonstrates that eNOS monomers, but not eNOS dimers, are degraded by ubiquitination. Additionally, the dimeric eNOS structure is the predominant condition for eNOS amino acid modification and activity regulation. Finally, destabilization of eNOS dimers not only results in eNOS degradation, but also causes changes in eNOS amino acid modifications that further affect eNOS activity.

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 μM) 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 β-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 LPS-induced 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.