Alicia N. Rizzo

Differential and opposing effects of imatinib on LPS- and ventilator-induced lung injury

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.

Pleiotropic effects of interleukin-6 in a “two-hit” murine model of acute respiratory distress syndrome

Patients with acute respiratory distress syndrome (ARDS) exhibit elevated levels of interleukin-6 (IL-6), which correlate with increased morbidity and mortality. The exact role of IL-6 in ARDS has proven difficult to study because it exhibits either pro- or anti-inflammatory actions in mouse models of lung injury, depending on the model utilized. In order to improve understanding of the role of this complex cytokine in ARDS, we evaluated IL-6 using the clinically relevant combination of lipopolysaccharide (LPS) and ventilator-induced lung injury (VILI) in IL-6−/– mice. Bronchoalveolar lavage fluid (BAL), whole-lung tissue, and histology were evaluated for inflammatory markers of injury. Transendothelial electrical resistance was used to evaluate the action of IL-6 on endothelial cells in vitro. In wild-type mice, the combination model showed a significant increase in lung injury compared to either LPS or VILI alone. IL-6−/– mice exhibited a statistically significant decrease in BAL cellular inflammation as well as lower histologic scores for lung injury, changes observed only in the combination model. A paradoxical increase in BAL total protein was observed in IL-6−/– mice exposed to LPS, suggesting that IL-6 provides protection from vascular leakage. However, in vitro data showed that IL-6, when combined with its soluble receptor, actually caused a significant increase in endothelial cell permeability, suggesting that the protection seen in vivo was likely due to complex interactions of IL-6 and other inflammatory mediators rather than to direct effects of IL-6. These studies suggest that a dual-injury model exhibits utility in evaluating the pleiotropic effects of IL-6 in ARDS on inflammatory cells and lung endothelium.

Targeting Abl Kinases to Regulate Vascular Leak During Sepsis and Acute Respiratory Distress Syndrome

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 Abl related gene (Arg, Abl2), phosphorylate several cytoskeletal effectors that mediate vascular permeability, including nonmuscle myosin light chain kinase, cortactin, vinculin, and &bgr;-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.Inflammation-induced vascular leak underlies the pathophysiology of multiple disorders that affect critically ill patients, including sepsis and Acute Respiratory Distress Syndrome (ARDS). These conditions are major causes of morbidity and mortality in the ICU affecting 750,000 and 200,000 patients/year in the US, respectively1, 2. The lack of medical therapies capable of attenuating vascular leak necessitates additional research into the mechanisms underlying EC barrier dysfunction and clinical trials of novel therapeutic strategies.

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

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.

Endothelial Nitric Oxide Synthase Dimerization Is Regulated by Heat Shock Protein 90 Rather than by Phosphorylation

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.

Connexin 43 Upregulation in Mouse Lungs during Ovalbumin-Induced Asthma.

Background Connexin (Cx)-based gap junction channels play important roles in the inflammatory response. Cx43 is involved in the pathogenesis of some lung diseases such as acute lung injury. However, the Cx43 expression in asthma is unclear. In the present study, we used a murine model of ovalbumin (OVA)-induced allergic airway disease to examine the levels of Cx43 and analyze the relationship between Cx43 and airway inflammation in allergic airway disease. Methods Asthma was induced in mice via sensitization and challenge with OVA. Cx43 mRNA and protein expression levels were investigated via QT-PCR, western blot, and immunohistochemistry 0 h, 8 h, 1 d, 2 d and 4 d after the first challenge. The relationship between Cx43 protein levels and inflammatory cell infiltration, cytokine levels was analyzed. Results The OVA-induced mice exhibited typical pathological features of asthma, including airway hyper-responsiveness; strong inflammatory cell infiltration surrounding the bronchia and vessels; many inflammatory cells in the bronchoalveolar lavage fluid (BALF); higher IL-4, IL-5 and IL-13 levels; and high OVA specific IgE levels. Low Cx43 expression was detected in the lungs of control (PBS) mice. A dramatic increase in the Cx43 mRNA and protein levels was found in the asthmatic mice. Cx43 mRNA and protein expression levels increased in a time-dependent manner in asthma mice, and Cx43 was mostly localized in the alveolar and bronchial epithelial layers. Moreover, lung Cx43 protein levels showed a significant positive correlation with inflammatory cell infiltration in the airway and IL-4 and IL-5 levels in the BALF at different time points after challenge. Interestingly, the increase in Cx43 mRNA and protein levels occurred prior to the appearance of the inflammatory cell infiltration. Conclusion Our data suggest that there is a strong upregulation of Cx43 mRNA and protein levels in the lungs in asthma. Cx43 levels also exhibited a positive correlation with allergic airway inflammation. Cx43 may represent a target to treat allergic airway diseases in the future.

The Importance of Bronchial Epithelial Junction Integrity in Asthma

Bronchial epithelial junctions provide not only a physical barrier, but also an immune barrier against the allergens of asthma. Epithelial junction integrity is closely related to the severity and progression of asthma. The bronchial epithelial barrier consists of tight junctions, adherens junctions, desmosomes, hemidesmosomes and gap junctions, all of which are potentially implicated in asthma pathophysiology. In tight junctions, claudins, occludens, ZO-1 and β-catenin expression have been shown to be decreased by asthma allergens, resulting tight junction disruption. Similarly, E-cadherin and α-catenin levels have also been reported to be dysregulated in response to asthma allergens, resulting in alterations in adherens junction structure. Asthma allergens also alter desmosome and hemidesmosome structure; however, no reports have shown that desmosome or hemidesmosome junction protein expression is altered in response to asthma allergens. Finally, in gap junctions, connexin 37 mRNA and protein were found to be decreased in the ovalbumin (OVA) induced allergic model. In summary, the regulation of bronchial junction protein expression and structure is an important and presently understudied component of asthma pathophysiology. We believe that further investigation in this area has the potential to aid in the development of novel asthma treatments.

The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain

In addition to superoxide (O2 .-) generation from nitric oxide synthase (NOS) oxygenase domain, a new O2 .- generation site has been identified in the reductase domain of inducible NOS (iNOS) and neuronal NOS (nNOS). Cysteine S-glutathionylation in eNOS reductase domain also induces O2 .- generation from eNOS reductase domain. However, the characteristics and regulatory mechanism of the O2 .- generation from NOS reductase domain remain unclear. We cloned and purified the wild type bovine eNOS (WT eNOS), a mutant of Serine 1179 replaced with aspartic acid eNOS (S1179D eNOS), which mimics the negative charge caused by phosphorylationand truncated eNOS reductase domain (eNOS RD). Both WT eNOS and S1179D eNOS generated significant amount of O2 .- in the absence of BH4 and L-arginine. The capacity of O2 .- generation from S1179D eNOS was significantly higher than that of WT eNOS (1.74:1). O2 .- generation from both WT eNOS and S1179D eNOS were not completely inhibited by 100nM tetrahydrobiopterin(BH4). This BH4 un-inhibited O2 .- generation from eNOS was blocked by 10mM flavoprotein inhibitor, diphenyleneiodonium (DPI). Purified eNOS reductase domain protein confirmed that this BH4 un-inhibited O2 .- generation originates at the FMN or FAD/NADPH binding site of eNOS reductase domain. DEPMPO-OOH adduct EPR signals and NADPH consumptions analyses showed that O2 .- generation from eNOS reductase domain was regulated by Serine 1179 phosphorylation and DPI, but not by L-arginine, BH4 or calmodulin (CaM). In addition to the heme center of eNOS oxygenase domain, we confirmed another O2 .- generation site in the eNOS reductase domain and characterized its regulatory properties.

Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury

Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK−/− mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK.

The ARP 2/3 complex mediates endothelial barrier function and recovery

Pulmonary endothelial cell (EC) barrier dysfunction and recovery is critical to the pathophysiology of acute respiratory distress syndrome. Cytoskeletal and subsequent cell membrane dynamics play a key mechanistic role in determination of EC barrier integrity. Here, we characterizAQe the actin related protein 2/3 (Arp 2/3) complex, a regulator of peripheral branched actin polymerization, in human pulmonary EC barrier function through studies of transendothelial electrical resistance (TER), intercellular gap formation, peripheral cytoskeletal structures and lamellipodia. Compared to control, Arp 2/3 inhibition with the small molecule inhibitor CK-666 results in a reduction of baseline barrier function (1,241 ± 53 vs 988 ± 64 ohm; p < 0.01), S1P-induced barrier enhancement and delayed recovery of barrier function after thrombin (143 ± 14 vs 93 ± 6 min; p < 0.01). Functional changes of Arp 2/3 inhibition on barrier integrity are associated temporally with increased intercellular gap area at baseline (0.456 ± 0.02 vs 0.299 ± 0.02; p < 0.05) and thirty minutes after thrombin (0.885 ± 0.03 vs 0.754 ± 0.03; p < 0.05). Immunofluorescent microscopy reveals reduced lamellipodia formation after S1P and during thrombin recovery in Arp 2/3 inhibited cells. Individual lamellipodia demonstrate reduced depth following Arp 2/3 inhibition vs vehicle at baseline (1.83 ± 0.41 vs 2.55 ± 0.46 µm; p < 0.05) and thirty minutes after S1P treatment (1.53 ± 0.37 vs 2.09 ± 0.36 µm; p < 0.05). These results establish a critical role for Arp 2/3 activity in determination of pulmonary endothelial barrier function and recovery through formation of EC lamellipodia and closure of intercellular gaps.