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Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX -silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX -silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX -silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1β, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin. NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model.

We previously reported that claudin-5, a tight junctional protein, mediates lung vascular permeability in a murine model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Recently, it has been reported that haloperidol, an antipsychotic medication, dose-dependently increases expression of claudin-5 in vitro and in vivo, in brain endothelium. Notably, claudin-5 is highly expressed in both brain and lung tissues. However, the effects of haloperidol on EC barrier function are unknown. We hypothesized that haloperidol increases lung EC claudin-5 expression and attenuates agonist-induced lung EC barrier disruption. Human pulmonary artery ECs were pretreated with haloperidol at variable concentrations (0.1-10 μM) for 24 h. Cell lysates were subjected to Western blotting for claudin-5, in addition to occludin and zona occludens-1 (ZO-1), two other tight junctional proteins. To assess effects on barrier function, EC monolayers were pretreated for 24 h with haloperidol (10 µM) or vehicle prior to treatment with thrombin (1 U/mL), with measurements of transendothelial electrical resistance (TER) recorded as a real-time assessment of barrier integrity. In separate experiments, EC monolayers grown in Transwell inserts were pretreated with haloperidol (10 µM) prior to stimulation with thrombin (1 U/mL, 1 h) and measurement of FITC-dextran flux. Haloperidol significantly increased claudin-5, occludin, and ZO-1 expression levels. Measurements of TER and FITC-dextran Transwell flux confirmed a significant attenuation of thrombin-induced barrier disruption associated with haloperidol treatment. Finally, mice pretreated with haloperidol (4 mg/kg, IP) prior to the intratracheal administration of LPS (1.25 mg/kg, 16 h) had increased lung claudin-5 expression with decreased lung injury as assessed by bronchoalveolar lavage (BAL) fluid protein content, total cell counts, and inflammatory cytokines, in addition to lung histology. Our data confirm that haloperidol results in increased claudin-5 expression levels and demonstrates lung vascular-protective effects both in vitro and in vivo in a murine ALI model. These findings suggest that haloperidol may represent a novel therapy for the prevention or treatment of ALI and warrants further investigation in this context.

Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. Previous studies showed that bovine eNOS serine 1179 (Serine 1177 for human eNOS) phosphorylation enhanced NO synthesis. Meanwhile, heat shock protein 90 (Hsp90) plays a critical role in maintenance of eNOS structure and function. However, the regulatory difference and importance between Serine 1179 phosphorylation and Hsp90 on eNOS activity have not been evaluated. In current studies, S1179D eNOS was employed to mimic phospho-eNOS and exhibited markedly increased enzyme activity than wild type eNOS (WT eNOS). Hsp90 showed a dose-dependent increase for both WT eNOS and S1179D eNOS activity at the presence of all eNOS cofactors, such as Calcium/Calmodulin (Ca2+ /CaM), BH4, and NADPH etc. The enhancement effects were abolished by dominant-negative mutant Hsp 90 protein. ENOS-cofactors dynamic assay showed that Hsp90 enhanced WT eNOS affinity to NADPH, L-arginine, and CaM but not to Ca2+ and BH4. The impact of eNOS Serine 1179 phosphorylation and Hsp90 on eNOS affinity to cofactors has also been compared. Different from the effect of Hsp90 on eNOS affinity to specific cofactors, Serine 1179 phosphorylation significantly increased eNOS affinity to all cofactors. Moreover, VEGF-induced eNOS phosphorylation in bovine aortic endothelial cells (BAECs) and more NO generation from eNOS compared to control. Inhibition of Hsp90 by geldanamycin decreased eNOS activity and decreased endothelial viability. In conclusion, by changing eNOS structure, Hsp90 profoundly affected eNOS functions, including change of affinity of eNOS to cofactors like Ca2+, L-arginine, BH4 and further affecting NO generation capability. These specific cofactors regulated by Hsp 90 could become potential therapeutic targets of the eNOS-related diseases in future.

We previously reported sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF) augment endothelial cell (EC) barrier function and attenuate murine acute lung inury (ALI). While the mechanisms underlying these effects are not fully understood, S1P and HGF both transactivate the S1P receptor, S1PR1 and integrin β4 (ITGB4) at membrane caveolin-enriched microdomains (CEMs). In the current study, we investigated the roles of S1PR2 and S1PR3 in S1P/HGF-mediated EC signaling and their associations with ITGB4. Our studies confirmed ITGB4 and S1PR2/3 are recruited to CEMs in human lung EC in response to either S1P (1 µM, 5 min) or HGF (25 ng/ml, 5 min). Co-immunoprecipitation experiments identified an S1P/HGF-mediated interaction of ITGB4 with both S1PR2 and S1PR3. We then employed an in situ proximity ligation assay (PLA) to confirm a direct ITGB4-S1PR3 association induced by S1P/HGF although a direct association was not detectable between S1PR2 and ITGB4. S1PR1 knockdown (siRNA), however, abrogated S1P/HGF-induced ITGB4-S1PR2 associations while there was no effect on ITGB4-S1PR3 associations. Moreover, PLA confirmed a direct association between S1PR1 and S1PR2 induced by S1P and HGF. Finally, silencing of S1PR2 significantly attenuated S1P/HGF-induced EC barrier enhancement as measured by transendothelial resistance while silencing of S1PR3 significantly augmented S1P/HGF-induced barrier enhancement. These results confirm an important role for S1PR2 and S1PR3 in S1P/HGF-mediated EC barrier responses that are associated with their complex formation with ITGB4. Our findings elucidate novel mechanisms of EC barrier regulation that may ultimately lead to new therapeutic targets for disorders characterized by increased vascular permeability including ALI., (© 2013 Wiley Periodicals, Inc.)

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met. We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.

We previously identified the marked upregulation of integrin beta4 in human lung endothelial cells (EC) treated with simvastatin, an HMG coA-reductase inhibitor with vascular-protective and anti-inflammatory properties in murine models of acute lung injury (ALI). We now investigate the role of integrin beta4 as a novel mediator of vascular inflammatory responses with a focus on mitogen-activated protein kinases (MAPK) signaling and the downstream expression of the inflammatory cytokines (IL-6 and IL-8) essential for the full elaboration of inflammatory lung injury. Silencing of integrin beta4 (siITGB4) in human lung EC resulted in significant increases in both basal and LPS-induced phosphorylation of ERK 1/2, JNK, and p38 MAPK, consistent with robust integrin beta4 regulation of MAPK activation. In addition, siITB4 increased both basal and LPS-induced expression of IL-6 and IL-8 mRNA and protein secretion into the media. We next observed that integrin beta4 silencing increased basal and LPS-induced phosphorylation of SHP-2, a protein tyrosine phosphatase known to modulate MAPK signaling. In contrast, inhibition of SHP-2 enzymatic activity (sodium stibogluconate) abrogated the increased ERK phosphorylation associated with integrin beta4 silencing in LPS-treated EC and attenuated the increases in levels of IL-6 and IL-8 in integrin-beta4-silenced EC. These findings highlight a novel negative regulatory role for integrin beta4 in EC inflammatory responses involving SHP-2-mediated MAPK signaling. Upregulation of integrin beta4 may represent an important element of the anti-inflammatory and vascular-protective properties of statins and provides a novel strategy to limit inflammatory vascular syndromes., ((c) 2010 Wiley-Liss, Inc.)

We previously reported integrin beta 4 (ITGB4) is an important mediator of lung vascular protection by simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitor. In this study, we report increased endothelial cell (EC) expression specifically of ITGB4E, an ITGB4 mRNA splice variant, by simvastatin with effects on EC protein expression and inflammatory responses. In initial experiments, human pulmonary artery ECs were treated using simvastatin (5 μM, 24 h) prior to immunoprecipitation of integrin alpha 6 (ITGA6), which associates with ITGB4, and Western blotting for full-length ITGB4 and ITGB4E, uniquely characterized by a truncated 114 amino acid cytoplasmic domain. These experiments confirmed a significant increase in both full-length ITGB4 and ITGB4E. To investigate the effects of increased ITGB4E expression alone, ECs were transfected with ITGB4E or control vector, and cells were seeded in wells containing Matrigel to assess effects on angiogenesis or used for scratch assay to assess migration. Decreased angiogenesis and migration were observed in ITGB4E transfected ECs compared with controls. In separate experiments, PCR and Western blots from transfected cells demonstrated significant changes in EC protein expression associated with increased ITGB4E, including marked decreases in platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin) as well as increased expression of E-cadherin and N-cadherin along with increased expression of the Slug and Snail transcription factors that promote endothelial-to-mesenchymal transition (EndMT). We, then, investigated the functional effects of ITGB4E overexpression on EC inflammatory responses and observed a significant attenuation of lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, including decreased phosphorylation of both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as reduced inflammatory cytokines (IL-6 and IL-8), expressed in the media of EC after either LPS or excessive cyclic stretch (CS). Finally, EC expression-increased ITGB4E demonstrated decreased barrier disruption induced by thrombin as measured by transendothelial electrical resistance. Our data support distinct EC phenotypic changes induced by ITGB4E that are also associated with an attenuation of cellular inflammatory responses. These findings implicate ITGB4E upregulation as an important mediator of lung EC protection by statins and may lead to novel therapeutic strategies for patients with or at risk for acute lung injury (ALI). [ABSTRACT FROM AUTHOR]

Cellular junctions play a critical role in structural connection and signal communication between cells in various tissues. Although there are structural and functional varieties, cellular junctions include tight junctions, adherens junctions, focal adhesion junctions, and tissue specific junctions such as PECAM-1 junctions in endothelial cells (EC), desmosomes in epithelial cells, and hemidesmosomes in EC. Cellular junction dysfunction and deterioration are indicative of clinical diseases. MicroRNAs (miRNA) are ~20 nucleotide, noncoding RNAs that play an important role in posttranscriptional regulation for almost all genes. Unsurprisingly, miRNAs regulate junction protein gene expression and control junction structure integrity. In contrast, abnormal miRNA regulation of junction protein gene expression results in abnormal junction structure, causing related diseases. The major components of tight junctions include zonula occluden-1 (ZO-1), claudin-1, claudin-5, and occludin. The miRNA regulation of ZO-1 has been intensively investigated. ZO-1 and other tight junction proteins such as claudin-5 and occludin were positively regulated by miR-126, miR-107, and miR21 in different models. In contrast, ZO-1, claudin-5, and occludin were negatively regulated by miR-181a, miR-98, and miR150. Abnormal tight junction miRNA regulation accompanies cerebral middle artery ischemia, brain trauma, glioma metastasis, and so forth. The major components of adherens junctions include VE-cadherin, β-catenin, plakoglobin, P120, and vinculin. VE-cadherin and β-catenin were regulated by miR-9, miR-99b, miR-181a, and so forth. These regulations directly affect VE-cadherin-β-catenin complex stability and further affect embryo and tumor angiogenesis, vascular development, and so forth. miR-155 and miR-126 have been shown to regulate PECAM-1 and affect neutrophil rolling and EC junction integrity. In focal adhesion junctions, the major components are integrin β4, paxillin, and focal adhesion kinase (FAK). Integrin β4 has been regulated by miR-184, miR-205, and miR-9. Paxillin has been regulated by miR-137, miR-145, and miR-218 in different models. FAK has been regulated by miR-7, miR-138, and miR-135. Deregulation of miRNAs is caused by viral infections, tumorigenesis, and so forth. By regulation of posttranscription, miRNAs manipulate junction protein expression in all cellular processes and further determine cellular fate and development. Elucidation of these regulatory mechanisms will become a new alternative therapy for many diseases, such as cancers and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Introduction: Acute lung injury (ALI) is mediated by the upregulation of endothelial cell integrin β4 (ITGB4). However, the mechanisms underlying these effects are unknown. Hypothesis: Both epigenetic regulation and promoter regulation involve in ITGB4 expression Methods: To explore this further, we investigated ITGB4 promoter regulation and epigenetic modifications by simvastatin. Human pulmonary artery EC were transfected with luciferase reporter promoter deletion constructs prior to treatment with simvastatin (5 μM) to identify specific 5' regions within the ITGB4 gene responsible for statin-mediated ITGB4 expression. Regions of interest were then subjected to chromatin immuoprecipitation (ChIP) to identify candidate transcription factors (TFs). Subsequently, EC were transfected with siRNA specific for TFs of interest prior to simvastatin treatment and Western blotting for ITGB4. In separate experiments, EC were pretreated with either a trichostain (TSA, 5 μM, 2h), or 5-aza-2'-deoxycytidine (5-Aza, 5 μM, 2h) prior to treatment with simvastatin (5 μM, 16 h) followed by RT-PCR and Western blotting for ITGB4 mRNA and protein levels, respectively. Finally, ChIP-qPCR assays were conducted to assess simvastatin effects on ITGB4 promoter methylation as well as specific histone modifications including H3K9 acetylation and H3K4 trimethylation associated with transcription activation. Results: ChIP assays identified simvastatin-induced NF-kB binding to the ITGB4 promoter. Subsequently, we confirmed that silencing of NF-kB2 specifically inhibits ITGB4 upregulation by simvastatin. Using CHIP-qPCR, we identified H3K9 acetylation and H3K4 trimethylation of ITGB4 promoter target regions by simvastatin. In addition, Inhibition of histone deacetylation (TSA) resulted in increased simvastatin-induced ITGB4 expression while inhibition of histone methylation (5-Aza) was associated with increased ITGB4 expression in response to simvastatin. Conclusions: These data confirm ITGB4 upregulation by simvastatin is mediated by both NF-kB activation of the ITGB4 promoter as well as specific epigenetic changes including DNA methylation and histone modifications. Our findings identify mechanisms of ITGB4 expression regulation which may ultimately lead to novel therapeutic strategies in patients with ALI. [ABSTRACT FROM AUTHOR]

Simvastatin, an HMG-CoA reductase inhibitor, has lung vascular-protective effects that are associated with decreased agonist-induced integrin β4 (ITGB4) tyrosine phosphorylation. Accordingly, we hypothesized that endothelial cell (EC) protection by simvastatin is dependent on these effects and sought to further characterize the functional role of ITGB4 as a mediator of EC protection in the setting of excessive mechanical stretch at levels relevant to ventilator-induced lung injury (VILI). Initially, early ITGB4 tyrosine phosphorylation was confirmed in human pulmonary artery EC subjected to excessive cyclic stretch (18% CS). EC overexpression of mutant ITGB4 with specific tyrosines mutated to phenylalanine (Y1440, Y1526 Y1640, or Y1422) resulted in significantly attenuated CS-induced cytokine expression (IL6, IL-8, MCP-1, and RANTES). In addition, EC overexpression of ITGB4 constructs with specific structural deletions also resulted in significantly attenuated CS-induced inflammatory cytokine expression compared to overexpression of wildtype ITGB4. Finally, mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were found to have attenuated lung injury after VILI-challenge (VT = 40 ml/kg, 4 h). Our results provide mechanistic insights into the anti-inflammatory properties of statins and may ultimately lead to novel strategies targeted at ITGB4 signaling to treat VILI. [ABSTRACT FROM AUTHOR]

Abstract Acute lung injury (ALI) is characterized by endothelial barrier disruption resulting in increased vascular permeability. As focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, is involved in endothelial cell (EC) barrier regulation, we hypothesized that FAK inhibition could attenuate agonist-induced EC barrier disruption relevant to ALI. Human lung EC were pretreated with one of three pharmacologic FAK inhibitors, PF-573,228 (PF-228, 10 μM), PF-562,271 (PF-271, 5 μM) or NVP-TAE226 (TAE226, 5 μM) for 30 min prior to treatment with thrombin (1 U/ml, 30 min). Western blotting confirmed attenuated thrombin-induced FAK phosphorylation associated with all three inhibitors. Subsequently, EC were pretreated with either PF-228 (10 μM), TAE226 (5 μM) or PF-271 (5 μM) for 30 min prior to thrombin stimulation (1 U/ml) followed by measurements of barrier integrity by transendothelial electrical resistance (TER). Separately, EC grown in transwell inserts prior to thrombin (1 U/ml) with measurements of FITC-dextran flux after 30 min confirmed a significant attenuation of thrombin-induced EC barrier disruption by PF-228 alone. Finally, in a murine ALI model induced by LPS (1.25 mg/ml, IT), rescue treatment with PF-228 was associated with significantly reduced lung injury. Our findings PF-228, currently being studied in clinical trials, may serve as a novel and effective therapeutic agent for ALI. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]