Bacterial pneumonia-induced shedding of epithelial heparan sulfate inhibits the bactericidal activity of cathelicidin in a murine model.

Bacterial pneumonia is a common clinical syndrome leading to significant morbidity and mortality worldwide. In the current study, we investigate a novel, multidirectional relationship between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Using an in vivo pneumonia model, we demonstrate that highly sulfated heparan sulfate (HS) oligosaccharides are shed into the airspaces in response to MRSA pneumonia. In vitro, these HS oligosaccharides do not directly alter MRSA growth or gene transcription. However, in the presence of an antimicrobial peptide (cathelicidin), increasing concentrations of HS inhibit the bactericidal activity of cathelicidin against MRSA as well as other nosocomial pneumonia pathogens (Klebsiella pneumoniae and Pseudomonas aeruginosa) in a dose-dependent manner. Surface plasmon resonance shows avid binding between HS and cathelicidin with a dissociation constant of 0.13 µM. These findings highlight a complex relationship in which shedding of airspace HS may hamper host defenses against nosocomial infection via neutralization of antimicrobial peptides. These findings may inform future investigation into novel therapeutic targets designed to restore local innate immune function in patients suffering from primary bacterial pneumonia.NEW & NOTEWORTHY Primary Staphylococcus aureus pneumonia causes pulmonary epithelial heparan sulfate (HS) shedding into the airspace. These highly sulfated HS fragments do not alter bacterial growth or transcription, but directly bind with host antimicrobial peptides and inhibit the bactericidal activity of these cationic polypeptides. These findings highlight a complex local interaction between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of bacterial pneumonia.

The role of the alveolar epithelial glycocalyx in acute respiratory distress syndrome.

The alveolar epithelial glycocalyx is a dense anionic layer of glycosaminoglycans (GAGs) and proteoglycans that lines the apical surface of the alveolar epithelium. In contrast to the pulmonary endothelial glycocalyx, which has well-established roles in vascular homeostasis and septic organ dysfunction, the alveolar epithelial glycocalyx is less understood. Recent preclinical studies demonstrated that the epithelial glycocalyx is degraded in multiple murine models of acute respiratory distress syndrome (ARDS), particularly those that result from inhaled insults (so-called "direct" lung injury), leading to shedding of GAGs into the alveolar airspaces. Epithelial glycocalyx degradation also occurs in humans with respiratory failure, as quantified by analysis of airspace fluid obtained from ventilator heat moisture exchange (HME) filters. In patients with ARDS, GAG shedding correlates with the severity of hypoxemia and is predictive of the duration of respiratory failure. These effects may be mediated by surfactant dysfunction, as targeted degradation of the epithelial glycocalyx in mice was sufficient to cause increased alveolar surface tension, diffuse microatelectasis, and impaired lung compliance. In this review, we describe the structure of the alveolar epithelial glycocalyx and the mechanisms underlying its degradation during ARDS. We additionally review the current state of knowledge regarding the attributable effect of epithelial glycocalyx degradation in lung injury pathogenesis. Finally, we address glycocalyx degradation as a potential mediator of ARDS heterogeneity, and the subsequent value of point-of-care quantification of GAG shedding to potentially identify patients who are most likely to respond to pharmacological agents aimed at attenuating glycocalyx degradation.

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 ß-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.

Imatinib attenuates inflammation and vascular leak in a clinically relevant two-hit model of acute lung injury.

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.

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 ß-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.

Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.

Advancing Precision Medicine for the Diagnosis and Treatment of Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a common and life-threatening cause of respiratory failure. Despite decades of research, there are no effective pharmacologic therapies to treat this disease process and mortality remains high. The shortcomings of prior translational research efforts have been increasingly attributed to the heterogeneity of this complex syndrome, which has led to an increased focus on elucidating the mechanisms underlying the interpersonal heterogeneity of ARDS. This shift in focus aims to move the field towards personalized medicine by defining subgroups of ARDS patients with distinct biology, termed endotypes, to quickly identify patients that are most likely to benefit from mechanism targeted treatments. In this review, we first provide a historical perspective and review the key clinical trials that have advanced ARDS treatment. We then review the key challenges that exist with regards to the identification of treatable traits and the implementation of personalized medicine approaches in ARDS. Lastly, we discuss potential strategies and recommendations for future research that we believe will aid in both understanding the molecular pathogenesis of ARDS and the development of personalized treatment approaches.

Relationship Between a State-Directed Lockdown on Non-COVID-19-related ICU Admissions and Mortality in a Multicenter Colorado Healthcare System.

Colorado issued a month long statewide lockdown on March 26, 2020, during the initial surge of the COVID-19 pandemic. The impact of this mandate on non-COVID-19 ICU admission rates and outcomes is unclear. DESIGN: We performed a retrospective analysis of all medical ICU admissions in the University of Colorado Health System in four predefined periods: 1) prepandemic (2 mo prior to lockdown period 1); 2) mandated lockdown from March 26 to April 26, 2020 (period 2); 3) between surges (period 3); and 4) nonmandated lockdown surge (between November 1, 2020, and March 31, 2021, period 4). SETTING: Nonsurgical ICU admissions at the University of Colorado Health Systems, including 10 hospitals throughout Colorado. SUBJECTS: All ICU admissions in four predefined time periods. MEASUREMENTS AND MAIN RESULTS: We included 13,787 patients who were admitted during the four study periods. The 28-day mortality rates for non-COVID-19 ICU admissions following index ICU admission were 13.6%, 18.0%, 13.5%, and 16.0% across periods 1-4, respectively. However, the increased odds in non-COVID-19 ICU mortality during the mandated lockdown period relative to prepandemic 1 (odds ratio [OR], 1.39; 95% CI, 1.11-1.72; p = 0.0.04) was attenuated and nonsignificant after adjustment for demographics, comorbidities, diagnosis flags, and severity (OR, 1.15; 95% CI, 0.89-1.48; p = 0.27). Similar results were found in time-to-event analyses. The most common diagnosis in each time period was acute respiratory failure (ARF), and we found it to have increased during lockdown (p < 0.001), whereas sepsis admissions increased during and decreased after lockdown (p = 0.004). Admissions for alcohol withdrawal syndrome (AWS) increased during lockdown and 6 months afterwards (p = 0.005). CONCLUSIONS: For non-COVID-19-related ICU admissions, mortality rate was similar before, during, and after Colorado's month long lockdown after confounder adjustment, including typical ICU admission flags. Primary admission diagnoses shifted throughout the predefined study periods with more admissions for severe critical diagnoses (i.e., ARF, sepsis, AWS) occurring during the mandated lockdown and nonmandated lockdown periods compared with the prepandemic and between surge period. This would suggest that the perceived increase in mortality during the lockdown for non-COVID-19 ICU admissions may be related to a shift inpatient demographics.

Alveolar epithelial glycocalyx degradation mediates surfactant dysfunction and contributes to acute respiratory distress syndrome.

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.

ABO blood type: a window into the genetics of acute respiratory distress syndrome susceptibility.

The genetic factors that determine a patient's risk for developing the acute respiratory distress syndrome (ARDS) remain understudied. In this issue of the JCI, Reilly and colleagues analyzed data from three cohorts of critically ill patients and observed an association between the ABO allele A1 and the onset of moderate-severe ARDS. This association was most notable in patients with non-pulmonary sepsis (an indirect, vasculature-targeted mechanism of lung injury) and persisted in patients who lacked epithelial expression of the A antigen, suggesting an endothelial mechanism of A1-associated ARDS susceptibility. Critically ill patients with blood type A had increased circulating concentrations of endothelium-derived glycoproteins such as von Willebrand factor and soluble thrombomodulin, and marginal lungs from blood type A donors were less likely to recover function during ex vivo perfusion. These findings implicate A antigen glycosylation of endothelial cells as a critical, genetically determined risk factor for indirect lung injury that may contribute to the mechanistic heterogeneity of ARDS.

Arg mediates LPS-induced disruption of the pulmonary endothelial barrier.

Acute Respiratory Distress Syndrome (ARDS) is a devastating disease process that involves dysregulated inflammation and decreased alveolar-capillary barrier function. Despite increased understanding of the pathophysiology, no effective targeted therapies exist to treat ARDS. Recent preclinical studies suggest that the multi-tyrosine kinase inhibitor, imatinib, which targets the Abl kinases c-Abl and Arg, has the potential to restore endothelial dysfunction caused by inflammatory agonists. Prior work demonstrates that imatinib attenuates LPS (lipopolysaccharide)-induced vascular leak and inflammation; however, the mechanisms underlying these effects remain incompletely understood. In the current study, we demonstrate that imatinib inhibits LPS-induced increase in the phosphorylation of CrkL, a specific substrate of Abl kinases, in human pulmonary endothelial cells. Specific silencing of Arg, and not c-Abl, attenuated LPS-induced pulmonary vascular permeability as measured by electrical cellular impedance sensing (ECIS) and gap formation assays. In addition, direct activation of Abl family kinases with the small molecule activator DPH resulted in endothelial barrier disruption that was attenuated by Arg siRNA. In complementary studies to characterize the mechanisms by which Arg mediates endothelial barrier function, Arg silencing was found to inhibit LPS-induced disruption of adherens junctions and phosphorylation of myosin light chains (MLC). Overall, these results characterize the mechanisms by which imatinib protects against LPS-induced endothelial barrier disruption and suggest that Arg inhibition may represent a novel strategy to enhance endothelial barrier function.

Single nucleotide polymorphisms in the MYLKP1 pseudogene are associated with increased colon cancer risk in African Americans.

INTRODUCTION: Pseudogenes are paralogues of functional genes historically viewed as defunct due to either the lack of regulatory elements or the presence of frameshift mutations. Recent evidence, however, suggests that pseudogenes may regulate gene expression, although the functional role of pseudogenes remains largely unknown. We previously reported that MYLKP1, the pseudogene of MYLK that encodes myosin light chain kinase (MLCK), is highly expressed in lung and colon cancer cell lines and tissues but not in normal lung or colon. The MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. In this study, we further validate MYLKP1 as an oncogene via elucidation of the functional role of MYLKP1 genetic variants in colon cancer risk. METHODS: Proliferation and migration assays were performed in MYLKP1-transfected colon and lung cancer cell lines (H441, A549) and commercially-available normal lung and colon cells. Fourteen MYLKP1 SNPs (MAFs >0.01) residing within the 4 kb MYLKP1 promoter region, the core 1.4 kb of MYLKP1 gene, and a 4 kb enhancer region were selected and genotyped in a colorectal cancer cohort. MYLKP1 SNP influences on activity of MYLKP1 promoter (2kb) was assessed by dual luciferase reporter assay. RESULTS: Cancer cell lines, H441 and A549, exhibited increased MYLKP1 expression, increased MYLKP1 luciferase promoter activity, increased proliferation and migration. Genotyping studies identified two MYLKP1 SNPs (rs12490683; rs12497343) that significantly increase risk of colon cancer in African Americans compared to African American controls. Rs12490683 and rs12497343 further increase MYLKP1 promoter activity compared to the wild type MYLKP1 promoter. CONCLUSION: MYLKP1 is a cancer-promoting pseudogene whose genetic variants differentially enhance cancer risk in African American populations.

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.

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 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.

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.

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.