Christine U. Vohwinkel

Elevated CO2 Levels Cause Mitochondrial Dysfunction and Impair Cell Proliferation

Background: Cells are exposed to elevated levels of CO2 (hypercapnia) in many diseases. Results: Hypercapnia decreased cell proliferation, which was prevented with α-ketoglutarate, IDH2 overexpression, and microRNA-183 inhibition. Conclusion: Hypercapnia causes mitochondrial dysfunction by up-regulation of microRNA-183, which decreases the levels of IDH2. Significance: Hypercapnia causes mitochondrial dysfunction, which is relevant for patients with lung diseases. Elevated CO2 concentrations (hypercapnia) occur in patients with severe lung diseases. Here, we provide evidence that high CO2 levels decrease O2 consumption and ATP production and impair cell proliferation independently of acidosis and hypoxia in fibroblasts (N12) and alveolar epithelial cells (A549). Cells exposed to elevated CO2 died in galactose medium as well as when glucose-6-phosphate isomerase was knocked down, suggesting mitochondrial dysfunction. High CO2 levels led to increased levels of microRNA-183 (miR-183), which in turn decreased expression of IDH2 (isocitrate dehydrogenase 2). The high CO2-induced decrease in cell proliferation was rescued by α-ketoglutarate and overexpression of IDH2, whereas proliferation decreased in normocapnic cells transfected with siRNA for IDH2. Also, overexpression of miR-183 decreased IDH2 (mRNA and protein) as well as cell proliferation under normocapnic conditions, whereas inhibition of miR-183 rescued the normal proliferation phenotype in cells exposed to elevated levels of CO2. Accordingly, we provide evidence that high CO2 induces miR-183, which down-regulates IDH2, thus impairing mitochondrial function and cell proliferation. These results are of relevance to patients with hypercapnia such as those with chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchopulmonary dysplasia, and muscular dystrophies.

Hypercapnia impairs lung neutrophil function and increases mortality in murine Pseudomonas pneumonia

Hypercapnia, an elevation of the level of carbon dioxide (CO2) in blood and tissues, is a marker of poor prognosis in chronic obstructive pulmonary disease and other pulmonary disorders. We previously reported that hypercapnia inhibits the expression of TNF and IL-6 and phagocytosis in macrophages in vitro. In the present study, we determined the effects of normoxic hypercapnia (10% CO2, 21% O2, and 69% N2) on outcomes of Pseudomonas aeruginosa pneumonia in BALB/c mice and on pulmonary neutrophil function. We found that the mortality of P. aeruginosa pneumonia was increased in 10% CO2-exposed compared with air-exposed mice. Hypercapnia increased pneumonia mortality similarly in mice with acute and chronic respiratory acidosis, indicating an effect unrelated to the degree of acidosis. Exposure to 10% CO2 increased the burden of P. aeruginosa in the lungs, spleen, and liver, but did not alter lung injury attributable to pneumonia. Hypercapnia did not reduce pulmonary neutrophil recruitment during infection, but alveolar neutrophils from 10% CO2-exposed mice phagocytosed fewer bacteria and produced less H2O2 than neutrophils from air-exposed mice. Secretion of IL-6 and TNF in the lungs of 10% CO2-exposed mice was decreased 7 hours, but not 15 hours, after the onset of pneumonia, indicating that hypercapnia inhibited the early cytokine response to infection. The increase in pneumonia mortality caused by elevated CO2 was reversible when hypercapnic mice were returned to breathing air before or immediately after infection. These results suggest that hypercapnia may increase the susceptibility to and/or worsen the outcome of lung infections in patients with severe lung disease.

Ubiquitination Participates in the Lysosomal Degradation of Na,K-ATPase in Steady-State Conditions

The alveolar epithelial cell (AEC) Na,K-ATPase contributes to vectorial Na(+) transport and plays an important role in keeping the lungs free of edema. We determined, by cell surface labeling with biotin and immunofluorescence, that approximately 30% of total Na,K-ATPase is at the plasma membrane of AEC in steady-state conditions. The half-life of the plasma membrane Na,K-ATPase was about 4 hours, and the incorporation of new Na,K-ATPase to the plasma membrane was Brefeldin A sensitive. Both protein kinase C (PKC) inhibition with bisindolylmaleimide (10 microM) and infection with an adenovirus expressing dominant-negative PKCzeta prevented Na,K-ATPase degradation. In cells expressing the Na,K-ATPase alpha1-subunit lacking the PKC phosphorylation sites, the plasma membrane Na,K-ATPase had a moderate increase in half-life. We also found that the Na,K-ATPase was ubiquitinated in steady-state conditions and that proteasomal inhibitors prevented its degradation. Interestingly, mutation of the four lysines described to be necessary for ubiquitination and endocytosis of the Na,K-ATPase in injurious conditions did not have an effect on its half-life in steady-state conditions. Lysosomal inhibitors prevented Na,K-ATPase degradation, and co-localization of Na,K-ATPase and lysosomes was found after labeling and chasing the plasma membrane Na,K-ATPase for 4 hours. Accordingly, we provide evidence suggesting that phosphorylation and ubiquitination are necessary for the steady-state degradation of the plasma membrane Na,K-ATPase in the lysosomes in alveolar epithelial cells.

Hypoxia signaling during acute lung injury

Acute lung injury (ALI) is an inflammatory lung disease that manifests itself in patients as acute respiratory distress syndrome and thereby contributes significantly to the morbidity and mortality of patients experiencing critical illness. Even though it may seem counterintuitive, as the lungs are typically well-oxygenated organs, hypoxia signaling pathways have recently been implicated in the resolution of ALI. For example, functional studies suggest that transcriptional responses under the control of the hypoxia-inducible factor (HIF) are critical in optimizing alveolar epithelial carbohydrate metabolism, and thereby dampen lung inflammation during ALI. In the present review we discuss functional roles of oxygenation, hypoxia and HIFs during ALI, mechanisms of how HIFs are stabilized during lung inflammation, and how HIFs can mediate lung protection during ALI.

Detrimental ELAVL-1/HuR-dependent GSK3β mRNA stabilization impairs resolution in acute respiratory distress syndrome

A hallmark of acute respiratory distress syndrome (ARDS) is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK) 3β during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3β could play a functional role in ARDS resolution. To address this hypothesis, we performed an in silico analysis to identify regulatory genes whose expression correlation to GSK3β messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3β, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like) as a central component in a likely GSK3β signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3β mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3β at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3β. Together, our findings indicate a previously unknown interaction between GSK3β and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3β pathways as a novel ARDS treatment approach.

Hydroxylation-independent HIF-1α stabilization through PKA: A new paradigm for hypoxia signaling

PKA-mediated stimulation and stabilization of HIF-1α may promote adaptive and anti-inflammatory tissue responses. In this issue of Science Signaling, Bullen et al. demonstrate that protein kinase A (PKA) phosphorylates and stimulates the transcriptional activity of hypoxia-inducible transcription factor–1α (HIF-1α). This finding may have implications in diseases processes that occur at the interface of hypoxia and inflammation, where HIF-1α stabilization can function to dampen hypoxia-driven inflammation.

Restoration of Megalin-Mediated Clearance of Alveolar Protein as a Novel Therapeutic Approach for Acute Lung Injury

Abstract Acute respiratory distress syndrome constitutes a significant disease burden with regard to both morbidity and mortality. Current therapies are mostly supportive and do not address the underlying pathophysiologic mechanisms. Removal of protein‐rich alveolar edema—a clinical hallmark of acute respiratory distress syndrome—is critical for survival. Here, we describe a transforming growth factor (TGF)‐&bgr;‐triggered mechanism, in which megalin, the primary mediator of alveolar protein transport, is negatively regulated by glycogen synthase kinase (GSK) 3&bgr;, with protein phosphatase 1 and nuclear inhibitor of protein phosphatase 1 being involved in the signaling cascade. Inhibition of GSK3&bgr; rescued transepithelial protein clearance in primary alveolar epithelial cells after TGF‐&bgr; treatment. Moreover, in a bleomycin‐based model of acute lung injury, megalin+/‐ animals (the megalin‐/‐ variant is lethal due to postnatal respiratory failure) showed a marked increase in intra‐alveolar protein and more severe lung injury compared with wild‐type littermates. In contrast, wild‐type mice treated with the clinically relevant GSK3&bgr; inhibitors, tideglusib and valproate, exhibited significantly decreased alveolar protein concentrations, which was associated with improved lung function and histopathology. Together, we discovered that the TGF‐&bgr;‐GSK3&bgr;‐megalin axis is centrally involved in disturbances of alveolar protein clearance in acute lung injury and provide preclinical evidence for therapeutic efficacy of GSK3&bgr; inhibition.

TGF-β inhibits alveolar protein transport by promoting shedding, regulated intramembrane proteolysis, and transcriptional downregulation of megalin

Disruption of the alveolar-capillary barrier is a hallmark of acute respiratory distress syndrome (ARDS) that leads to the accumulation of protein-rich edema in the alveolar space, often resulting in comparable protein concentrations in alveolar edema and plasma and causing deleterious remodeling. Patients who survive ARDS have approximately three times lower protein concentrations in the alveolar edema than nonsurvivors; thus the ability to remove excess protein from the alveolar space may be critical for a positive outcome. We have recently shown that clearance of albumin from the alveolar space is mediated by megalin, a 600-kDa transmembrane endocytic receptor and member of the low-density lipoprotein receptor superfamily. In the currents study, we investigate the molecular mechanisms by which transforming growth factor-β (TGF-β), a key molecule of ARDS pathogenesis, drives downregulation of megalin expression and function. TGF-β treatment led to shedding and regulated intramembrane proteolysis of megalin at the cell surface and to a subsequent increase in intracellular megalin COOH-terminal fragment abundance resulting in transcriptional downregulation of megalin. Activity of classical protein kinase C enzymes and γ-secretase was required for the TGF-β-induced megalin downregulation. Furthermore, TGF-β-induced shedding of megalin was mediated by matrix metalloproteinases (MMPs)-2, -9, and -14. Silencing of either of these MMPs stabilized megalin at the cell surface after TGF-β treatment and restored normal albumin transport. Moreover, a direct interaction of megalin with MMP-2 and -14 was demonstrated, suggesting that these MMPs may function as novel sheddases of megalin. Further understanding of these mechanisms may lead to novel therapeutic approaches for the treatment of ARDS.

Acute respiratory distress syndrome following cardiovascular surgery: current concepts and novel therapeutic approaches

Purpose of review This review gives an update on current treatment options and novel concepts on the prevention and treatment of the acute respiratory distress syndrome (ARDS) in cardiovascular surgery patients. Recent findings The only proven beneficial therapeutic options in ARDS are those that help to prevent further ventilator-induced lung injury, such as prone position, use of lung-protective ventilation strategies, and extracorporeal membrane oxygenation. In the future also new approaches like mesenchymal cell therapy, activation of hypoxia-elicited transcription factors or targeting of purinergic signaling may be successful outside the experimental setting. Owing to the so far limited treatment options, it is of great importance to determine patients at risk for developing ARDS already perioperatively. In this context, serum biomarkers and lung injury prediction scores could be useful. Summary Preventing ARDS as a severe complication in the cardiovascular surgery setting may help to reduce morbidity and mortality. As cardiovascular surgery patients are of greater risk to develop ARDS, preventive interventions should be implemented early on. Especially, use of low tidal volumes, avoiding of fluid overload and restrictive blood transfusion regimes may help to prevent ARDS.

Capturing the multifactorial nature of ARDS – “Two-hit” approach to model murine acute lung injury

Severe acute respiratory distress syndrome (ARDS) presents typically with an initializing event, followed by the need for mechanical ventilation. Most animal models of ALI are limited by the fact that they focus on a singular cause of acute lung injury (ALI) and therefore fail to mimic the complex, multifactorial pathobiology of ARDS. To better capture this scenario, we provide a comprehensive characterization of models of ALI combining two injuries: intra tracheal (i.t.) instillation of LPS or hypochloric acid (HCl) followed by ventilator‐induced lung injury (VILI). We hypothesized, that mice pretreated with LPS or HCl prior to VILI and thus receiving a (“two‐hit injury”) will sustain a superadditive lung injury when compared to VILI. Mice were allocated to following treatment groups: control with i.t. NaCl, ventilation with low peak inspiratory pressure (PIP), i.t. HCl, i.t. LPS, VILI (high PIP), HCl i.t. followed by VILI and LPS i.t. followed by VILI. Severity of injury was determined by protein content and MPO activity in bronchoalveolar lavage (BAL), the expression of inflammatory cytokines and histopathology. Mice subjected to VILI after HCl or LPS instillation displayed augmented lung injury, compared to singular lung injury. However, mice that received i.t. LPS prior to VILI showed significantly increased inflammatory lung injury compared to animals that underwent i.t. HCl followed by VILI. The two‐hit lung injury models described, resulting in additive but differential acute lung injury recaptures the clinical relevant multifactorial etiology of ALI and could be a valuable tool in translational research.