Background: Proteolytic degradation of epithelial sodium channels (ENaC) assists in regulating net salt and water balance in lung epithelia. Results: H 2 O 2 increases surface expression of ␣-ENaC, transepithelial Na transport, and alveolar fluid clearance via redoxsensitive Nedd8. Conclusion: Redox-sensitive Nedd8 is involved in the ubiquitination of lung ENaC. Significance: Understanding ROS-mediated signaling of lung ENaC is crucial for understanding pulmonary physiology and pathology.

Background Varying strategies are currently being evaluated to develop tissue-engineered constructs for the treatment of ischemic heart disease. This study examines an angiogenic and biodegradable cardiac construct seeded with neonatal cardiomyocytes for the treatment of chronic heart failure (CHF). Methods We evaluated a neonatal cardiomyocyte (NCM)-seeded three-dimensional fibroblast construct (3DFC) in vitro for the presence of functional gap junctions and the potential of the NCM-3DFC to restore left ventricular (LV) function in an in vivo rat model of CHF at 3 weeks after permanent left coronary artery ligation. Results The NCM-3DFC demonstrated extensive cell-to-cell connectivity following dye injection. At 5 days in culture, the patch contracted spontaneously in a rhythmic and directional fashion, at 43±3 beats/min with a mean displacement of 1.3±0.3 mm and contraction velocity of 0.8±0.2 mm/sec. The seeded patch could be electrically paced at near physiological rates (270±30 beats/min) while maintaining coordinated, directional contractions. Three weeks after implantation, the NCM-3DFC improved LV function by increasing (p<0.05) ejection fraction 26%, cardiac index 33%, dP/dt(+) 25%, dP/dt(−) 23%, and peak developed pressure (PDP) 30%, while decreasing (p<0.05) LV end diastolic pressure 38% and the time constant of relaxation (Tau) 16%. Eighteen weeks post implantation, the NCM-3DFC improved LV function by increasing (p<0.05) ejection fraction 54%, mean arterial pressure 20%, dP/dt(+) 16%, dP/dt(−) 34% and PDP 39%. Conclusion This study demonstrates that a multicellular, electromechanically organized, cardiomyocyte scaffold, constructed in vitro by seeding NCM onto 3DFC, can improve LV function long-term when implanted in rats with CHF.

Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS). However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC) via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH) would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a ‘pro-injury’ environment in the alcohol lung.

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91 phox signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 6 3.03 mg versus 2.48 6 0.53 mg), lower sRAGE (1,205 6 292 pg/ml versus 1,910 6 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 6 14 ml versus 133 6 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 6 0.08 to 0.83 6 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 6 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 6 0.05 to 0.53 6 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 6 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47 phox and increased gp91 phox -dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91 phox abrogated the response.Keywords: acute respiratory distress syndrome; chronic obstructive pulmonary disease; pulmonary edema; alveolar microenvironment; lung injury Clinical RelevanceReceptor for advanced glycation end-products (RAGE) plays a critical role in regulating inflammation in the lung and may be a therapeutic target in the treatment of acute and chronic lung diseases. Herein we describe the signal transduction pathway through which RAGE regulates the epithelial sodium channel.

Exosomes are nano-sized vesicles that are secreted into the extracellular environment. These vesicles contain various biological effector molecules that can regulate intracellular signaling pathways in recipient cells. The aim of this study was to examine a correlation between exosomal cathepsin B activity and the receptor for advanced glycation end-products (RAGE). Type 1 alveolar epithelial (R3/1) cells were treated with or without hydrogen peroxide and exosomes isolated from the cell conditioned media were characterized by NanoSight analysis. Lipidomic and proteomic analysis showed exosomes released from R3/1 cells exposed to oxidative stress induced by hydrogen peroxide or vehicle differ in their lipid and protein content, respectively. Cathepsin B activity was detected in exosomes isolated from hydrogen peroxide treated cells. The mRNA and protein expression of RAGE increased in cultured R3/1 cells treated with exosomes containing active cathepsin B while depletion of exosomal cathepsin B attenuated RAGE mRNA and protein expression. These results suggest exosomal cathepsin B regulates RAGE in type 1 alveolar cells under conditions of oxidative stress. J. Cell. Biochem. 119: 599-606, 2018. © 2017 Wiley Periodicals, Inc.