Immature phenotypes of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) limit the utility of these cells in clinical application and basic research. During cardiac development, postnatal cardiomyocytes experience high oxygen tension along with a concomitant downregulation of hypoxia-inducible factor 1α (HIF-1α), leading to increased fatty acid oxidation (FAO). We hypothesized that targeting HIF-1α alone or in combination with other metabolic regulators could promote the metabolic maturation of hiPSC-CMs. We examined the effect of HIF-1α inhibition on the maturation of hiPSC-CMs and investigated a multipronged approach to promote hiPSC-CM maturation by combining HIF-1α inhibition with molecules that target key pathways involved in the energy metabolism. Cardiac spheres of highly-enriched hiPSC-CMs were treated with a HIF-1α inhibitor alone or in combination with an agonist of peroxisome proliferator activated receptor α (PPARα) and three postnatal factors (triiodothyronine hormone T3, insulin-like growth factor-1 and dexamethasone). HIF-1α inhibition significantly increased FAO and basal and maximal respiration of hiPSC-CMs. Combining HIF-1α inhibition with PPARα activation and the postnatal factors further increased FAO and improved mitochondrial maturation in hiPSC-CMs. Compared with mock-treated cultures, the cultures treated with the five factors had increased mitochondrial content and contained more cells with mitochondrial distribution throughout the cells, which are features of more mature cardiomyocytes. Consistent with these observations, a number of transcriptional regulators of mitochondrial metabolic processes were upregulated in hiPSC-CMs treated with the five factors. Furthermore, these cells had significantly increased Ca2+ transient kinetics and contraction and relaxation velocities, which are functional features for more mature cardiomyocytes. Therefore, targeting HIF-1α in combination with other metabolic regulators significantly improves the metabolic maturation of hiPSC-CMs.

Background: We hypothesized that maternal alcohol use occurs in pregnancies that end prematurely and that in utero alcohol exposure is associated with an increased risk of morbidities of premature newborns. Methods: In an observational study of mothers who delivered very low birth weight newborns (VLBW) ≤1,500 g, maternal alcohol use was determined via a standardized administered questionnaire. We compared the effect of maternal drinking on the odds of developing late-onset sepsis (LOS), bronchopulmonary dysplasia (BPD), death, BPD or Death days on oxygen or any morbidity (either LOS, BPD or death). The effect of drinking amounts (light versus heavy) was also evaluated. Results: A total of 129 subjects who delivered 143 VLBW newborns were enrolled. Approximately 1 in 3 (34%) subjects reported drinking alcohol during the first trimester ("exposed"). Within the exposed group, 15% reported drinking ≥7. drinks/week ("heavy") and 85% of the subjects reported drinking < 7. drinks/week ("light"). When controlling for maternal age, drug or tobacco use during pregnancy and neonatal gestational age, any drinking increased the odds of BPD or Death and any morbidity. Furthermore, light or heavy drinking increased the odds of BPD or Death and any morbidity, whereas heavy drinking increased the odds of LOS. Conclusions: In utero alcohol exposure during the first trimester occurred in 34% of VLBW newborns. Maternal drinking in the first trimester was associated with significantly increased odds of neonatal morbidity. Further studies are warranted to determine the full effect of . in utero alcohol exposure on the adverse outcomes of VLBW premature newborns.

Background Alterations in inflammatory mediators are an important finding in neonates who develop bronchopulmonary dysplasia (BPD); however, there is a lack of research examining the relationship between multiple inflammatory mediators in premature neonates and the development of BPD. Objective This study investigated whether the distribution of 12 inflammatory mediators detected in the tracheal aspirate (TA) of neonates within 24 hours of birth could differentiate between neonates who did and did not develop BPD. Study Design TA samples were collected from 27 very low birth weight neonates (BPD+ =11) and the concentrations of 12 biomarkers associated with BPD were determined. Linear discriminate analysis (LDA) was used to classify neonates into two outcome groups. Result LDA based on the 12 measured biomarkers displayed a significant level of discriminant function (p=0.007). Conclusion Using linear discriminant analysis, predictive models of BPD can be generated. Our results suggest that multiple inflammatory mediators collected within 24 hours of birth may be used to classify neonates into who will and will not develop BPD.

In utero alcohol exposure dramatically increases the risk of premature delivery. However, the majority of premature and term newborns exposed to alcohol remain undetected by medical caregivers. There is a desperate need for reliable and accurate biomarkers of alcohol exposure for the term and premature newborn population. The inability to identify the exposed newborn severely limits our understanding of alcohol's pathophysiological effects on developing organs such as the lung. This chapter will review potential advancements in future biomarkers of alcohol exposure for the newborn population. We will discuss alcohol's effects on redox homeostasis and cellular development of the neonatal lung. Finally, we will present the evidence describing in utero alcohol's derangement of innate and adaptive immunity and risk for infectious complications in the lung. Continued investigations into the identification and understanding of the mechanisms of alcohol-induced alterations in the premature lung will advance the care of this vulnerable patient population.

Bronchopulmonary dysplasia (BPD) is a frequent complication of premature newborns, particularly very low birth-weight babies (< 1500 g). Undoubtedly multiple mechanisms contribute to the adverse outcomes associated with BPD but oxidative stress is one causative factor. In this issue of Free Radical Biology & Medicine, Lavoie et al describe the increased peroxide generation when the multivitamin solution used for nutritional support, total perenteral nutrition (TPN), is exposed to ambient light. Since the premature newborn has limited antioxidant capacity, this increased oxidative burden from the TPN becomes increasingly significant. Infusion of this light-exposed solution in a newborn guinea pig decreased lung tissue vitamin C but vitamin E. When the multivitamin and lipid solutions were mixed and then exposed to light, alveolarization of the developing lung was decreased. This study by Lavoie et al highlights simple measures that may potentially decrease the oxidant burden delivered to this vulnerable population and improve alveolarization.

Background Alcohol consumption is associated with oxidative stress in multiple tissues in vivo, yet the effect of chronic alcohol intake on intestinal redox state has received little attention. In this study, we investigated the redox status of 2 major intracellular redox regulating couples: glutathione (GSH)/glutathione disulfide (GSSG) and cysteine (Cys)/cystine (CySS) in a rat model of chronic alcohol ingestion. Methods Sprague-Dawley rats were fed the liquid Lieber-DeCarli diet consisting of 36% ethanol of total calories for 6 weeks. Control rats were pair-fed with an isocaloric, ethanol-free liquid diet. Defined mucosal samples from the jejunum, ileum, and colon were obtained and analyzed by high-performance liquid chromatography (HPLC) for GSH and Cys pool redox status. Mucosal free malondialdehyde (MDA) was measured as an indicator of lipid peroxidation. Results In the ethanol-fed rats, Cys and mixed disulfide (GSH-Cys) were significantly decreased in all 3 segments of intestinal mucosa. Free MDA was increased in jejunal but not in ileal or colonic mucosa. Chronic ethanol ingestion significantly increased mucosal GSH concentration in association with a more reducing GSH/GSSG redox potential in the jejunum, but these indices were unchanged in the ileum. In the colon, chronic ethanol ingestion increased oxidant stress as suggested by decreased GSH and oxidized GSH/GSSG redox potential. Conclusions Chronic alcohol intake differentially alters the mucosal redox status in proximal to distal intestinal segments in rats. Such changes may reflect different adaptability of these intestinal segments to the oxidative stress challenge induced by chronic ethanol ingestion.

Summary Background Increased systemic oxidant stress contributes to a variety of maternal complications of pregnancy. Although the antioxidant glutathione (GSH) and its oxidized component glutathione disulfide (GSSG) have been demonstrated to be significantly altered in the adult alcoholic, the effects of maternal alcohol use during pregnancy on oxidant stress in the post partum female remain under investigation. We hypothesized that maternal alcohol use would increase systemic oxidant stress in the pregnant female, evidenced by an oxidized systemic GSH redox potential. Methods As a subset analysis of a larger maternal language study, we evaluated the effects of alcohol consumption during pregnancy on the systemic GSH redox status of the post partum female. Using an extensive maternal questionnaire, post partum women where queried regarding their alcohol consumption during pregnancy. Any drinking, the occurrence of drinking > 3 drinks/occasion, and excessive drinking of >5 drinks/occasion during pregnancy were noted. Using HPLC, maternal plasma samples were analyzed for GSH, oxidized GSSH and the redox potential of the GSH/GSSG antioxidant pair calculated. Results Maternal alcohol use occurred in 25% (83/321) of our study sample. Two in ten women reported consuming > 3 drinks/occasion during pregnancy, while one in ten women reported consuming excessive alcohol at > 5 drinks/occasion. Any alcohol use during pregnancy significantly decreased plasma GSH (p<.05), while alcohol at >3 drinks/occasion or > 5 drinks/occasion significantly decreased plasma GSH concentration (p<0.05), increased the percent of oxidized GSSG (p<0.05), and substantially oxidized the plasma GSH redox potential (p<0.05). Conclusions Alcohol use during pregnancy, particularly at levels of more than 3 drinks/occasion, caused significant oxidation of the systemic GSH system in the post partum women. The clinical ramifications of the observed alcohol-induced oxidation of the GSH redox system on high risk pregnancies or on the exposed offspring require more accurate identification and further investigation.

Alcoholic patients have an increased risk of respiratory infections, which is partially due to an impaired immune response of alveolar macrophages. The mechanisms by which alcohol impairs alveolar macrophage function are poorly understood. In this study, we demonstrated in a guinea pig model that chronic ethanol ingestion significantly impaired alveolar macrophage differentiation and function. Isolated alveolar macrophages were separated into 4 different subpopulations with varying densities and levels of maturation. Compared to control values, chronic ethanol ingestion decreased the percentage of alveolar macrophages in the mature fractions by ~60%. Alveolar macrophage function in each subpopulation was determined by measuring phagocytosis of FITC-labeled S. aureus. Alveolar macrophages from ethanol-fed animals had ~80% decrease in the phagocytic index. Western blot and immunohistochemical analysis of the differential markers GM-CSF receptor α (GM-CSFR-α), PU.1, CD11c, and CD11b verified that alcoholic macrophages displayed impaired terminal differentiation. While oral supplementation with the glutathione precursor S-adenosyl-methionine (SAM) did not alter the maturational status of control animals, SAM s supplementation shifted the distribution of macrophages to more mature fractions, normalized the phagocytic index; as well as normalized expression of CD11c, CD11b, PU.1, and GM-CSFR-α. Chronic ethanol ingestion also impaired the differentiation status of interstitial macrophages which was normalized by SAM supplementation. This improvement in the maturational status suggested that ethanol-induced oxidant stress is a central feature in impaired terminal differentiation of macrophages in the interstitial and alveolar space. Therefore, strategies targeting pulmonary oxidant stress may restore macrophage differentiation and function even after chronic ethanol ingestion.

Background Sepsis is the most common risk factor associated with acute respiratory distress syndrome (ARDS) and results in a 40–60% mortality rate due to respiratory failure. Furthermore, recent epidemiological studies have demonstrated that a history of alcohol abuse increases the risk of ARDS by 3.6-fold. More recently, group B streptococcus (GBS) infections in non-pregnant adults have been increasing, particularly in alcoholics where there is an increased risk of lobular invasion and mortality. We have shown in an established rat model that chronic ethanol ingestion impaired macrophage internalization of inactivated infectious particles in vitro and enhanced bidirectional protein flux across the alveolar epithelial-endothelial barriers, both of which were attenuated when glutathione precursors were added to the diet. We hypothesized that chronic ethanol ingestion would increase the risk of infection even though GBS is less pathogenic but that dietary N-acetylcysteine (NAC), a glutathione precursor, would improve in vivo clearance of infectious particles and reduce systemic infection. Methods After 6 weeks of ethanol feeding, rats were given GBS intratracheally and sacrificed 24 hours later. GBS colony-forming units were counted in the lung, liver, spleen and bronchoalveolar lavage fluid. Acute lung injury in response to GBS was also assessed. Results Chronic ethanol exposure decreased GBS clearance from the lung indicating an active lung infection. In addition, increased colonies formed within the liver and spleen indicating that ethanol increased the risk of systemic infection. Ethanol also exacerbated the acute lung injury induced by GBS. NAC supplementation normalized GBS clearance by the lung, prevented the appearance of GBS systemically and attenuated acute lung injury. Conclusions These data suggested that chronic alcohol ingestion increased the susceptibility of the lung to bacterial infections from GBS as well as systemic infections. Furthermore, dietary NAC improved in vivo clearance of GBS particles, attenuated acute lung injury and disseminated infection.

Summary Background The effects of fetal alcohol exposure on the risks of neonatal lung injury and infection remain under investigation. The resident alveolar macrophage (AM) is the first line of immune defense against pulmonary infections. In utero ethanol (ETOH) exposure deranges the function of both premature and term guinea pig AM. We hypothesized that fetal ETOH exposure would increase the risk of pulmonary infection in vivo. Methods We developed a novel in vivo model of group B streptococcus (GBS) pneumonia using our established guinea pig model of fetal ETOH exposure. Timed-pregnant guinea pigs were pair fed ± ETOH and some were supplemented with the glutathione (GSH) precursor S-adenosyl-methionine (SAM-e). Term pups were given GBS intratracheally while some were pre-treated with inhaled GSH prior to the experimental GBS. Neonatal Lung and whole blood were evaluated for GBS while isolated AM were evaluated using fluorescent microscopy for GBS phagocytosis. Results ETOH-exposed pups demonstrated increased lung infection and sepsis while AM phagocytosis of GBS was deficient compared to control. When SAM-e was added to the maternal diet containing ETOH, neonatal lung and systemic infection from GBS was attenuated and AM phagocytosis was improved. Inhaled GSH therapy prior to GBS similarly protected the ETOH-exposed pup from lung and systemic infection. Conclusions In utero ETOH exposure impaired the neonatal lung’s defense against experimental GBS, while maintaining GSH availability protected the ETOH-exposed lung. This study suggested that fetal alcohol exposure deranges the neonatal lung’s defense against bacterial infection, and support further investigations into the potential therapeutic role for exogenous GSH to augment neonatal AM function.