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Perinatal nicotine exposure via tobacco smoking results in increased proclivity to chronic lung disease (CLD); however, the underlying molecular mechanisms remain incompletely understood. We previously demonstrated that in addition to nicotine's direct effects on the developing lung, there are also adverse molecular alterations in bone marrow-derived mesenchymal stem cells (BMSCs), which are vital to lung injury repair. Whether perinatal nicotine exposure via electronic-cigarette (e-cig) vaping also adversely affects BMSCs is unknown. This is highly relevant due to marked increase in e-cig vaping including by pregnant women. Hypothesizing that perinatal nicotine exposure via e-cig vaping predisposes BMSCs to a pro-myofibroblastic phenotype, pregnant rat dams were exposed to fresh air (control), vehicle (e-cig without nicotine), or e-cig (e-cig with nicotine) daily during pregnancy and lactation. At postnatal day 21, offspring BMSCs were isolated and studied for cell proliferation, migration, wound healing response, and expression of key Wnt and PPARγ signaling intermediates (β-catenin, LEF-1, PPARγ, ADRP and C/EBPα) and myogenic markers (fibronectin, αSMA, calponin) proteins using immunoblotting. Compared to controls, perinatal e-cig exposure resulted in significant decrease in BMSC proliferation, migration, and wound healing response. The expression of key Wnt signaling intermediates (β-catenin, LEF-1) and myogenic markers (fibronectin, αSMA, calponin) increased significantly, while PPARγ signaling intermediates (PPARγ, ADRP, and C/EBPα) decreased significantly. Based on these data, we conclude that perinatally e-cig exposed BMSCs demonstrate pro-myofibroblastic phenotype and impaired injury-repair potential, indicating a potentially similar susceptibility to CLD following perinatal nicotine exposure via vaping as seen following parenteral perinatal nicotine exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Reproduction is a remarkably intricate process involving the interaction of multiple cell types and organ systems unfolding over long periods of time and that culminates with the production of gametes. The initiation of germ cell development takes place during embryogenesis but only completes decades later in humans. The complexity inherent to reproduction and its study has long hampered our ability to decipher how environmental agents disrupt this process. Single-cell approaches provide an opportunity for a deeper understanding of the action of toxicants on germline function and analyze how the response to their exposure is differentially distributed across tissues and cell types. In addition to single-cell RNA sequencing, other single-cell or nucleus level approaches such as ATAC-sequencing and multi-omics have expanded the strategies that can be implemented in reproductive toxicological studies to include epigenomic and the nuclear transcriptomic data. Here we will discuss the current state of single-cell technologies and how they can best be utilized to advance reproductive toxicological studies. We will then discuss case studies in two model organisms ( Caenorhabditis elegans and mouse) studying different environmental exposures (alcohol and e-cigarettes respectively) to highlight the value of single-cell and single-nucleus approaches for reproductive biology and reproductive toxicology., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Objectives: To observe the effects of electroacupuncture(EA) on memory, cognitive impairment, and the brain-derived neurotrophic factor(BDNF)/N-methyl-D-aspartate receptor subtype 1(NMDAR1) pathway in the brains of offspring rat with intrauterine growth restriction(IUGR) induced by perinatal nicotine exposure(PNE), so as to explore the underlying mechanism., Methods: SD rats were randomly divided into normal, model, and EA groups, with 4 mothers and 10 offspring rats of each mother in each group. The IUGR model was established by subcutaneous injection of nicotine during pregnancy and lactation. From the 6 th day of pregnancy in the mothers until the 21 st day after birth of the offspring rats, EA (2 Hz/15 Hz, 1 mA) was administered bilaterally at the "Zusanli"(ST36) of mothers, once daily for 20 min. The brain organ coefficient was used to evaluate the brain development of the offspring rats. The Y-maze test and novel object recognition experiments were performed to assess memory and cognitive function. HE staining was used to observe the development and cellular morphology of the hippocampus and prefrontal cortex in the offspring rats. UV spectrophotometry was used to measure the glutamate(Glu) content in the hippocampus. ELISA was used to detect the BDNF content in the hippocampus. Western blot was performed to measure the protein expression of NMDAR1 in the hippocampus. Immunohistochemistry was used to count the number of BDNF-positive cells in the hippocampus and prefrontal cortex., Results: Compared with the normal group, the brain organ coefficient, exploration time of the novel arm, spontaneous alternation rate, and novel object recognition index, contents of BDNF and expression of NMDAR1 proteins in the hippocampus, the number of BDNF-positive cells in the CA1 and CA3 regions of the hippocampus and prefrontal cortex were significantly reduced( P <0.01), while the Glu content in the hippocampus was significantly increased( P <0.01) in the model group of offspring rats；decreased cell number, scattered arrangement, and disrupted cellular structure were observed in the hippocampus and prefrontal cortex of offspring rats in the model group. Compared with the model group, the brain organ coefficient, exploration time of the novel arm, spontaneous alternation rate, and novel object recognition index, the BDNF contents and NMDAR1 protein expression in the hippocampus, the number of BDNF-positive cells in the hippocampal CA1 and CA3 regions and prefrontal cortex significantly increased( P <0.01, P <0.05), while the Glu content in the hippocampus was significantly decreased ( P <0.01) in offspring rats of the EA group；increased cell number, neat arrangement, and reduced cellular damage were observed in the hippocampus and prefrontal cortex in the EA group., Conclusions: EA has an improving effect on memory and cognitive function impairment in offspring rats with IUGR induced by PNE, and this mechanism may be associated with the regulation of BDNF/NMDAR1 pathway, thereby improving the neuronal quantity and structure of the hippocampus and prefrontal cortex in offspring rats.

Objective: Many factors during pregnancy can induce intrauterine growth restriction (IUGR), resulting in various adverse perinatal outcomes such as low birth weight and multiple organ disorders. Among these factors, prenatal smoke/nicotine exposure is a common cause of IUGR, often associated with altered fetal lung development. The classical Wnt signaling pathway plays a vital role in lung development, and its alterations are commonly associated with developmental lung pathologies. The purpose of this study was to determine whether electroacupuncture (EA) at "Zusanli" (ST 36) points protects perinatal nicotine exposure (PNE)-induced offspring lung dysplasia through Wnt/β-catenin signaling pathway and to identify specific Wnt signaling pathway targets of EA., Methods: Following a well-established protocol, nicotine (1 mg/kg/ body weight) was administered subcutaneously to pregnant Sprague Dawley rat dams from gestational day 6 to postnatal day 21. In the EA group, dams were treated with EA at both ST 36 acupoints, while in another experimental group, Wnt/β-catenin signaling pathway agonist was injected subcutaneously (2 mg/kg/ body weight). Offspring body weight (PND 1, 7, 14, and 21), lung weight, Wnt signaling markers, pulmonary function, and lung morphology were determined at sacrifice on PND 21. Specifically, Western blotting and Real-time PCR were used to detect the protein and mRNA levels of critical Wnt signaling markers Wnt2, Wnt7b, FZD4, FZD7, LRP5, and LRP6 in the offspring lung. The protein levels of β-catenin in lung tissue of offspring rats were detected by ELISA that of LEF-1 by Western blotting., Results: Compared to the control group, the body and lung weights of the offspring rats were significantly decreased in the nicotine-only exposed group. The pulmonary function determined as FVC, PEF, TV, and Cdyn was also significantly decreased, while PIF was significantly increased. The protein levels and mRNA expression of Wnt2, Wnt7b, FZD4, FZD7, LRP5, and LRP6 in the lung tissue of the PNE offspring rats were significantly increased. With EA administration at ST 36 acupoints concomitant with nicotine administration, the body and lung weights, pulmonary function (FVC, PEF, PIF, TV, and Cdyn), protein and mRNA levels Wnt signaling pathway markers (Wnt2, Wnt7b, FZD4, FZD7, LRP5, LRP6, β-catenin, and LEF-1) normalized and were not different from the control group. Notably, Wnt agonists agonist administration blocked the protective effects of EA against PNE-induced lung morphological, molecular, and function changes, highlighting the central significance of Wnt pathway signaling in PNE-induced offspring pulmonary pathology and its modulation by EA at ST 36 acupoints., Conclusion: Concomitant maternal EA at ST 36 acupoints from gestational day 6 to PND 21 protects against offspring PNE-induced lung phenotype. The protective effect is achieved by regulating the expression of Wnt ligand proteins (Wnt2 and Wnt7b) and receptor proteins (FZD4, FZD7, LRP5, and LRP6) upstream of the Wnt/β-catenin signaling pathway intermediates β-catenin, and LEF-1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical disorders that mainly develop from viral respiratory infections, sepsis, and chest injury. Antigen-presenting cells play a pivotal role in propagating uncontrolled inflammation and injury through the excess secretion of pro-inflammatory cytokines and recruitment of immune cells. Autophagy, a homeostatic process that involves the degradation of cellular components, is involved in many processes including lung inflammation. Here, we use a polyinosinic-polycytidylic acid (poly(I:C))-induced lung injury mouse model to mimic viral-induced ALI/ARDS and show that disruption of autophagy in macrophages exacerbates lung inflammation and injury, whereas autophagy induction attenuates this process. Therefore, induction of autophagy in macrophages can be a promising therapeutic strategy in ALI/ARDS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Hyperoxia-induced lung injury is characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. Although a combination of aerosolized pioglitazone (PGZ) and a synthetic lung surfactant (B-YL peptide, a surfactant protein B mimic) prevents hyperoxia-induced neonatal rat lung injury, whether it is also effective in preventing hyperoxia-induced adult lung injury is unknown., Method: Using adult mice lung explants, we characterize the effects of 24 and 72-h (h) exposure to hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of lung injury, 2) aberrations of lung homeostasis and injury repair pathways, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination., Results: Our study reveals that hyperoxia exposure to adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1, and PECAM-1) markers. All of these changes were largely mitigated by the PGZ + B-YL combination., Conclusion: The effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo is promising to be an effective therapeutic approach for adult lung injury in vivo., Competing Interests: Declaration of competing interest Authors Drs. Rehan, Walther, and Waring hold a patent on the composition and methods of administering PPARγ agonists, surfactant peptides, and phospholipids for preventing and treating neonatal lung injury (Patent #:20220047681). No other competing interest or conflict of interest to declare. All authors completed the ICMJE Form., (Copyright © 2023. Published by Elsevier Ltd.)

This review delineates the main pulmonary issues related to preterm birth, perinatal tobacco/nicotine exposure, and its effects on offspring, focusing on respiratory health and its possible transmission to subsequent generations. We review the extent of the problem of preterm birth, prematurity-related pulmonary effects, and the associated increased risk of asthma later in life. We then review the impact of developmental tobacco/nicotine exposure on offspring asthma and the significance of transgenerational pulmonary effects following perinatal tobacco/nicotine exposure, possibly via its effects on germline epigenetics.

Perinatal exposure to smoking has been associated with childhood asthma, one of the most common pediatric conditions affecting millions of children globally. Of great interest, this disease phenotype appears heritable as it can persist across multiple generations even in the absence of persistent exposure to smoking in subsequent generations. Although the molecular mechanisms underlying childhood asthma induced by perinatal exposure to smoking or nicotine remain elusive, an epigenetic mechanism has been proposed, which is supported by the data from our earlier analyses on germline DNA methylation (5mC) and histone marks (H3 and H4 acetylation). To further investigate the potential epigenetic inheritance of childhood asthma induced by perinatal nicotine exposure, we profiled both large and small RNAs in the sperm of F1 male rats. Our data revealed that perinatal exposure to nicotine leads to alterations in the profiles of sperm-borne RNAs, including mRNAs and small RNAs, and that rosiglitazone, a PPARγ agonist, can attenuate the effect of nicotine and reverse the sperm-borne RNA profiles of F1 male rats to close to placebo control levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Liu, Gao, Yan and Rehan.)

Stem cells, including the resident lung mesenchymal stem cells (LMSCs), are critically important for injury repair. Compelling evidence links perinatal vitamin D (VD) deficiency to reactive airway disease; however, the effects of perinatal VD deficiency on LMSC function is unknown. We tested the hypothesis that perinatal VD deficiency alters LMSC proliferation, differentiation, and function, leading to an enhanced myogenic phenotype. We also determined whether LMSCs' effects on alveolar type II (ATII) cell function are paracrine. Using an established rat model of perinatal VD deficiency, we studied the effects of four dietary regimens (0, 250, 500, or 1,000 IU/kg cholecalciferol-supplemented groups). At Postnatal Day 21, LMSCs were isolated, and cell proliferation and differentiation (under basal and adipogenic induction conditions) were determined. LMSC paracrine effects on ATII cell proliferation and differentiation were determined by culturing ATII cells in LMSC-conditioned media from different experimental groups. Using flow cytometry, >95% of cells were CD45-ve, >90% were CD90 + ve, >58% were CD105 + ve, and >64% were Stro-1 + ve, indicating their stem cell phenotype. Compared with the VD-supplemented groups, LMSCs from the VD-deficient group demonstrated suppressed PPARγ, but enhanced Wnt signaling, under basal and adipogenic induction conditions. LMSCs from 250 VD- and 500 VD-supplemented groups effectively blocked the effects of perinatal VD deficiency. LMSC-conditioned media from the VD-deficient group inhibited ATII cell proliferation and differentiation compared with those from the 250 VD- and 500 VD-supplemented groups. These data support the concept that perinatal VD deficiency alters LMSC proliferation and differentiation, potentially contributing to increased respiratory morbidity seen in children born to mothers with VD deficiency.

Maternal food restriction (MFR) during pregnancy leads to pulmonary dysplasia in the newborn period and increases susceptibility to diseases, such as asthma and chronic lung disease, later in life. Previous studies have shown that maternal electro-acupuncture (EA) applied to "Zusanli" (ST 36) could prevent the abnormal expression of key lung developmental signaling pathways and improve the lung morphology and function in perinatal nicotine exposed offspring. There is a significant overlap in lung developmental signaling pathways affected by perinatal nicotine exposure and MFR during pregnancy; however, whether maternal EA at ST 36 also blocks the MFR-induced lung phenotype is unknown. Here, we examined the effects of EA applied to maternal ST 36 on lung morphology and function and the expression of key lung developmental signaling pathways, and the hypercorticoid state associated with MFR during pregnancy. These effects were compared with those of metyrapone, an intervention known to block MFR-induced offspring hypercorticoid state and the resultant pulmonary pathology. Like metyrapone, maternal EA at ST 36 blocked the MFR-induced changes in key developmental signaling pathways and protected the MFR-induced changes in lung morphology and function. These results offer a novel and safe, nonpharmacologic approach to prevent MFR-induced pulmonary dysplasia in offspring., (© 2021 Wiley Periodicals LLC.)

Perinatal smoke/nicotine exposure alters lung development and causes asthma in exposed offspring, transmitted transgenerationally. The mechanism underlying the transgenerational inheritance of perinatal smoke/nicotine-induced asthma remains unknown, but germline epigenetic modulations may play a role. Using a well-established rat model of perinatal nicotine-induced asthma, we determined the DNA methylation pattern of spermatozoa of F1 rats exposed perinatally to nicotine in F0 gestation. To identify differentially methylated regions (DMRs), reduced representation bisulfite sequencing was performed on spermatozoa of F1 litters. The top regulated gene body and promoter DMRs were tested for lung gene expression levels, and key proteins involved in lung development and repair were determined. The overall CpG methylation in F1 sperms across gene bodies, promoters, 5'-UTRs, exons, introns, and 3'-UTRs was not affected by nicotine exposure. However, the methylation levels were different between the different genomic regions. Eighty one CpG sites, 16 gene bodies, and 3 promoter regions were differentially methylated. Gene enrichment analysis of DMRs revealed pathways involved in oxidative stress, nicotine response, alveolar and brain development, and cellular signaling. Among the DMRs, Dio1 and Nmu were the most hypermethylated and hypomethylated genes, respectively. Gene expression analysis showed that the mRNA expression and DNA methylation were incongruous. Key proteins involved in lung development and repair were significantly different (FDR < 0.05) between the nicotine and placebo-treated groups. Our data show that DNA methylation is remodeled in offspring spermatozoa upon perinatal nicotine exposure. These epigenetic alterations may play a role in transgenerational inheritance of perinatal smoke/nicotine induced asthma., (© 2021 Federation of American Societies for Experimental Biology.)

Eligibility of ventilated preterm rabbit model to investigate extreme pulmonary immaturity at birth transition is unknown. By extending this model to early saccular stage of fetal lung development, we evaluated efficacy in survival, lung maturation, and underlying mechanisms of contemporary perinatal therapies. Pregnant New Zealand White rabbit does were given dexamethasone (DEX), or sham injection as control (NDEX), 48 and 24 h before delivery at gestational age (GA) of 25-28 days. At birth, newborn rabbits were anesthetized and randomly allocated to four groups receiving either surfactant or nonsurfactant for both DEX and NDEX, and mechanically ventilated within low tidal volumes. Ranges of time to maintain survival rate ≥ 50% in GA 25-28 days were 59-136, 138-259, 173-288, and 437 to ≥600 min, respectively, each across the four groups. The benefits of DEX and/or surfactant for survival were more obvious in GA 25-26 days, as judged by improved lung mechanics, lower lung injury scores, higher lung surfactant phospholipid pools, and surfactant protein mRNA expression, with DEX-surfactant combination being the most optimal for the outcome. In contrast, those of GA 27-28 days had variable but meaningful responses to the treatment. Cox regression analysis revealed GA, DEX, and surfactant being independently protective factors whereas pneumothorax was a risk factor. The extremely preterm rabbits at GA 25-26 days markedly responded to the perinatal therapies for longer survival, lung maturation and injury alleviation, and were relevant for study of preterm birth transition-associated morbidities and underlying mechanisms. NEW & NOTEWORTHY An extremely preterm rabbit model with gestational age of 25-26 ( term 31 ) days was established by mechanical ventilation with individually adjusted tidal volume at lower ranges. The administration of antenatal glucocorticoids and/or postnatal surfactant achieved significantly longer duration to maintain 50% survival and facilitated lung maturation and protection at early saccular stage. The usefulness of this model should be validated in future investigation of perinatal and neonatal morbidity and mortality at extremely preterm birth transition.

Intrauterine growth restriction (IUGR) leads to offspring obesity. In a maternal food restriction (MFR) during pregnancy-related IUGR rat model, bone marrow stem cells showed enhanced adipogenic programming; however, the effect of IUGR on white adipose tissue (WAT) progenitors is unknown. Here, by mRNA and functional profiling, we determined sex-specific adipogenic programming of WAT progenitors isolated from pups on the postnatal day (PND) 1 and 21. On PND1, PPARγ and Pref-1 expression was significantly downregulated in preadipocytes of both MFR males and females; however, at PND21, preadipocytes of MFR males showed upregulation in these genes. Even following adipogenic induction, both male and female MFR adipocytes exhibited lower PPARγ, ADRP, and adiponectin levels at PND1; however, at PND21 MFR male adipocytes showed an upward trend in the expression of these genes. An adipogenesis-specific RT-PCR array showed that male MFR adipocytes were programmed to exhibit stronger adipogenic propensity than females. Last, serum sex hormone and adipocyte estrogen/testosterone receptor expression profiles provide preliminary insights into the possible mechanism underlying sex-specific adipogenic programming in the IUGR offspring. In summary, IUGR programs WAT preadipocytes to greater adipogenic potential in males. Although the altered adipogenic programming following MFR was detectable at PND1, the changes were more pronounced at PND21, suggesting a potential role of postnatal nutrition in facilitating the sex-specific adipogenic programming in the IUGR offspring., (© 2020 Federation of American Societies for Experimental Biology.)

Electronic-cigarette (e-cig) vaping is a serious concern, as many pregnant women who vape consider it safe. However, little is known about the harmful effects of prenatal e-cig exposure on adult offspring, especially on extracellular-matrix (ECM) deposition and myogenesis in the lungs of offspring. We evaluated the biochemical and molecular implications of maternal exposure during pregnancy to e-cig aerosols on the adult offspring of both sexes, with a particular focus on pulmonary ECM remodeling and myogenesis. Pregnant CD-1 mice were exposed to e-cig aerosols with or without nicotine, throughout gestation, and lungs were collected from adult male and female offspring. Compared with the air-exposed control group, female mice exposed to e-cig aerosols, with or without nicotine, demonstrated increased lung protein abundance of LEF-1 (lymphoid enhancer-binding factor 1), fibronectin, and E-cadherin, whereas altered E-cadherin and PPARγ (peroxisome proliferator-activated receptor γ) levels were observed only in males exposed to e-cig aerosols with nicotine. Moreover, lipogenic and myogenic mRNAs were dysregulated in adult offspring in a sex-dependent manner. PAI-1 (plasminogen activator inhibitor-1), one of the ECM regulators, was significantly increased in females exposed prenatally to e-cig aerosols with nicotine and in males exposed to e-cig aerosols compared with control animals exposed to air. MMP9 (matrix metalloproteinase 9), a downstream target of PAI-1, was downregulated in both sexes exposed to e-cig aerosols with nicotine. No differences in lung histology were observed among any of the treatment groups. Overall, adult mice exposed prenatally to e-cig aerosols could be predisposed to developing pulmonary disease later in life. Thus, these findings suggest that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

Antenatal steroids (ANS) accelerate fetal lung maturation and reduce the incidence of respiratory distress syndrome. However, sex specificity, i.e., being less effective in males, and potential long-term neurodevelopmental sequelae, particularly with repeated courses, remain significant limitations. The differential sex response to ANS is likely mediated via the inhibitory effect of fetal androgens on steroid's stimulatory effect on alveolar epithelial-mesenchymal interactions. Since peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists accelerate lung maturation by stimulating alveolar epithelial-mesenchymal interactions, independent of fetal sex, we hypothesized that the effect of PPAR-γ agonist pioglitazone (PGZ) would be sex-independent. Pregnant Sprague-Dawley rat dams were intraperitoneally administered dexamethasone (DEX) or PGZ on embryonic day (e) 18 and e19. At e20, pups were delivered by cesarean section, and fetal lungs and brains were examined for markers of lung maturation and apoptosis, respectively. Mixed epithelial-fibroblast cell cultures were examined to gain mechanistic insights. Antenatal PGZ increased alveolar epithelial and mesenchymal maturation markers equally in males and females; in contrast, antenatal DEX had sex-specific effects. Additionally, unlike DEX, antenatal PGZ did not increase hippocampal apoptosis. We conclude that PPAR-γ agonist administration is an effective, and probably even a superior, alternative to ANS for accelerating fetal lung maturity equally in both males and females.

Although increased predisposition to cardiac fibrosis and cardiac dysfunction has been demonstrated in the perinatally nicotine-exposed heart, the underlying mechanisms remain unclear. With the use of a well-established rat model and cultured primary neonatal rat cardiac fibroblasts, the effect of perinatal nicotine exposure on offspring heart extracellular matrix deposition and the likely underlying mechanisms were investigated. Perinatal nicotine exposure resulted in increased collagen type I (COL1A1) and III (COL3A1) deposition along with a decrease in miR-29 family and an increase in long noncoding RNA myocardial infarction-associated transcript (MIAT) levels in offspring heart. Nicotine treatment of isolated primary neonatal rat cardiac fibroblasts suggested that these effects were mediated via nicotinic acetylcholine receptors including α7 and the induced collagens accumulation was reversed by a gain-of function of miR-29 family. Knockdown of MIAT resulted in increased miR-29 family and decreased COL1A1 and COL3A1 levels, suggesting nicotine-mediated MIAT induction as the underlying mechanism for nicotine-induced collagen deposition. Luciferase reporter assay and RNA immunoprecipitation studies showed an intense physical interaction between MIAT, miR-29 family, and argonaute 2, corroborating the mechanistic link between perinatal nicotine exposure and increased extracellular matrix deposition. Overall, perinatal nicotine exposure resulted in lower miR-29 family levels in offspring heart, while it elevated cardiac MIAT and collagen type I and III levels. These findings provide mechanistic basis for cardiac dysfunction in perinatal nicotine-exposed offspring and offer multiple novel potential therapeutic targets. NEW & NOTEWORTHY Using an established rat model and cultured primary neonatal cardiac fibroblasts, we show that nicotine mediated MIAT induction as the underlying mechanism for the excessive cardiac collagen deposition. These observations provide mechanistic basis for the increased predisposition to cardiac dysfunction following perinatal cigarette/nicotine exposure and offer novel potential therapeutic targets.

Prevention of bronchopulmonary dysplasia (BPD) in premature-birth babies continues to be an unmet medical need. Intramuscular vitamin A is currently employed in preterm neonates to prevent BPD but requires intramuscular injections in fragile neonates. We hypothesized that noninvasive inhaled delivery of vitamin A, targeted to lung, would be a more effective and tolerable strategy. We employed our well-established hyperoxia-injury neonatal rat model, exposing newborn rats to 7 days of constant extreme (95% O 2 ) hyperoxia, comparing vitamin A dosed every 48 h via either aerosol inhalation or intramuscular injection with normoxic untreated healthy animals and vehicle-inhalation hyperoxia groups as positive and negative controls, respectively. Separately, similar vitamin A dosing of normoxia-dwelling animals was performed. Analyses after day 7 included characterization of alveolar histomorphology and protein biomarkers of alveolar maturation [surfactant protein C (SP-C), peroxisome proliferator-activated receptor (PPAR) γ, cholinephosphate cytidylyl transferase, vascular endothelial growth factor and its receptor, FLK-1, and retinoid X receptors (RXR-α, -β, and -γ], apoptosis (Bcl2 and Bax) key injury repair pathway data including protein markers (ALK-5 and β-catenin) and neutrophil infiltration, and serum vitamin A levels. Compared with intramuscular dosing, inhaled vitamin A significantly enhanced biomarkers of alveolar maturation, mitigated hyperoxia-induced lung damage, and enhanced surfactant protein levels, suggesting that it may be more efficacious in preventing BPD in extremely premature infants than the traditionally used IM dosing regimen. We speculate lung-targeted inhaled vitamin A may also be an effective therapy against other lung damaging conditions leading to BPD or, more generally, to acute lung injury.

In a rat model, perinatal nicotine exposure results in an epigenetically driven multi- and trans-generationally transmitted asthmatic phenotype that tends to wane over successive generations. However, the effect of repeat nicotine exposure during the F1 (Filial 1) gestational period on the transmitted phenotype is unknown. Using a well-established rat model, we compared lung function, mesenchymal markers of airway reactivity, and global gonadal DNA methylation changes in F2 offspring in a sex-specific manner following perinatal exposure to nicotine in only the F0 gestation, in both F0 and F1 (F0/F1) gestations, and in neither (control group). Both F0 only and F0/F1 exposure groups showed an asthmatic phenotype, an effect that was more pronounced in the F0/F1 exposure group, especially in males. Testicular global DNA methylation increased, while ovarian global DNA methylation decreased in the F0/F1 exposed group. Since the offspring of smokers are more likely to smoke than the offspring of nonsmokers, this sets the stage for more severe asthma if both mother and grandmother had smoked during their pregnancies. Increased gonadal DNA methylation changes following nicotine reexposure in the F1 generation suggests that epigenetic mechanisms might well underlie the transgenerational inheritance of acquired phenotypic traits in general and nicotine-induced asthma in particular., (© 2020 Federation of American Societies for Experimental Biology.)

N-myristoyltransferase-1 (NMT1) catalyzes protein myristoylation, a lipid modification that is elevated in cancer cells. NMT1 sustains proliferation and/or survival of cancer cells through mechanisms that are not completely understood. We used genetic and pharmacological inhibition of NMT1 to further dissect the role of this enzyme in cancer, and found an unexpected essential role for NMT1 at promoting lysosomal metabolic functions. Lysosomes mediate enzymatic degradation of vesicle cargo, and also serve as functional platforms for mTORC1 activation. We show that NMT1 is required for both lysosomal functions in cancer cells. Inhibition of NMT1 impaired lysosomal degradation leading to autophagy flux blockade, and simultaneously caused the dissociation of mTOR from the surface of lysosomes leading to decreased mTORC1 activation. The regulation of lysosomal metabolic functions by NMT1 was largely mediated through the lysosomal adaptor LAMTOR1. Accordingly, genetic targeting of LAMTOR1 recapitulated most of the lysosomal defects of targeting NMT1, including defective lysosomal degradation. Pharmacological inhibition of NMT1 reduced tumor growth, and tumors from treated animals had increased apoptosis and displayed markers of lysosomal dysfunction. Our findings suggest that compounds targeting NMT1 may have therapeutic benefit in cancer by preventing mTORC1 activation and simultaneously blocking lysosomal degradation, leading to cancer cell death.

Introduction . Environmental exposure of the developing offspring to cigarette smoke or nicotine is an important predisposing factor for many chronic respiratory conditions, such as asthma, emphysema, pulmonary fibrosis, and so forth, in the exposed offspring. Studies showed that electroacupuncture (EA) applied to maternal "Zusanli" (ST36) acupoints during pregnancy and lactation protects against perinatal nicotine exposure- (PNE-) induced lung damage. However, the most effective time period, that is, prenatal vs. postnatal, to attain this effect has not been determined., Objective: To determine the most effective developmental timing of EA's protective effect against PNE-induced lung phenotype in the exposed offspring., Methods: Pregnant rats were given (1) saline ("S" group); (2) nicotine ("N" group); (3) nicotine + EA, exclusively prenatally ("Pre-EA" group); (4) nicotine + EA, exclusively postnatally ("Post-EA," group); and (5) nicotine + EA, administered both prenatally and postnatally ("Pre- and Post-EA" group). Nicotine was injected once daily (1 mg/kg, 100  μ l) and EA was administered to bilateral ST36 acupoints once daily during the specified time-periods. At the end of the experimental periods, key hypothalamic pituitary adrenal (HPA) axis markers in pups and dams, and lung function, morphometry, and the central molecular markers of lung development in the offspring were determined., Results: After nicotine exposure, alveolar mean linear intercept (MLI) increased, but mean alveolar number (MAN) decreased and lung PPAR γ level decreased, but glucocorticoid receptor (GR) and serum corticosterone (Cort) levels increased, in line with the known PNE-induced lung phenotype. In the nicotine exposed group, maternal hypothalamic corticotropin releasing hormone (CRH) level decreased, but pituitary adrenocorticotropic hormone (ACTH) and serum Cort levels increased. In the "Pre- and Post-EA" groups, PNE-induced alterations in lung morphometry, lung development markers, and HPA axis were blocked. In the "Pre-EA" group, PNE-induced changes in lung morphometry, GR, and maternal HPA axis improved; lung PPAR γ level decreased, but glucocorticoid receptor (GR) and serum corticosterone (Cort) levels increased, in line with the known PNE-induced lung phenotype. In the nicotine exposed group, maternal hypothalamic corticotropin releasing hormone (CRH) level decreased, but pituitary adrenocorticotropic hormone (ACTH) and serum Cort levels increased. In the "Pre- and Post-EA" groups, PNE-induced alterations in lung morphometry, lung development markers, and HPA axis were blocked. In the "Pre-EA" group, PNE-induced changes in lung morphometry, GR, and maternal HPA axis improved; lung PPAR., Conclusions: Maternal EA applied to ST36 acupoints during both pre- and postnatal periods preserves offspring lung structure and function despite perinatal exposure to nicotine. EA applied during the "prenatal period" affords only limited benefits, whereas EA applied during the "postnatal period" is ineffective, suggesting that the EA's effects in modulating PNE-induced lung phenotype are limited to specific time-periods during lung development., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Jian Dai et al.)

Background: Maternal smoking and/or exposure to environmental tobacco smoke continue to be significant factors in fetal and childhood morbidity and are a serious public health issue worldwide. Nicotine passes through the placenta easily with minimal biotransformation, entering fetal circulation, where it results in many harmful effects on the developing offspring, especially on the developing respiratory system., Objectives: Recently, in a rat model, electroacupuncture (EA) at maternal acupoints ST 36 has been shown to block perinatal nicotine-induced pulmonary damage; however, the underlying mechanism and the specificity of ST 36 acupoints for this effect are unknown. Here, we tested the hypothesis that compared with EA at ST 36, EA at LU 5 acupoints, which are on lung-specific meridian, will be equally or more effective in preventing perinatal nicotine-induced pulmonary changes., Methods: Twenty-four pregnant rat dams were randomly divided into 4 groups: saline ("S"), nicotine ("N"), nicotine + ST 36 (N + ST 36), and nicotine + LU 5 (N + LU 5) groups. Nicotine (1 mg/kg, subcutaneously) and EA (at ST 36 or LU 5 acupoints, bilaterally) were administered from embryonic day 6 to postnatal day 21 once daily. The "S" group was injected saline. As needed, using ELISA, western analysis, q-RT-PCR, lung histopathology, maternal and offspring hypothalamic pituitary adrenal axes, offspring key lung developmental markers, and lung morphometry were determined., Results: With nicotine exposure, alveolar count decreased, but mean linear intercept and septal thickness increased. It also led to a decrease in pulmonary function and PPAR γ and an increase of β -catenin and glucocorticoid receptor expression in lung tissue and corticosterone in the serum of offspring rats. Electroacupuncture at ST 36 normalized all of these changes, whereas EA at LU 5 had no obvious effect., Conclusion: Electroacupuncture applied to ST 36 acupoints provided effective protection against perinatal nicotine-induced lung changes, whereas EA applied at LU 5 acupoints was ineffective, suggesting mechanistic specificity and HPA axis' involvement in mediating EA at ST 36 acupoints' effects in mitigating perinatal nicotine-induced pulmonary phenotype. This opens the possibility that other acupoints, besides ST 36, can have similar or even more robust beneficial effects on the developing lung against the harmful effect of perinatal nicotine exposure. The approach proposed by us is simple, cheap, quick, easy to administer, and is devoid of any significant side effects., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Yawen Lu et al.)

Maternal smoking during pregnancy remains as a significant public health crisis as it did decades ago. Although its prevalence is decreasing in high-income countries, it has worsened globally, along with a concerning emergence of electronic-cigarette usage within the last two decades. Extensive epidemiologic and experimental evidence exists from both human and animal studies, demonstrating the detrimental long-term pulmonary outcomes in the offspring of mothers who smoke during pregnancy. Even secondhand and thirdhand smoke exposure to the developing lung might be as or even more harmful than firsthand smoke exposure. Furthermore, these effects are not limited only to the exposed progeny, but can also be transmitted transgenerationally. There is compelling evidence to support that the majority of the effects of perinatal smoke exposure on the developing lung, including the transgenerational transmission of asthma, is mediated by nicotine. Nicotine exposure induces cell-specific molecular changes in lungs, which offers a unique opportunity to prevent, halt, and/or reverse the resultant damage through targeted molecular interventions. Experimentally, the proposed interventions, such as administration of peroxisome proliferator-activated receptor gamma (PPARγ) agonists can not only block but also potentially reverse the perinatal nicotine exposure-induced respiratory morbidity in the exposed offspring. However, the development of a safe and effective intervention is still many years away. In the meantime, electropuncture at specific acupoints appears to be emerging as a more practical and safe physiologic approach to block the harmful pulmonary consequences of perinatal nicotine exposure., (© 2019 Wiley Periodicals, Inc.)

Nicotine inhalation via electronic cigarettes (e-cigs) is an emerging concern. However, little is known about the acute toxicity in the lungs following inhalation of nicotine-containing e-cig aerosols. We hypothesized that acute exposure to aerosolized nicotine causes lung toxicity by eliciting inflammatory and dysregulated repair responses. Adult C57BL/6J mice were exposed 2 h daily for 3 days to e-cig aerosols containing propylene glycol (PG) with or without nicotine. Acute exposure to nicotine-containing e-cig aerosols induced inflammatory cell influx (neutrophils and CD8a + T-lymphocytes), and release of pro-inflammatory cytokines in bronchoalveolar lavage fluid in a sex-dependent manner. Inhalation of e-cig aerosol containing PG alone significantly augmented the lung levels of various homeostasis/repair mediators (PPARγ, ADRP, ACTA2, CTNNB1, LEF1, β-catenin, E-cadherin, and MMP2) in a sex-dependent manner when compared to air controls. These findings were accompanied by an increase in protein abundance and altered gene expression of lipogenic markers (PPARγ, ADRP) and myogenic markers (fibronectin, α-smooth muscle actin and β-catenin), suggesting a dysregulated repair response in mouse lungs. Furthermore, exposure to nicotine containing e-cig aerosols or PG alone differentially affected the release of pro-inflammatory cytokines in healthy and COPD human 3D EpiAirway tissues. Overall, acute exposure to nicotine containing e-cig aerosols was sufficient to elicit a pro-inflammatory response and altered mRNA and protein levels of myogenic, lipogenic, and extracellular matrix markers in mouse lung in a sex-dependent manner. Thus, acute exposure to inhaled nicotine via e-cig leads to dysregulated repair and inflammatory responses, which may lead to airway remodeling in the lungs., Competing Interests: Conflicting financial interests: The authors have declared that no conflict of interest exists.

Group 2 Innate lymphoid cells (ILC2) contribute significantly to allergic inflammation. However, the role of microbiota on ILC2s remains to be unraveled. Here we show that short chain fatty acids (SCFAs), such as butyrate, derived from fermentation of dietary fibers by the gut microbiota inhibit pulmonary ILC2 functions and subsequent development of airway hyperreactivity (AHR). We further show that SCFAs modulate GATA3, oxidative phosphorylation, and glycolytic metabolic pathways in pulmonary ILC2s. The observed phenotype is associated with increased IL-17a secretion by lung ILC2s and linked to enhanced neutrophil recruitment to the airways. Finally, we show that butyrate-producing gut bacteria in germ-free mice effectively suppress ILC2-driven AHR. Collectively, our results demonstrate a previously unrecognized role for microbial-derived SCFAs on pulmonary ILC2s in the context of AHR. The data suggest strategies aimed at modulating metabolomics and microbiota in the gut, not only to treat, but to prevent lung inflammation and asthma., (Copyright © 2019 Lewis, Wang, Shafiei Jahani, Hurrell, Banie, Aleman Muench, Maazi, Howard, Galle-Treger, Lo, Santosh, Baltus, Bongers, San-Mateo, Gilliland, Rehan, Soroosh and Akbari.)

Objective: To describe the clinical approach used by neonatologists for diagnosis of congenital/perinatal infections (CPI); no such data currently exist., Study Design: A national survey regarding the diagnosis of toxoplasma, syphilis, rubella, cytomegalovirus, and herpes simplex virus (HSV) infection in neonates., Result: We received 553 (11%) responses. Central nervous system calcification or hydrocephalus was the commonest trigger to pursue a CPI diagnosis (98%); maternal history was the least frequent (67%). Four hundred twenty-two (76%) used general screening such as "TORCH titer screen" (57%) or total IgG or IgM (39%). Further evaluation targeted known clinical sequelae; but cerebrospinal fluid testing was used in only 65% of those suspected of having HSV or syphilis. Fifty-six percent chose a treponemal instead of a non-treponemal test for syphilis. Multivariable analyses did not identify factors associated with the clinical diagnostic approach., Conclusion: We observed clinically important deviations from CPI diagnostic test recommendations in a national cohort of neonatologists.

Objective: To compare the different effect of electroacupuncture (EA) at "Zusanli" (ST36) and "Chize" (LU5) of mother rats exposed to Nicotine during pregnancy and lactation on lung function and morphological changes in offspring rats, so as to explore the most effective acupoint for improving the development of lung in neonatal rats., Methods: A total of 24 female pregnancy SD rats were randomly divided into 4 groups: normal control, model, EA-ST36 and EA-LU5 ( n ＝6 rats in each group). Rats of the normal group were treated by subcutaneous injection of normal saline, and those of the other 3 groups treated by subcutaneous injection of nicotine (1 mg•kg －1 •d －1 ) beginning from the 6 th day to about the 21 st day of pregnancy (childbirth day) for nicotine exposure during pregnancy and lactation. The daily EA treatment (2 Hz /15 Hz，1 mA) was applied to bilateral ST36 and LU5 for 20 min, beginning from the 6 th day of pregnancy to the 21 st day (childbirth day). The lung function of the offspring rats including the peak inspiratory flow (PIF), peak expiratory flow (PEF), lung resistance (RL), exhalation resistance (RE)and lung dynamic compliance (Cdyn) was detected by using a lung function analysis system. Histopathological changes (severity of alveolarization) of the offspring rats' lung tissue were observed under microscope after H．E. stain., Results: Compared with the normal group, the PIF, RL and RE values were significantly increased ( P <0.01), and PEF and Cdyn values significantly decreased in the model group ( P <0.01). The alveolar diameter in the model group was evidently increased relevant to the normal group ( P <0.01). Following the intervention, modeling induced increase of PIF, RL, RE and alveolar diameter and decrease of PEF and Cdyn values in the EA-ST36 group, and the increased PIF, RL and RE levels in the EA-LU5 group were obviously suppressed relevant to the model group ( P <0.01, P <0.05). Additionally, modeling induced obvious congestion and edema of the alveolar wall, alveolar deformation, rupture and fusion, and reduction of the number of the pulmonary alveoli were evidently milder in both EA-ST36 and EA-LU5 groups. No significant differences were found between the EA-ST36 and EA-LU5 groups in the abovementioned 5 indexes of pulmonary function and alveolar diameter ( P >0.05)．., Conclusion: EA of ST36 and LU5 of mother rats experiencing nicotine exposure during pregnancy and lactation can improve the lung function and morphological changes in neonatal rats, and the effect of ST36 is relatively better.

Metabolic syndrome is characterized by disturbances in glucose homeostasis and the development of low-grade systemic inflammation, which increase the risk to develop type 2 diabetes mellitus (T2DM). Type-2 innate lymphoid cells (ILC2s) are a recently discovered immune population secreting Th2 cytokines. While previous studies show how ILC2s can play a critical role in the regulation of metabolic homeostasis in the adipose tissue, a therapeutic target capable of modulating ILC2 activation has yet to be identified. Here, we show that GITR, a member of the TNF superfamily, is expressed on both murine and human ILC2s. Strikingly, we demonstrate that GITR engagement of activated, but not naïve, ILC2s improves glucose homeostasis, resulting in both protection against insulin resistance onset and amelioration of established insulin- resistance. Together, these results highlight the critical role of GITR as a novel therapeutic molecule against T2DM and its fundamental role as an immune checkpoint for activated ILC2s.

Perinatal nicotine exposure drives the differentiation of alveolar lipofibroblasts (LIFs), which are critical for lung injury repair, to myofibroblasts (MYFs), which are the hallmark of chronic lung disease. Bone marrow-derived mesenchymal stem cells (BMSCs) are important players in lung injury repair; however, how these cells are affected with perinatal nicotine exposure and whether these can be preferentially driven to a lipofibroblastic phenotype are not known. We hypothesized that perinatal nicotine exposure would block offspring BMSCs lipogenic differentiation, driving these cells toward a MYF phenotype. Since peroxisome proliferator activated-receptor γ (PPARγ) agonists can prevent nicotine-induced MYF differentiation of LIFs, we further hypothesized that the modulation of PPARγ expression would inhibit nicotine's myogenic effect on BMSCs. Sprague Dawley dams were perinatally administered nicotine (1 mg/kg bodyweight) with or without the potent PPARγ agonist rosiglitazone (RGZ), both administered subcutaneously. At postnatal day 21, BMSCs were isolated and characterized morphologically, molecularly, and functionally for their lipogenic and myogenic potentials. Perinatal nicotine exposure resulted in decreased oil red O staining, triolein uptake, expression of PPARγ, and its downstream target gene adipocyte differentiation-related protein by BMSCs, but enhanced α-smooth muscle actin and fibronectin expression, and activated Wnt signaling, all features indicative of their inhibited lipogenic, but enhanced myogenic potential. Importantly, concomitant treatment with RGZ virtually blocked all of these nicotine-induced morphologic, molecular, and functional changes. Based on these data, we conclude that BMSCs can be directionally induced to differentiate into the lipofibroblastic phenotype, and PPARγ agonists can effectively block perinatal nicotine-induced MYF transdifferentiation, suggesting a possible molecular therapeutic approach to augment BMSC's lung injury/repair potential., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Considerable epidemiological and experimental evidence supports the concept that the adult chronic lung disease (CLD), is due, at least in part, to aberrations in early lung development in response to an abnormal intrauterine environment; however, the underlying molecular mechanisms remain unknown. We used a well-established rat model of maternal undernutrition (MUN) during pregnancy that results in offspring intrauterine growth restriction (IUGR) and adult CLD to test the hypothesis that in response to MUN, excess maternal glucocorticoids (GCs) program offspring lung development to a CLD phenotype by altering microRNA (miR)-29 expression, which is a key miR in regulating extracellular matrix (ECM) deposition during development and injury-repair. At postnatal day 21 and 5 mo, compared with the control offspring lung, MUN offspring lung miR-29 expression was significantly decreased in conjunction with an elevated expression of multiple downstream target ECM proteins [collagen (COL)1A1, COL3A1, COL4A5, and elastin], at both mRNA and protein levels. Importantly, MUN-induced changes in miR-29 and target gene expressions were at least partially blocked in the lungs of offspring of MUN dams treated with metyrapone, a selective GC synthesis inhibitor. Furthermore, dexamethasone treatment of cultured fetal rat lung fibroblasts significantly induced miR-29 expression along with the suppression of target ECM proteins. These data, along with the previously known role of miR-29 in regulating ECM deposition in vascular tissue in the MUN offspring, suggest miR-29 to be a common mechanistic denominator for the vascular and pulmonary phenotypes in the IUGR offspring, providing a novel potential therapeutic target.

Perinatal nicotine exposure can not only lead to lung dysplasia in offspring, but also cause epigenetic changes and induce transgenerational asthma. Previous studies have shown that electro-acupuncture (EA) applied to "Zusanli" (ST 36) can improve the lung morphology and correct abnormal expression of lung development-related protein in perinatal nicotine exposure offspring. However, it is still unclear whether ST 36 has a specific therapeutic effect and how maternal acupuncture can protect the offspring from pulmonary dysplasia. In this study, we compared the different effect of ST 36 and "Fenglong" (ST 40), which belong to the same meridian, in terms of lung pulmonary function and morphology, PPARγ, β-catenin, GR levels in the lung tissues and CORT in the serum of perinatal nicotine exposure offspring, and explored the mechanism of acupuncture based on the maternal hypothalamus-pituitary-adrenal (HPA) axis. It is shown that EA applied to ST 36 could restore the normal function of maternal HPA axis and alleviate maternal glucocorticoid overexposure in offspring, thereby it can up-regulate the PTHrP/PPARγ and down-regulate the Wnt/β-catenin signaling pathways, and protects perinatal nicotine exposure-induced pulmonary dysplasia in offspring. Its effect is better than that of ST 40. These results are of great significance in preventing perinatal nicotine exposure-induced pulmonary dysplasia in offspring., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bronchopulmonary dysplasia (BPD) is potentially one of the most devastating conditions in premature infants with longstanding consequences involving multiple organ systems including adverse effects on pulmonary function and neurodevelopmental outcome. Here we review recent studies in the field to summarize the progress made in understanding in the pathophysiology, prognosis, prevention, and treatment of BPD in the last decade. The work reviewed includes the progress in understanding its pathobiology, genomic studies, ventilatory strategies, outcomes, and therapeutic interventions. We expect that this review will help guide clinicians to treat premature infants at risk for BPD better and lead researchers to initiate further studies in the field.

Background: Despite improvements in perinatal care, bronchopulmonary dysplasia (BPD) in extremely premature infants has not decreased. Postnatal surfactant therapy provides symptomatic relief from respiratory distress syndrome, but does not translate into a reduction in BPD. Therefore, the search for effective interventions to prevent BPD continues., Objectives: Since PPAR-γ agonists have been demonstrated to promote neonatal lung maturation and injury repair, we hypothesized that a formulation of a PPAR-γ agonist, pioglitazone (PGZ) and a synthetic lung surfactant (a surfactant protein B peptide mimic, B-YL) combined would stimulate lung maturation and block hyperoxia-induced neonatal lung injury more effectively than either modality alone., Methods: One-day-old Sprague-Dawley rat pups were administered PGZ + B-YL via nebulization every 24 h for up to 72 h. The pups were exposed to either 21 or 95% O2, and then sacrificed. Their lungs were examined for markers of lung maturation (levels of PPAR-γ, SP-C and choline-phosphate cytidylyltransferase [CCT-α] and [3H]triolein uptake) and injury repair (bronchoalveolar lavage cell count and protein content, and levels of LEF-1, fibronectin, ALK5, and β-catenin) by Western blot analysis., Results: Markers of alveolar epithelial/mesenchymal maturation (PPAR-γ, SP-C, CCT-α, and triolein uptake) increased significantly in the PGZ + B-YL group, more than with either drug alone. Similarly, markers of hyperoxia-induced lung injury were blocked effectively with PGZ + B-YL treatment., Conclusions: Nebulized PPAR-γ agonist PGZ with a synthetic lung surfactant accelerates lung maturation and prevents neonatal hyperoxia-induced lung injury more than either modality alone, with the potential to provide more effective prevention of BPD., (© 2018 S. Karger AG, Basel.)

There is increasing concern that early-life exposure to endocrine disruptors affects male offspring reproduction. However, whether di-n-butyl phthalate (DBP), a widely used endocrine disruptor, has transgenerational effects and, if so, the exact underlying molecular mechanisms involved remain unknown. In our study, 5 of time-mated pregnant SD rats were exposed to 0 and 500 mg/kg DBP with corn oil as the vehicle via oral gavage from embryonic days (E8-E14). Epigenetic and metabolomic of testis were analyzed after post-natal 60 days. Sperm and testicular cell functions were examined to confirm the transgenerational effects. DBP exposure significantly decreased the sperm counts in F1 through F3 generation. We found distinct metabolic changes in the testis of both F1 and F3 generation offspring, specifically, a significantly increased level of betaine, which is an important methyl donor. In contrast, the expression of betaine homocysteine S-methyltransferase (BHMT), which catalyzes the transfer of methyl moiety from betaine to homocysteine, significantly decreased. There was accompanying global DNA hypomethylation, along with a reduction in follistatin-like 3 (Fstl3) promoter hypomethylation, which is a known modulator of Sertoli cell number and spermatogenesis. In summary, we conclude that metabolomic and epigenetic changes induced by the aberrant expression of BHMT represent a novel mechanism linking in utero DBP exposure to transgenerational spermatogenesis failure., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: A head to head comparison study on renal function and ductal response between indomethacin and ibuprofen has rarely been conducted in extremely low birth weight (ELBW) infants., Objectives: The aim was to compare renal function and ductal response between indomethacin and ibuprofen in ELBW infants., Methods: We performed a double-blind randomized control trial to compare renal function and ductal response between indomethacin (0.2, 0.1, and 0.1 mg/kg i.v. every 24 h for 3 doses) and ibuprofen lysine (10, 5, and 5 mg/kg i.v. every 24 h for 3 doses) in ELBW infants with significant hemodynamic patent ductus arteriosus (cardiovascular dysfunction score >3 and LA/AO ratio ≥1.3)., Results: A total of 144 infants were enrolled: 73 received indomethacin and 71 received ibuprofen lysine. Significant decreases in urine output were seen in 30 infants (41%) in the indomethacin group and 15 (21%) in the ibuprofen group (p = 0.02). The indomethacin group was associated with a significantly higher chance of persistent ductal response than the ibuprofen group (66 vs. 49%, p = 0.046), but with a lower glomerular filtration rate on day 1, higher serum creatinine on days 1, 2, and 7, and lower urinary prostaglandin on days 2-7. Both groups were comparable in mortality and in bronchopulmonary dysplasia, intraventricular hemorrhage, necrotizing enterocolitis, and retinopathy of prematurity morbidity., Conclusions: With the current dosage, ibuprofen had fewer renal side effects but was associated with a lower rate of persistent ductal closure in ELBW infants. The precise role of prostaglandin on renal tubular function in ELBW infants remains to be further investigated., (© 2016 S. Karger AG, Basel.)

Introduction: The physiologic vitamin D (VD), 1α,25(OH) 2 D 3 (1,25D) is a local paracrine/autocrine effecter of fetal lung maturation. By stimulating alveolar type II cell and lipofibroblast proliferation and differentiation, parenterally administered 1,25D has been shown to enhance neonatal lung maturation; but due to the potential systemic side effects of the parenteral route, the translational value of these findings might be limited. To minimize the possibility of systemic toxicity, we examined the effects of VD on neonatal lung maturation, when delivered directly to lungs via nebulization., Methods: One-day-old rat pups were administered three different doses of 1,25D and its physiologic precursor 25(OH)D (25D), or the diluent, via nebulization daily for 14 days. Pups were sacrificed for lung, kidneys, and blood collection to determine markers of lung maturation, and serum 25D and calcium levels., Results: Compared to controls, nebulized 25D and 1,25D enhanced lung maturation as evidenced by the increased expression of markers of alveolar epithelial (SP-B, leptin receptor), mesenchymal (PPARγ, C/EBPα), and endothelial (VEGF, FLK-1) differentiation, surfactant phospholipid synthesis, and lung morphology without any significant increases in serum 25D and calcium levels., Conclusions: Inhaled VD is a potentially safe and effective novel strategy to enhance neonatal lung maturation., Competing Interests: Compliance with Ethical Standards Conflicts of Interest The authors declare that they have nothing to declare.

Objective: Although intrauterine nutritional stress is known to result in offspring obesity and the metabolic phenotype, the underlying cellular/molecular mechanisms remain incompletely understood. We tested the hypothesis that compared with the controls, the bone marrow-derived mesenchymal stem cells (BMSCs) of the intrauterine growth-restricted (IUGR) offspring exhibit a more adipogenic phenotype., Methods: A well-established rat model of maternal food restriction (MFR), that is, 50% global caloric restriction during the later-half of pregnancy and ad libitum diet following birth that is known to result in an obese offspring with a metabolic phenotype was used. BMSCs at 3 weeks of age were isolated, and then molecularly and functionally profiled., Results: BMSCs of the intrauterine nutritionally-restricted offspring demonstrated an increased proliferation and an enhanced adipogenic molecular profile at miRNA, mRNA and protein levels, with an overall up-regulated PPARγ (miR-30d, miR-103, PPARγ, C/EPBα, ADRP, LPL, SREBP1), but down-regulated Wnt (LRP5, LEF-1, β-catenin, ZNF521 and RUNX2) signaling profile. Following adipogenic induction, compared with the control BMSCs, the already up-regulated adipogenic profile of the MFR BMSCs, showed a further increased adipogenic response., Conclusions: Markedly enhanced adipogenic molecular profile and increased cell proliferation of MFR BMSCs suggest a possible novel cellular/mechanistic link between the intrauterine nutritional stress and offspring metabolic phenotype. This provides new potential predictive and therapeutic targets against these conditions in the IUGR offspring.

Objective: Perinatal nicotine exposure alters offspring lung structure and function; however, the underlying mechanisms remain incompletely understood. Whether epithelial-mesenchymal transition (EMT), a known contributor to pulmonary pathology, occurs following moderate perinatal nicotine exposure is not known., Methods: Pregnant, pair-fed Sprague Dawley rat dams received either placebo (diluent) or nicotine [1 mg/kg, subcutaneously] once daily from embryonic day (e) 6 to postnatal day (PND) 21. Generation 1 (F1) and 3 (F3) offspring lungs were isolated at PND 21, and using Western analysis, q-RT-PCR and immunohistochemistry examined for evidence of EMT. To gain further supportive evidence for nicotine-induced EMT, embryonic day 19 primary rat lung alveolar type II (ATII) cells were cultured and treated with nicotine for 24 hr., Results: Protein levels of α-smooth muscle actin, fibronectin, and calponin (myogenic differentiation markers) increased significantly. However, surfactant proteins B and C, and cholinephosphate cytidylyltransferase-α (epithelial cell markers), as well as the typical markers of EMT, E-cadherin, N-cadherin, and fibroblast specific protein (FSP)-1, in both F1 and F3 generation lungs, showed no significant change between the nicotine exposed and control dams. Immunostaining of lung sections and data from in vitro treated ATII cells strongly supported the Western data., Conclusions: Enhanced myogenic molecular profile, without evidence of EMT, as evidenced by the absence of the loss of E-cadherin or gains in N-cadherin and FSP-1, suggest that perinatal nicotine exposure does not result in EMT, but it leads to myogenesis, which predominantly accounts for the lung phenotype seen in perinatally nicotine exposed rat offspring. Pediatr Pulmonol. 2016;51:1142-1150. © 2016 Wiley Periodicals, Inc., Competing Interests: None., (© 2016 Wiley Periodicals, Inc.)

Introduction: Pregnant women exposed to tobacco smoke predispose the offspring to many adverse consequences including an altered lung development and function. There is no effective therapeutic intervention to block the effects of smoke exposure on the developing lung. Clinical and animal studies demonstrate that acupuncture can modulate a variety of pathophysiological processes, including those involving the respiratory system; however, whether acupuncture affects the lung damage caused by perinatal smoke exposure is not known., Methods: To determine the effect of acupuncture on perinatal nicotine exposure on the developing lung, pregnant rat dams were administered (1) saline, (2) nicotine, or (3) nicotine + electroacupuncture (EA). Nicotine was administered (1 mg/kg subcutaneously) once a day and EA was applied to both "Zusanli" (ST 36) points. Both interventions were administered from gestational day 6 to postnatal day 21 (PND21), following which pups were sacrificed. Lungs, blood, and brain were collected to examine markers of lung injury, repair, and hypothalamic pituitary adrenal (HPA) axis., Results: Concomitant EA application blocked nicotine-induced changes in lung morphology, lung peroxisome proliferator-activated receptor γ and wingless-int signaling, two key lung developmental signaling pathways, hypothalamic pituitary adrenal axis (hypothalamic corticotropic releasing hormone and lung glucocorticoid receptor levels), and plasma β-endorphin levels., Conclusions: Electroacupuncture blocks the nicotine-induced changes in lung developmental signaling pathways and the resultant myogenic lung phenotype, known to be present in the affected offspring. We conclude that EA is a promising novel intervention against the smoke exposed lung damage to the developing lung.

Perinatal smoke/nicotine exposure predisposes to chronic lung disease and morbidity. Mitochondrial abnormalities may contribute as the PPARγ pathway is involved in structural and functional airway deficits after perinatal nicotine exposure. We hypothesized perinatal nicotine exposure results in lung mitochondrial dysfunction that can be rescued by rosiglitazone (RGZ; PPARγ receptor agonist). Sprague-Dawley dams received placebo (CON), nicotine (NIC, 1 mg kg(-1)), or NIC + RGZ (3 mg kg(-1)) daily from embryonic day 6 to postnatal day 21. Parenchymal lung (~10 mg) was taken from adult male offspring for mitochondrial assessment in situ. ADP-stimulated O2 consumption was less in NIC and NIC + RGZ compared to CON (F[2,14] = 17.8; 4.5 ± 0.8 and 4.1 ± 1.4 vs. 8.8 ± 2.5 pmol s mg(-1); p < 0.05). The respiratory control ratio for ADP, an index of mitochondrial coupling, was reduced in NIC and remediated in NIC + RGZ (F[2,14] = 3.8; p < 0.05). Reduced mitochondrial oxidative capacity and abnormal coupling were evident after perinatal nicotine exposure. RGZ improved mitochondrial function through tighter coupling of oxidative phosphorylation.

The pulmonary alveolar lipofibroblast was first reported in 1970. Since then its development, structure, function and molecular characteristics have been determined. Its capacity to actively absorb, store and 'traffic' neutral lipid for protection of the alveolus against oxidant injury, and for the active supply of substrate for lung surfactant phospholipid production have offered the opportunity to identify a number of specialized functions of these strategically placed cells. Namely, Parathyroid Hormone-related Protein (PTHrP) signaling, expression of Adipocyte Differentiation Related Protein, leptin, peroxisome proliferator activator receptor gamma, and the prostaglandin E2 receptor EP2- which are all stretch-regulated, explaining how and why surfactant production is 'on-demand' in service to ventilation-perfusion matching. Because of the central role of the lipofibroblast in vertebrate lung physiologic evolution, it is a Rosetta Stone for understanding how and why the lung evolved in adaptation to terrestrial life, beginning with the duplication of the PTHrP Receptor some 300 mya. Moreover, such detailed knowledge of the workings of the lipofibroblast have provided insight to the etiology and effective treatment of Bronchopulmonary Dysplasia based on physiologic principles rather than on pharmacology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10(+) progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development., (© 2015. Published by The Company of Biologists Ltd.)

Background: Fetal exposure to nicotine is not limited to maternal tobacco smoke, as electronic cigarettes have an increased prevalence of use among reproductive aged women. Animal models have shown that nicotine exposure in utero is associated with increased risk of asthma and cognitive deficits, as well as increased expression of the hippocampal glucocorticoid receptor. We hypothesized that in utero nicotine exposure is associated with epigenetic changes in the offspring lung and brain which may contribute to a memory of this exposure, Methods: Sprague-Dawley rat dams received either saline or 2 mg/kg of nicotine by intraperitoneal injection once daily from embryonic day 6 (e6) to e22. Pups were killed on day 1 of life, and brain and lung tissues were harvested (N = 3/ group)., Results: We found that nicotine exposed offspring have altered histone modifications in the brain. Dimethylation of lysine 9 of histone H3 is decreased (0.43-fold; p = 0.03) while acetylation is increased (1.79-fold; p = 0.031). Histone deacetylase activity is significantly decreased with nicotine exposure in brain and lung (0.11-fold; p < 0.001; 0.12-fold; p < 0.001, respectively). Expression of splice variant 1.7 of the glucocorticoid receptor is reduced in the nicotine exposed offspring lung (0.25-fold; p = 0.038)., Conclusion: We conclude that nicotine exposure is associated with epigenetic alterations in the offspring and may lead to susceptibility to adult disease,. Our finding that in utero exposure to nicotine is associated with inhibition of histone deacetylase activity in the brain of offspring is of importance as a similar inhibition has been suggested as a mechanism for the potentiation of addiction., (© 2015 Wiley Periodicals, Inc.)

In a rat model, downregulation of homeostatic mesenchymal peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling following perinatal nicotine exposure contributes to offspring asthma, which can be effectively prevented by concomitant administration of PPAR-γ agonist rosiglitazone (RGZ). However, whether perinatal nicotine exposure-induced asthma can be reversed is not known. We hypothesized that perinatal nicotine exposure-induced asthma would be reversed by PPAR-γ agonist RGZ. Pregnant rat dams received either placebo or nicotine from embryonic day 6 until term. Following spontaneous delivery at term, dams were continued on the assigned treatments, up to postnatal day 21 (PND21). However, at delivery, pups were divided into two groups; one group received placebo, and the other group received RGZ from PND1 to PND21. At PND21, pulmonary function and the expression of mesenchymal markers of airway contractility (α-smooth muscle actin, calponin, fibronectin, collagen I, and collagen III) were determined by immunoblotting and immunostaining for the evidence of reversibility of perinatal nicotine exposure-induced lung effects. Compared with controls, perinatal nicotine exposure caused 1) a significant increase in airway resistance and a decrease in airway compliance following methacholine challenge, 2) a significant increase in acetylcholine-induced tracheal constriction, and 3) increased pulmonary and tracheal expression of the mesenchymal markers of contractility. Treatment with RGZ, starting on PND1, reversed all of the nicotine-induced molecular and functional pulmonary effects, virtually normalizing the pulmonary phenotype of the treated animals. We conclude that perinatal nicotine exposure-induced functional and molecular alterations in upper and lower airways can be reversed by PPAR-γ agonist RGZ, allowing an effective intervention even when started postnatally., (Copyright © 2015 the American Physiological Society.)

Maternal food restriction (MFR) causes intrauterine growth restriction, a known risk factor for developing chronic lung disease. However, it is unknown whether this negative outcome is gender specific or preventable by blocking the MFR-induced hyperglucocorticoidism. Using a well-established rat model, we used metyrapone (MTP), an inhibitor of glucocorticoid synthesis, to study the MFR-induced lung changes on postnatal day (p) 21 in a gender-specific manner. From embryonic day 10 until delivery, pregnant dams were fed either an ad libitum diet or a 50% caloric restricted diet with or without MTP supplementation. Postnatally, the offspring were fed ad libitum from healthy dams until p21. Morphometric, Western blot, and immunohistochemical analysis of the lungs demonstrated that MTP mitigated the MFR-mediated decrease in alveolar count, decrease in adipogenic protein peroxisome proliferator-activated receptor γ, increase in myogenic proteins (fibronectin, α-smooth muscle actin, and calponin), increase in Wnt signaling intermediates (lymphoid enhancer-binding factor 1 and β-catenin), and increase in glucocorticoid receptor (GR) levels. The MFR-induced lung phenotype and the effects of MTP were similar in both genders. To elucidate the mechanism of MFR-induced shift of the adipogenic-to-myogenic phenotype, lung fibroblasts were used to independently study the effects of (1) nutrient restriction and (2) excess steroid exposure. Nutrient deprivation increased myogenic proteins, Wnt signaling intermediates, and GR, all changes blocked by protein supplementation. MTP also blocked, likely by normalizing nicotinamide adenine dinucleotide phosphate levels, the corticosterone-induced increase in myogenic proteins, but had no effect on GR levels. In summary, protein restriction and increased glucocorticoid levels appear to be the key players in MFR-induced lung disease, affecting both genders., (© The Author(s) 2014.)

Whereas epidemiological data strongly link vitamin D (VD) deficiency to childhood asthma, the underlying molecular mechanisms remain unknown. Although VD is known to stimulate alveolar epithelial-mesenchymal interactions, promoting perinatal lung maturation, whether VD supplementation during this period protects against childhood asthma has not been demonstrated experimentally. Using an in vivo rat model, we determined the effects of perinatal VD deficiency on overall pulmonary function and the tracheal contraction as a functional marker of airway contractility. One month before pregnancy, rat dams were put on either a no cholecalciferol-added or a 250, 500, or 1,000 IU/kg cholecalciferol-added diet, which was continued throughout pregnancy and lactation. At postnatal day 21, offspring plasma 25(OH)D levels and pulmonary function (whole body plethysmography and tracheal contraction response to acetylcholine) were determined. 25(OH)D levels were lowest in the no cholecalciferol-supplemented group, increasing incrementally in response to cholecalciferol supplementation. Compared with the 250 and 500 IU/kg VD-supplemented groups, the no cholecalciferol-supplemented group demonstrated a significant increase in airway resistance following methacholine challenge. However, the cholecalciferol deficiency-mediated increase in tracheal contractility in the cholecalciferol-depleted group was only blocked by supplementation with 500 IU/kg cholecalciferol. Therefore, in addition to altering alveolar epithelial-mesenchymal signaling, perinatal VD deficiency also alters airway contractility, providing novel insights to asthma pathogenesis in perinatally VD-deficient offspring. Perinatal VD supplementation at 500 IU/kg appears to effectively block these effects of perinatal VD deficiency in the rat model used, providing a strong clinical rationale for effective perinatal VD supplementation for preventing childhood asthma., (Copyright © 2014 the American Physiological Society.)

Significance: Oxygen, the main mode of support for premature infants with immature lungs, can cause toxicity by producing reactive oxygen species (ROS) that disrupt homeostasis; yet, these same molecules were entrained to promote vertebrate lung phylogeny. By providing a deeper understanding of this paradox, we propose physiologically rational strategies to prevent chronic lung disease (CLD) of prematurity., Recent Advances: To prevent neonatal hyperoxic lung damage biologically, we have exploited the alveolar defense mechanism(s) that evolutionarily evolved to combat increased atmospheric oxygen during the vertebrate water to land transition., Critical Issues: Over the course of vertebrate lung evolution, ROS promoted the formation of lipofibroblasts, specialized adepithelial cells, which protect the alveoli against oxidant injury; peroxisome proliferator-activated receptor gamma (PPARγ), the master switch for lipofibroblast differentiation, prevents such oxidant lung injury, both by directly promoting mesodermal differentiation and its antioxidant defenses, and indirectly by stimulating the developmental epithelial-mesenchymal paracrine interactions that have physiologically determined lung surfactant production in accord with the lung's phylogenetic adaptation to atmospheric oxygen, preventing Respiratory Distress Syndrome at birth., Future Directions: The molecular strategy (PPARγ agonists) to prevent CLD of prematurity, proposed by us, although seems to be robust, effective, and safe under experimental conditions, it awaits detailed pharmacokinetic and pharmacodynamic studies for its safe and effective clinical translation to human infants. Antioxid. Redox Signal. 21, 1893-1904. "I have procured air [oxygen]…between five and six times as good as the best common air that I have ever met with." -Joseph Priestley, 1775.