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Hyperoxia-induced inflammation contributes significantly to developmental lung injury and bronchopulmonary dysplasia (BPD) in preterm infants. Platelet activating factor (PAF) is known to be a major driver of inflammation in lung diseases such as asthma and pulmonary fibrosis, but its role in BPD has not been previously investigated. Therefore, to determine whether PAF signaling independently modulates neonatal hyperoxic lung injury and BPD pathogenesis, lung structure was assessed in 14 day-old C57BL/6 wild-type (WT) and PAF receptor knockout (PTAFR KO) mice that were exposed to 21% (normoxia) or 85% O2(hyperoxia) from postnatal day 4. Lung morphometry showed that PTAFR KO mice had attenuated hyperoxia-induced alveolar simplification when compared to WT mice. Functional analysis of gene expression data from hyperoxia-exposed vs. normoxia-exposed lungs of WT and PTAFR KO showed that the most upregulated pathways were thehypercytokinemia/hyperchemokinemiapathway in WT mice,NAD signalingpathway in PTAFR KO mice, andagranulocyte adhesion and diapedesisas well as other pro-fibrotic pathways such astumor microenvironmentandoncostatin-M signalingin both mice strains, indicating that PAF signaling may contribute to inflammation but may not be a significant mediator of fibrotic processes during hyperoxic neonatal lung injury. Gene expression analysis also indicated increased expression of pro-inflammatory genes such as CXCL1, CCL2 and IL-6 in the lungs of hyperoxia-exposed WT mice and metabolic regulators such as HMGCS2 and SIRT3 in the lungs of PTAFR KO mice, suggesting that PAF signaling may modulate BPD risk through changes in pulmonary inflammation and/or metabolic reprogramming in preterm infants.

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants. Hyperoxia exposure and microbial dysbiosis are contributors to BPD development. However, the mechanisms linking pulmonary microbial dysbiosis to worsening lung injury are unknown. Nuclear factor-erythroid factor 2-related factor 2 (Nrf2) is a transcription factor that regulates oxidative stress responses and modulates hyperoxia-induced lung injury. We hypothesized that airway dysbiosis would attenuate Nrf2-dependent antioxidant function, resulting in a more severe phenotype of BPD. Here, we show that preterm infants with a Gammaproteobacteria-predominant dysbiosis have increased endotoxin in tracheal aspirates, and mice monocolonized with the representative Gammaproteobacteria, Escherichia coli, show increased expression of Nrf2-regulated genes Hmox1 and Nqo1. Furthermore, we show Nrf2 deficient mice have worse lung structure and function after exposure to hyperoxia when the airway microbiome is augmented with E. coli. To confirm the disease-initiating potential of airway dysbiosis, we developed a novel humanized mouse model by colonizing germ-free mice with tracheal aspirates from human infants with or without severe BPD, producing gnotobiotic mice with BPD-associated and non-BPD-associated lung microbiomes. After hyperoxia exposure, BPD-associated mice demonstrated a more severe BPD phenotype and increased expression of Nrf2-regulated genes, as compared to germ-free and non-BPD-associated mice. Furthermore, augmenting Nrf2-mediated antioxidant activity by supporting colonization with Lactobacillus species improved dysbiotic-augmented lung injury. Our results demonstrate that pulmonary microbial dysbiosis in the lung produces an Nrf2-mediated antioxidant response which is augmented by a respiratory probiotic blend. We anticipate antioxidant pathways will be major targets of future microbiome-based therapeutics for respiratory disease.

Objective Work-related pain and disability have been reported in the literature among surgeons. This national survey was designed to identify the prevalence and severity of these symptoms in vascular surgeons. Methods A survey was emailed to the 2910 members of the Society for Vascular Surgery. Physical pain was evaluated based on body part, and type of vascular procedure performed using the Borg 0 to 10 pain scale. Wellness questions were also queried. Results A total of 775 of Society for Vascular Surgery members responded, with a 26.6% response rate. Retirees were excluded from the study (n = 39). Among those actively working (n = 736), surgeons have been practicing surgery, on average, for 17.2 ± 11.6 years, with a mean age of 51.4 ± 10.9 years, and 83.6% are male. After a full day of open surgery, the majority of the responding vascular surgeons are in a moderately strong amount of pain (mean score, 4.4 ± 2.3). After a full day of endovascular procedures, most vascular surgeons are in a moderately strong amount of pain (mean score, 3.9 ± 2.4). Pain after open surgery is greatest in the neck, and after endovascular surgery pain is highest in the lower back. Surgeons performing endovenous procedures demonstrated the lowest pain scores (2.0 ± 2.0). In total, 36.9% (242/655, 81 missing responses) have sought medical care for work-related pain, with 8.3% (61/736) taking time away from the operating room. Of those, 26.2% (193/736) report pain severe enough that it interferes with sleep. Seventy-two (10%) required surgery or other significant medical procedures. Of the 39 retirees, 26% ended their careers owing to physical disabilities from work-related pain. Out of the entire cohort, 52.7% (334/633,103 missing responses) feel that physical discomfort will affect the longevity of their careers. Additionally, we found that high work-related physical discomfort is significantly associated with burnout (burnout vs no burnout; P Conclusions Our study shows that the majority of practicing vascular surgeons responding to the survey are in pain after a day of operating. Addressing work-related pain serves to improve the lives and careers of vascular surgeons and enhance surgical longevity.

BackgroundMicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD).MethodsHere we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants.ResultsmiR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situhybridizationof human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR‐219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR‐219 increased significantly after miR‐219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation.ConclusionsTaken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

Supplemental Digital Content is available in the text., OBJECTIVES: Cardiopulmonary bypass triggers systemic inflammation, resulting in lung injury, and frequently leads to prolonged mechanical ventilation. Biomarkers of systemic inflammation are required to predict the risk of such complications. We hypothesize that specific serum proteins can be used as biomarkers to predict the severity of lung injury following cardiac surgery. DESIGN: Retrospective chart review study. SETTING: Clinical variables were collected and used in conjuncture with unbiased proteomic analysis using mass spectrometry that was performed on frozen plasma samples from a study group (patients with mechanical ventilation > 48 hr post surgery) and a control group (patients with mechanical ventilation < 48 hr post surgery). SUBJECTS: Subjects included were infants who underwent cardiac surgery with similar complexity (Society of Thoracic Surgeons-European Association for Cardiothoracic Surgery 3 or 4) using cardiopulmonary bypass. Patients in both groups were matched for their weight, age, and duration of cardiopulmonary bypass. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Four-hundred eighty-three proteins were identified (99% minimum confidence and two peptides minimum, protein false discovery rate 0.1%) on proteomic analysis of four control and four study patients at precardiopulmonary bypass, 0, and 48 hours postcardiopulmonary bypass samples. Thirty-six of 178 proteins were significantly different (≥ 1.5-fold; p < 0.05) at precardiopulmonary bypass (top increased: tenascin; top decreased: tetranectin), 18 of 140 proteins at 0 hour (top increased: hemoglobin beta; top decreased: C8 beta), and 25 of 166 proteins at 48 hours post surgery (top increased: proteoglycan 4; top decreased: galectin-3–binding protein). The top pathway involved cytoskeleton remodeling. Other pathways involved immune response and blood coagulation. Proteoglycan 4 was validated by enzyme-linked immunosorbent assay in a different set of samples (n = 20/group; mean ± sd: 128 ± 67 vs 195 ± 160 ng/mL) (p = 0.037). CONCLUSIONS: Multiple proteomic biomarkers were associated with worse respiratory outcomes. Precardiopulmonary bypass biomarkers might indicate risk factors (e.g., abnormalities of coagulation), whereas those identified at 0 hour and post cardiopulmonary bypass may reflect mechanisms of ongoing pathobiology.

BACKGROUND Our understanding of the normative concentrations of urine biomarkers in premature neonates is limited. METHODS We evaluated urine from 750 extremely low gestational age (GA) neonates without severe acute kidney injury (AKI) to determine how GA affects ten different urine biomarkers at birth and over the first 30 postnatal days. Then, we investigated if the urine biomarkers changed over time at 27, 30, and 34 weeks postmenstrual age (PMA). Next, we evaluated the impact of sex on urine biomarker concentrations at birth and over time. Finally, we evaluated if urine biomarkers were impacted by treatment with erythropoietin (Epo). RESULTS We found that all ten biomarker concentrations differ at birth by GA and that some urine biomarker concentrations increase, while others decrease over time. At 27 weeks PMA, 7/10 urine biomarkers differed by GA. By 30 weeks PMA, 5/10 differed, and by 34 weeks PMA, only osteopontin differed by GA. About half of the biomarker concentrations differed by sex, and 4/10 showed different rates of change over time between males vs. females. We found no differences in urine biomarkers by treatment group. CONCLUSIONS The temporal patterns, GA, and sex differences need to be considered in urine AKI biomarker analyses. IMPACT Urine biomarker concentrations differ by GA at birth. Some urine biomarkers increase, while others decrease, over the first 30 postnatal days. Most urine biomarkers differ by GA at 27 weeks PMA, but are similar by 34 weeks PMA. Some urine biomarkers vary by sex in premature neonates. Urine biomarkers did not differ between neonates randomized to placebo vs. Epo.

Physician burnout has been linked to medical errors, decreased patient satisfaction, and decreased career longevity. In light of the increasing prevalence of cardiovascular disease, vascular surgeon burnout presents a legitimate public health concern owing to the impact on the adequacy of the vascular surgery workforce. The aims of this study were to define the prevalence of burnout among practicing vascular surgeons and identify factors that contribute to burnout to facilitate future Society for Vascular Surgery (SVS) initiatives to mitigate this crisis.In 2018, active SVS members were surveyed electronically and confidentially using the Maslach Burnout Inventory. The survey was tailored to explore specialty-specific issues, and to capture demographic and practice-related characteristics. Emotional exhaustion (EE) and depersonalization (DP) were analyzed as dimensions of burnout. Consistent with convention, surgeons with a high score on the DP and/or EE subscales of the Maslach Burnout Inventory were considered to have at least one manifestation of professional burnout. Risk factors associated with symptoms of burnout were identified using bivariate analyses (χOf 2905 active SVS members, 960 responded to the survey (34% participation rate). After excluding retired surgeons and incomplete submissions, responses from 872 practicing vascular surgeons were analyzed. The mean age was 49.7 ± 11.0 years; the majority of respondents (81%) were male. Primary practice settings were academic (40%), community practice (41%), veteran's hospital (3.3%), active military practice (1.5%), or other. Years in practice averaged 15.7 ± 11.7. Overall, 41% of respondents had at least one symptoms of burnout (ie, high EE and/or high DP), 37% endorsed symptoms of depression in the past month, and 8% indicated they had considered suicide in the last 12 months. In unadjusted analysis, factors significantly associated with burnout (P .05) included clinical work hours, on-call frequency, electronic medical record and documentation requirements, work-home conflict, and work-related physical pain. On multivariate analysis, age, work-related physical pain and work-home conflict were independent predictors for burnout.Symptoms of burnout and depression are common among vascular surgeons. Advancing age, work-related physical pain, and work-home conflict are independent predictors for burnout among vascular surgeons. Efforts to promote vascular surgeon well-being must address specialty-specific challenges, including the high prevalence of work-home conflict and occupational factors that contribute to work-related pain.

Cyclooxygenase-2 (COX-2/PTGS2) mediates hyperoxia-induced impairment of lung development in newborn animals and is increased in the lungs of human infants with bronchopulmonary dysplasia (BPD). COX-2 catalyzes the production of cytoprotective prostaglandins, such as prostacyclin (PGI2), as well as proinflammatory mediators, such as thromboxane A2. Our objective was to determine whether iloprost, a synthetic analog of PGI2, would attenuate hyperoxia effects in the newborn mouse lung. To test this hypothesis, newborn C57BL/6 mice along with their dams were exposed to normoxia (21% O2) or hyperoxia (85% O2) from 4 to 14 days of age in combination with daily intraperitoneal injections of either iloprost 200 µg·kg−1·day−1, nimesulide (selective COX-2 antagonist) 100 mg·kg−1·day−1, or vehicle. Alveolar development was estimated by radial alveolar counts and mean linear intercepts. Lung function was determined on a flexiVent, and multiple cytokines and myeloperoxidase (MPO) were quantitated in lung homogenates. Lung vascular and microvascular morphometry was performed, and right ventricle/left ventricle ratios were determined. We determined that iloprost (but not nimesulide) administration attenuated hyperoxia-induced inhibition of alveolar development and microvascular density in newborn mice. Iloprost and nimesulide both attenuated hyperoxia-induced, increased lung resistance but did not improve lung compliance that was reduced by hyperoxia. Iloprost and nimesulide reduced hyperoxia-induced increases in MPO and some cytokines (IL-1β and TNF-α) but not others (IL-6 and KC/Gro). There were no changes in pulmonary arterial wall thickness or right ventricle/left ventricle ratios. We conclude that iloprost improves lung development and reduces lung inflammation in a newborn mouse model of BPD.

Background Glioblastomas (GBMs), the most common and most lethal of the primary brain tumors, are characterized by marked intra-tumor heterogeneity. Several studies have suggested that within these tumors a restricted population of chemoresistant glioma cells is responsible for recurrence. However, the gene expression patterns underlying chemoresistance are largely unknown. Numerous efforts have been made to block IGF-1R signaling pathway in GBM. However, those therapies have been repeatedly unsuccessful. This failure may not only be due to the complexity of IGF receptor signaling, but also due to complex cell-cell interactions in the tumor mass. We hypothesized that differential expression of proteins in the insulin-like growth factor (IGF) system underlie cell-specific differences in the resistance to temozolomide (TMZ) within GBM tumors. Methods Expression of IGF-1R was analyzed in cell lines, patient-derived xenograft cell lines and human biopsies by cell surface proteomics, flow cytometry, immunofluorescence and quantitative real time polymerase chain reaction (qRT-PCR). Using gain-of-function and loss-of-function strategies, we dissected the molecular mechanism responsible for IGF-binding protein 6 (IGFBP6) tumor suppressor functions both in in vitro and in vivo. Site direct mutagenesis was used to study IGFBP6-IGF2 interactions. Results We determined that in human glioma tissue, glioma cell lines, and patient-derived xenograft cell lines, treatment with TMZ enhances the expression of IGF1 receptor (IGF-1R) and IGF2 and decreases the expression of IGFBP6, which sequesters IGF2. Using chemoresistant and chemosensitive wild-type and transgenic glioma cells, we further found that a paracrine mechanism driven by IGFBP6 secreted from TMZ-sensitive cells abrogates the proliferation of IGF-1R-expressing TMZ-resistant cells in vitro and in vivo. In mice bearing intracranial human glioma xenografts, overexpression of IGFBP6 in TMZ-resistant cells increased survival. Finally, elevated expression of IGF-1R and IGF2 in gliomas associated with poor patient survival and tumor expression levels of IGFBP6 directly correlated with overall survival time in patients with GBM. Conclusions Our findings support the view that proliferation of chemoresistant tumor cells is controlled within the tumor mass by IGFBP6-producing tumor cells; however, TMZ treatment eliminates this population and enriches the TMZ-resistant cell populationleading to accelerated growth of the entire tumor mass.

Rationale: MicroRNAs (miRNAs) destabilize mRNA transcripts and inhibit protein translation. miR-145 is of particular interest in cystic fibrosis (CF) as it has a direct binding site in the 3′-untranslated region of CFTR (cystic fibrosis transmembrane conductance regulator) and is upregulated by the CF genetic modifier TGF (transforming growth factor)-β. Objectives: To demonstrate that miR-145 mediates TGF-β inhibition of CFTR synthesis and function in airway epithelia. Methods: Primary human CF (F508del homozygous) and non-CF airway epithelial cells were grown to terminal differentiation at the air–liquid interface on permeable supports. TGF-β (5 ng/ml), a miR-145 mimic (20 nM), and a miR-145 antagonist (20 nM) were used to manipulate CFTR function. In CF cells, lumacaftor (3 μM) and ivacaftor (10 μM) corrected mutant F508del CFTR. Quantification of CFTR mRNA, protein, and function was done by standard techniques. Measurements and Main Results: miR-145 is increased fourfold in CF BAL fluid compared with non-CF (P

Background Vascular groin wounds have higher than expected surgical site infection (SSI) rates and some patients are at enhanced risk. The Wiseman et al. paper suggests an objective scoring system that identifies patients at enhanced risk of postdischarge SSI. We hypothesize that prophylactic groin wound vacuum-assisted closure (VAC) therapy in enhanced risk patients will decrease SSI and readmission and the Wiseman model provides potential evidence that enhanced risk patients can be objectively identified. Methods A single institution, retrospective analysis was conducted from January 2013 to September 2016 utilizing procedure codes to identify patients with wound VACs placed in the operating room. Two distinct groups were identified. The first was a wound complication patient group with 15 limbs (13 patients) with a groin wound VAC placed within 45 days postoperatively for groin wound complications. Eleven of these limbs had the VAC placed at readmission. The second group was a prophylactic patient group that included 8 limbs (7 patients) who received a VAC prophylactically placed in the enhanced risk wounds. These wounds were determined to be enhanced risk based on clinical criteria judged by the operating surgeon such as a large overhanging panniculus and/or one of several ongoing medical issues. We calculated a Wiseman score for all patients, determined total cost of the readmissions, and determined 30-day postsurgical SSI incidence for the prophylactic VAC group. Results Per the Wiseman scores, 9 limbs with postoperative complications were high risk and 3 limbs were moderate/high risk. Eleven limbs had a VAC placed at readmission with an average readmission cost of $8876.77. For the prophylactic group, 8 limbs were high risk with no observed postdischarge SSI in the first 30 days from surgery. The Wiseman scores showed close correlation between the retrospective high and moderate/high risk groups versus the prophylactic VAC group (31.5 ± 7.3 vs. 32 ± 5.5, P = 0.87). Conclusions The Wiseman scores showed objective validation in the prognosis of anticipating groin wound breakdown. Our initial results suggest that prophylactic groin wound VAC placement for enhanced risk vascular surgery patients may proactively decrease wound morbidity, decrease readmission secondary to groin wound complications, and provide some cost benefit.

Background Bronchopulmonary dysplasia (BPD) portends lifelong organ impairment and death. Our ability to predict BPD in first days of life is limited, but could be enhance using novel biomarkers. Methods Using an available clinical and urine biomarker database obtained from a prospective 113 infant cohort (birth weight ≤1200 g and/or gestational age ≤31 weeks), we evaluated the independent association of 14 urine biomarkers with BPD/mortality. Results Two of the 14 urine biomarkers were independently associated with BPD/mortality after controlling for gestational age (GA), small for gestational age (SGA), and intubation status. The best performing protein was clusterin, a ubiquitously expressed protein and potential sensor of oxidative stress associated with lung function in asthma patients. When modeling for BPD/mortality, the independent odds ratio for maximum adjusted urine clusterin was 9.2 (95% CI 3.3 - 32.8, P

Serum creatinine (SCr)-based AKI definitions have important limitations, particularly in very low-birth-weight (VLBW) neonates. Urine biomarkers may improve our ability to detect kidney damage. We assessed the association between 14 different urine biomarkers and AKI in VLBW infants.We performed a prospective cohort study on 113 VLBW infants (weight ≤1200 g or31 weeks' gestation) admitted to a regional neonatal intensive care unit at the University of Alabama at Birmingham between February 2012 and June 2013. SCr was measured on postnatal days 1, 2, 3, and 4 and was combined with clinically measured SCr to determine AKI according to Kidney Disease Improving Global Outcomes AKI definition (increase in SCr ≥0.3 mg/dl or ≥50% increase from previous lowest value). Urine was collected on the first 4 days (average number of urine collections, 3; range, 1-4). The maximum urine biomarkers and urine biomarker/creatinine levels were calculated for 12 urine biomarkers, and the minimum urine biomarker and biomarker/creatinine levels were assessed for two urine biomarkers. We compared these values between infants with and those without AKI. Ideal cutoffs, area under the receiver-operating characteristic curve , and area under the curve adjusted for gestational age were calculated.Cumulative incidence of AKI during the first 2 postnatal weeks was 28 of 113 (25%). Infants with AKI had higher maximum levels of urine cystatin C, neutrophil gelatinase-associated lipocalin, osteopontin, clusterin, and α glutathione S-transferase (2.0, 1.8, 1.7, 1.7, and 3.7 times higher, respectively) than infants without AKI. In addition, infants with AKI had lower minimum levels of epithelial growth factor and uromodulin than those without AKI (1.4 and 1.6 times lower, respectively). Most but not all participants had their maximum (or minimum) biomarker values preceding AKI. These associations remained after adjustment for gestational age.Urine biomarkers measured in the first 4 days of life are associated with AKI during the first postnatal weeks. Further evaluations are necessary to determine whether these biomarkers can predict important clinical outcomes. In addition, intervention studies that use biomarkers to stratify enrollment groups are needed before bedside evaluations can be incorporated into care.

MicroRNAs (miRs) are small conserved RNA that regulate gene expression. Bioinformatic analysis of miRNA profiles during mouse lung development indicated a role for multiple miRNA, including miRNA-489. miR-489 increased on completion of alveolar septation [postnatal day 42 (P42)], associated with decreases in its conserved target genes insulin-like growth factor-1 ( Igf1) and tenascin C ( Tnc). We hypothesized that dysregulation of miR-489 and its target genes Igf1 and Tnc contribute to hyperoxia-induced abnormal lung development. C57BL/6 mice were exposed to normoxia (21%) or hyperoxia (85% O2) from P4 to P14, in combination with intranasal locked nucleic acid against miR-489 to inhibit miR-489, cytomegalovirus promoter (pCMV)-miR-489 to overexpress miR-489, or empty vector. Hyperoxia reduced miR-489 and increased Igf1 and Tnc. Locked nucleic acid against miR-489 improved lung development during hyperoxia and did not alter it during normoxia, whereas miR-489 overexpression inhibited lung development during normoxia. The 3′ untranslated region in vitro reporter studies confirmed Igf1 and Tnc as targets of miR-489. While miR-489 was of epithelial origin and present in exosomes, its targets Igf1 and Tnc were produced by fibroblasts. Infants with bronchopulmonary dysplasia (BPD) had reduced lung miR-489 and increased Igf1 and Tnc compared with normal preterm or term infants. These results suggest increased miR-489 is an inhibitor of alveolar septation. During hyperoxia or BPD, reduced miR-489 and increased Igf1 and Tnc may be inadequate attempts at compensation. Further inhibition of miR-489 may permit alveolar septation to proceed. The use of specific miRNA antagonists or agonists may be a therapeutic strategy for inhibited alveolarization, such as in BPD.

Preterm infants often require prolonged oxygen supplementation and are at high risk of neurodevelopmental impairment. We recently reported that adult mice exposed to neonatal hyperoxia (postnatal day [P] 2 to 14) had spatial navigation memory deficits associated with hippocampal shrinkage. The mechanisms by which early oxidative stress impair neurodevelopment are not known. Our objective was to identify early hyperoxia-induced alterations in hippocampal receptors and signaling pathways necessary for memory formation. We evaluated C57BL/6 mouse pups at P14, exposed to either 85% oxygen or air from P2 to 14. We performed targeted analysis of hippocampal ligand-gated ion channels and proteins necessary for memory formation, and global bioinformatic analysis of differentially expressed hippocampal genes and proteins. Hyperoxia decreased hippocampal mGLU7, TrkB, AKT, ERK2, mTORC1, RPS6, and EIF4E and increased α3, α5, and ɤ2 subunits of GABAA receptor and PTEN proteins, although changes in gene expression were not always concordant. Bioinformatic analysis indicated dysfunction in mitochondria and global protein synthesis and translational processes. In conclusion, supraphysiological oxygen exposure reduced proteins necessary for hippocampus-dependent memory formation and may adversely impact hippocampal mitochondrial function and global protein synthesis. These early hippocampal changes may account for memory deficits seen in preterm survivors following prolonged oxygen supplementation.

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product–induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.

Background Heme Oxygenase 1 (HO1) catalyzes heme degradation, and offers protection for several organs, including the kidney. Genetic polymorphisms of HO-1 are associated with poor clinical outcomes in several populations. Methods Population: We prospectively enrolled 117 premature infants (birth weight ≤1200 gm. or post gestational age ≤31 weeks) and evaluated 2 DNA genetic variants proximal to the promoter region of HO-1 (GT(n) repeats, and–413T>A SNP). We evaluated how these polymorphisms affect 2 clinical outcomes i) AKI - rise in serum creatinine (SCr) ≥ 0.3 mg/dl or ≥ 150–200% from lowest previous value, ii) bronchopulmonary dysplasia (BPD) defined as receipt of oxygen at 36 weeks post menstrual age (PMA) / mortality. Results AKI occurred in 34/117 (29%) of neonates; 12/117 (10%) died; 29/105 (28%) survivors had BPD. Neonates with TT genotype at – 413T>A before the HO-1 promoter) had higher rates of AKI (pA genetic alteration had lower incidence of AKI. Further studies using larger cohorts are needed to better understand these relationships.

Vascular endothelial growth factor (VEGF), a well-characterized regulator of angiogenesis, has been mechanistically implicated in retinal neovascularization and in the pathogenesis of retinopathy of prematurity. However, the ontogeny of VEGF expression in the human fetal retina is not well known. Because retinal vasculature grows with gestational maturation, we hypothesized that VEGF expression also increases in the midgestation human fetal eye as a function of gestational age. To identify changes in VEGF gene expression during normal human development, we measured VEGF mRNA by quantitative PCR and measured VEGF protein by enzyme-linked immunosorbent assay and western blots in 10–24 wk gestation fetal vitreous, retina, and serum. VEGF mRNA expression in the retina increased with gestational age. VEGF isoform A, particularly its VEGF121 splice variant, contributed to this positive correlation. Consistent with these findings, we detected increasing VEGF121 protein concentrations in vitreous humor from fetuses of 10–24 wk gestation, while VEGF concentrations decreased in fetal serum. VEGF121 mRNA and protein concentrations increase with increasing gestational age in the developing human retina. We speculate that VEGF plays an important role in normal retinal vascular development, and that preterm delivery affects production of this vascular growth factor.

The halogen bromine (Br2) is used extensively in industry and stored and transported in large quantities. Its accidental or malicious release into the atmosphere has resulted in significant casualties. The pathophysiology of Br2-induced lung injury has been studied in adult animals, but the consequences of Br2 exposure to the developing lung are completely unknown. We exposed neonatal mouse littermates on postnatal day 3 (P3) to either Br2 at 400 ppm for 30 min (400/30), to Br2 at 600 ppm for 30 min (600/30), or to room air, then returned them to their dams and observed until P14. Mice exposed to Br2 had decreased survival (S) and had decreased weight (W) at P14 in the 400/30 group (S = 63.5%, W = 6.67 ± 0.08) and in the 600/30 group (S = 36.1%, W = 5.13 ± 0.67) as compared with air breathing mice (S = 100%, W = 7.96 ± 0.30). Alveolar development was impaired, as evidenced by increased mean linear intercept at P14. At P14, Br2 exposed mice also exhibited a decrease of arterial partial pressure of oxygen, decreased quasi-static lung compliance, as well as increased alpha smooth muscle actin mRNA and protein and increased mRNA for IL-1β, IL-6, CXCL1, and TNFα. Global gene expression, evaluated by RNA sequencing and Ingenuity Pathway Analysis, revealed persistent abnormalities in gene expression profiles at P14 involving pathways of “formation of lung” and “pulmonary development.” The data indicate that Br2 inhalation injury early in life results in severe lung developmental consequences, wherein persistent inflammation and global altered developmental gene expression are likely mechanistic contributors.

Endoglin (ENG) regulates signaling by transforming growth factor‐β (TGF‐β), a genetic modifier of cystic fibrosis (CF) lung disease severity. We hypothesized that ENG mediates TGF‐β pathobiology in CF airway epithelia. Comparing CF and non‐CF human lungs, we measured ENG by qPCR, immunoblotting and ELISA. In human bronchial epithelial cell lines (16HBE), we used CFTR siRNA knockdown and functional inhibition (CFTRINH‐172) to connect loss of CFTR to ENG synthesis. Plasmid overexpression of ENG assessed the direct effect of ENG on TGF‐β transcription and signal amplification in 16HBE cells. We found ENG protein to be increased more than fivefold both in human CF bronchoalveolar fluid (BALF) and human CF lung homogenates. ENG transcripts were increased threefold in CF, with a twofold increase in TGF‐β signaling. CFTR knockdown in 16HBE cells tripled ENG transcription and doubled protein levels with corresponding increases in TGF‐β signaling. Plasmid overexpression of ENG alone nearly doubled TGF‐β1 mRNA and increased TGF‐β signaling in 16HBE cells. These experiments identify that loss of CFTR function increases ENG expression in CF epithelia and amplifies TGF‐β signaling. Targeting ENG may offer a novel therapeutic opportunity to address TGF‐β associated pathobiology in CF.

Nanoparticles are used in an increasing number of biomedical, industrial, and food applications, but their safety profiles in developing organisms, including the human fetus and infant, have not been evaluated. Titanium oxide (TiO2) nanoparticles, which are commonly used in cosmetics, sunscreens, paints, and food, have been shown to induce emphysema and lung inflammation in adult mice. We hypothesized that exposure of newborn mice to TiO2 would induce lung inflammation and inhibit lung development. C57BL/6 mice were exposed to TiO2 (anatase; 8–10 nm) nanoparticles by intranasal instillation as a single dose on postnatal day 4 (P4) or as three doses on postnatal days 4, 7, and 10 (each dose = 1 μg/g body wt). Measurements of lung function (compliance and resistance), development (morphometry), inflammation (histology; multiplex analysis of bronchoalveolar lavage fluid for cytokines; PCR array and multiplex analysis of lung homogenates for cytokines) was performed on postnatal day 14. It was observed that a single dose of TiO2 nanoparticles led to inflammatory cell influx, and multiple doses led to increased inflammation and inhibition of lung development without significant effects on lung function. Macrophages were noted to take up the TiO2 nanoparticles, followed by polymorphonuclear infiltrate. Multiple cytokines and matrix metalloproteinase-9 were increased in lung homogenates, and VEGF was reduced. These results suggest that exposure of the developing lung to nanoparticles may lead to ineffective clearance by macrophages and persistent inflammation with resulting effects on lung development and may possibly impact the risk of respiratory disorders in later life.

Alterations of pulmonary microbiome have been recognized in multiple respiratory disorders. It is critically important to ascertain if an airway microbiome exists at birth and if so, whether it is associated with subsequent lung disease. We found an established diverse and similar airway microbiome at birth in both preterm and term infants, which was more diverse and different from that of older preterm infants with established chronic lung disease (bronchopulmonary dysplasia). Consistent temporal dysbiotic changes in the airway microbiome were seen from birth to the development of bronchopulmonary dysplasia in extremely preterm infants. Genus Lactobacillus was decreased at birth in infants with chorioamnionitis and in preterm infants who subsequently went on to develop lung disease. Our results, taken together with previous literature indicating a placental and amniotic fluid microbiome, suggest fetal acquisition of an airway microbiome. We speculate that the early airway microbiome may prime the developing pulmonary immune system and dysbiosis in its development may set the stage for subsequent lung disease.

Rationale: DNA methylation is an important epigenetic mechanism, which often occurs in response to environmental stimuli and is crucial in regulating gene expression. It is likely that epigenetic alterations contribute to pathogenesis in idiopathic pulmonary fibrosis (IPF).Objectives: To determine the DNA methylation changes in IPF and their effects on gene expression.Methods: Total DNA methylation and DNA methyltransferase expression were compared in IPF and normal control lung tissues. IPF and normal tissues were subjected to comparative analysis of genome-wide DNA methylation and RNA expression using DNA hybridization to the Illumina HumanMethylation27 BeadChip and RNA hybridization to Illumina HumanHT-12 BeadChip. Functional analyses of differentially expressed and differentially methylated genes were done. Selected genes were validated at DNA, RNA, and protein levels.Measurements and Main Results: DNA methylation status was altered in IPF. IPF samples demonstrated higher DNA methyltransferase express...

Division of large, immature alveolar structures into smaller, more numerous alveoli increases the surface area available for gas exchange. Alveolar division requires precise epithelial-mesenchymal interactions. However, few experimental models exist for studying how these cell-cell interactions produce changes in 3-dimensional structure. Here we report an epithelial-mesenchymal cell co-culture model where 3-dimensional peaks form with similar cellular orientation as alveolar structures in vivo. Co-culturing fetal mouse lung mesenchyme with A549 epithelial cells produced tall peaks of cells covered by epithelia with cores of mesenchymal cells. These structures did not form when using adult lung fibroblasts. Peak formation did not require localized areas of cell proliferation or apoptosis. Mesenchymal cells co-cultured with epithelia adopted an elongated cell morphology closely resembling myofibroblasts within alveolar septa in vivo. Because inflammation inhibits alveolar formation, we tested the effects of E. coli lipopolysaccharide on 3-dimensional peak formation. Confocal and time-lapse imaging demonstrated that lipopolysaccharide reduced mesenchymal cell migration, resulting in fewer, shorter peaks with mesenchymal cells present predominantly at the base. This epithelial-mesenchymal co-culture model may therefore prove useful in future studies of mechanisms regulating alveolar morphogenesis.

DNA methylation, a major epigenetic mechanism, may regulate coordinated expression of multiple genes at specific time points during alveolar septation in lung development. The objective of this study was to identify genes regulated by methylation during normal septation in mice and during disordered septation in bronchopulmonary dysplasia. In mice, newborn lungs (preseptation) and adult lungs (postseptation) were evaluated by microarray analysis of gene expression and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-Seq). In humans, microarray gene expression data were integrated with genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm and term lung. Genes with reciprocal changes in expression and methylation, suggesting regulation by DNA methylation, were identified. In mice, 95 genes with inverse correlation between expression and methylation during normal septation were identified. In addition to genes known to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its extracellular matrix (Tnc, Eln, etc.), genes involved with immune and antioxidant defense (Stat4, Sod3, Prdx6, etc.) were also observed. In humans, 23 genes were differentially methylated with reciprocal changes in expression in bronchopulmonary dysplasia compared with preterm or term lung. Genes of interest included those involved with detoxifying enzymes (Gstm3) and transforming growth factor-β signaling (bone morphogenetic protein 7 [Bmp7]). In terms of overlap, 20 genes and three pathways methylated during mouse lung development also demonstrated changes in methylation between preterm and term human lung. Changes in methylation correspond to altered expression of a number of genes associated with lung development, suggesting that DNA methylation of these genes may regulate normal and abnormal alveolar septation.

We tested whether 2-aminoethoxydiphenyl borate (2-APB) induces arrhythmia in perfused rat hearts and whether this arrhythmia might result from the activation of voltage-independent calcium channels. Rat hearts were Langendorff perfused and beat under sinus rhythm. An isovolumic balloon inserted into the left ventricle was used to record mechanical function while bipolar electrograms were recorded from electrodes sutured to the base and the apex of hearts. Western and immunofluorescence analyses were performed on rat left ventricular protein extracts and left ventricular frozen sections, respectively. Rat ventricular myocytes express Orai 1 and Orai 3, and ventricle also contains the Orai regulator Stim1. Rat hearts (n=5) perfused with Krebs-Henseleit (KH) alone maintained sinus rhythm at 4.8 ± 0.1 Hz and stable mechanical function. By contrast, perfusing hearts (n=5) with (KH+22 μM 2-APB) provoked a period of tachycardic ectopy at rates of up to 10.8 ± 0.2 Hz. As perfusion with (KH+22 μM 2-APB) continued, the rate of spontaneous ventricular depolarization increased to 21.8 ± 1.2 Hz and became disorganized. Heart mechanical function collapsed as developed pressure decreased from 87 ± 8.8 to 3.5 ± 1.9 mm Hg. Flow rate did not change between normal (16.6 ± 0.9 ml/min) and fibrillating (17.4 ± 0.8 ml/min) hearts. The addition of 20 μM 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) to (KH+22 μM 2-APB) perfusates (n=4) restored sinus rhythm and heart mechanical output. These data indicate that activating myocardial voltage-independent calcium channels, possibly the Orais, may be a novel cause of ventricular arrhythmia.

Nanostructured diamond coatings improve the smoothness and wear characteristics of the metallic component of total hip replacements and increase the longevity of these implants, but the effect of nanodiamond wear debris on macrophages needs to be determined to estimate the long-term inflammatory effects of wear debris. The objective was to investigate the effect of the size of synthetic nanodiamond particles on macrophage proliferation (BrdU incorporation), apoptosis (Annexin-V flow cytometry), metabolic activity (WST-1 assay) and inflammatory cytokine production (qPCR). RAW 264.7 macrophages were exposed to varying sizes (6, 60, 100, 250 and 500 nm) and concentrations (0, 10, 50, 100 and 200 μg ml(-1)) of synthetic nanodiamonds. We observed that cell proliferation but not metabolic activity was decreased with nanoparticle sizes of 6-100 nm at lower concentrations (50 μg ml(-1)), and both cell proliferation and metabolic activity were significantly reduced with nanodiamond concentrations of 200 μg ml(-1). Flow cytometry indicated a significant reduction in cell viability due to necrosis irrespective of particle size. Nanodiamond exposure significantly reduced gene expression of tumor necrosis factor-α, interleukin-1β, chemokine Ccl2 and platelet-derived growth factor compared to serum-only controls or titanium oxide (anatase 8 nm) nanoparticles, with variable effects on chemokine Cxcl2 and vascular endothelial growth factor. In general, our study demonstrates a size and concentration dependence of macrophage responses in vitro to nanodiamond particles as possible wear debris from diamond-coated orthopedic joint implants.

Hypoxia enhances transforming growth factor-β (TGF-β) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-β signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-β signaling, and collagen content were made. To determine whether excessive TGF-β signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-β-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-β signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-β signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-β signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-β signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-β signaling and improving alveolar development.

Calcium transport through plasma membrane voltage-independent calcium channels is vital for signaling events in non-excitable and excitable cells. Following up on our earlier work, we tested the hypothesis that this type of calcium transport can disrupt myocardial electromechanical stability. Our Western and immunofluorescence analyses show that left atrial and ventricular myocytes express the Orai1 and the Orai3 calcium channels. Adding the Orai activator 2-aminoethoxydiphenyl borate (2-APB) to the superfusate of rat left atria causes these non-automatic muscles to contract spontaneously and persistently at rates of up to 10 Hz, and to produce normal action potentials from normal resting potentials, all in the absence of external stimulation. 2-APB likewise induces such automatic activity in superfused rat left ventricular papillary muscles, and the EC(50)s at which 2-APB induces this activity in both muscles are similar to the concentrations which activate Orais. Importantly, the voltage-independent calcium channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) suppresses this automaticity with an IC(50) of 11 ± 0.6 μM in left atria and 6 ± 1.6 μM in papillary muscles. 1-(5-Iodonaphthalene-1-sulfonyl)-hexahydro-1,4-diazepine (ML-7), a second voltage-independent calcium channel inhibitor, and two calmodulin inhibitors also prevent 2-APB automaticity while two calmodulin-dependent protein kinase II inhibitors do not. Thus an activator of the Orai calcium channels provokes a novel type of high frequency automaticity in non-automatic heart muscle.

SummaryIL-10R2 is a shared cell surface receptor required for the activation of five class 2 cytokines (IL-10, IL-22, IL-26, IL-28, and IL-29) that play critical roles in host defense. To define the molecular mechanisms that regulate its promiscuous binding, we have determined the crystal structure of the IL-10R2 ectodomain at 2.14 Å resolution. IL-10R2 residues required for binding were identified by alanine scanning and used to derive computational models of IL-10/IL-10R1/IL-10R2 and IL-22/IL-22R1/IL-10R2 ternary complexes. The models reveal a conserved binding epitope that is surrounded by two clefts that accommodate the structural and chemical diversity of the cytokines. These results provide a structural framework for interpreting IL-10R2 single nucleotide polymorphisms associated with human disease.

Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin alpha8beta1 is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin alpha8beta1 in lung development using integrin alpha8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and in vitro that alpha8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin alpha8beta1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggest that disruption of the interactions between extracellular matrix and integrin alpha8beta1 may contribute to the pathogenesis of BPD.

Many extremely preterm infants continue to suffer from bronchopulmonary dysplasia, which results from abnormal saccular-stage lung development. Here, we show that fibroblast growth factor-10 (FGF-10) is required for saccular lung development and reduced in the lung tissue of infants with bronchopulmonary dysplasia. Although exposure to bacteria increases the risk of bronchopulmonary dysplasia, no molecular target has been identified connecting inflammatory stimuli and abnormal lung development. In an experimental mouse model of saccular lung development, activation of Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) inhibited FGF-10 expression, leading to abnormal saccular airway morphogenesis. In addition, Toll-mediated FGF-10 inhibition disrupted the normal positioning of myofibroblasts around saccular airways, similar to the mislocalization of myofibroblasts seen in patients with bronchopulmonary dysplasia. Reduced FGF-10 expression may therefore link the innate immune system and impaired lung development in bronchopulmonary dysplasia.