Type 1 and type 2 diabetes result from an absolute or relative reduction in functional β-cell mass. One approach to replacing lost β-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying factors that enhance β-cell differentiation and proliferation in vivo or in vitro. Connective tissue growth factor (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and embryonic β cells that we previously showed is required for β-cell proliferation, differentiation, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or β cells results in decreased embryonic β-cell proliferation, making CTGF unique as an identified β cell-derived factor that regulates embryonic β-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in β-cell proliferation. Furthermore, CTGF overexpression in β cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature β cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature β cells in directed differentiation or regeneration protocols.

Pulmonary hypertension (PH) frequently occurs in infants with bronchopulmonary dysplasia (BPD), causing increased mortality and right ventricular (RV) dysfunction that persists into adulthood. A first step in developing better therapeutic options is identifying and characterizing an appropriate animal model. Previously, we characterized the short-term morbidities of a model in which C57BL/6J wild-type (WT) mice were exposed to 70% O (hyperoxia) during the neonatal period. Here, we aimed to determine the long-term morbidities using lung morphometry, echocardiography (Echo), and cardiac magnetic resonance imaging (cMRI). The major highlight of this study is the use of the state-of-the art imaging technique, cMRI, in mice to characterize the long-term cardiac effects of neonatal hyperoxia exposure. To this end, WT mice were exposed to 21% O (normoxia) or hyperoxia for two weeks of life, followed by recovery in normoxia for six weeks. Alveolarization, pulmonary vascularization, pulmonary hypertension, and RV function were quantified at eight weeks. We found that hyperoxia exposure resulted in persistent alveolar and pulmonary vascular simplification. Furthermore, the Echo and cMRI studies demonstrated that hyperoxia-exposed mice had signs of PH and RV dysfunction as indicated by increased RV pressure, mass, and end-systolic and -diastolic volumes, and decreased RV stroke volume and ejection fractions. Taken together, our results demonstrate that neonatal hyperoxia exposure in mice cause cardiopulmonary morbidities that persists into adulthood and provides evidence for the use of this model to develop novel therapies for BPD infants with PH.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of infants that is characterized by interrupted lung development. Postnatal sepsis causes BPD, yet the contributory mechanisms are unclear. To address this gap, studies have used lipopolysaccharide (LPS) during the alveolar phase of lung development. However, the lungs of infants who develop BPD are still in the saccular phase of development, and the effects of LPS during this phase are poorly characterized. We hypothesized that chronic LPS exposure during the saccular phase disrupts lung development by mechanisms that promote inflammation and prevent optimal lung development and repair. Wild-type C57BL6J mice were intraperitoneally administered 3, 6, or 10 mg/kg of LPS or a vehicle once daily on postnatal days (PNDs) 3–5. The lungs were collected for proteomic and genomic analyses and flow cytometric detection on PND6. The impact of LPS on lung development, cell proliferation, and apoptosis was determined on PND7. Finally, we determined differences in the LPS effects between the saccular and alveolar lungs. LPS decreased the survival and growth rate and lung development in a dose-dependent manner. These effects were associated with a decreased expression of proteins regulating cell proliferation and differentiation and increased expression of those mediating inflammation. While the lung macrophage population of LPS-treated mice increased, the T-regulatory cell population decreased. Furthermore, LPS-induced inflammatory and apoptotic response and interruption of cell proliferation and alveolarization was greater in alveolar than in saccular lungs. Collectively, the data support our hypothesis and reveal several potential therapeutic targets for sepsis-mediated BPD in infants.

Glucose metabolism was compared in dogs consuming a chow/meat diet throughout pregnancy (P group, n = 6) and dogs switched to a high-fat/high-fructose (HFF) diet during the 4th-5th gestational week (gestation ≃9 wk; P-HFF group; n = 6). An oral glucose tolerance test (OGTT; 0.9 g/kg) was administered in the 6th-7th gestational week, and a hyperinsulinemic [0-120 min: 1.8 pmol·kg(-1)·min(-1) (low insulin); 120-240 min: 9 pmol·kg(-1)·min(-1) (high insulin)] euglycemic clamp was performed the following week. Nonpregnant (NP) female dogs underwent OGTTs but not clamp studies. All P-HFF dogs exhibited impaired glucose tolerance (IGT) or gestational diabetes (GDM), but only one P dog had IGT. Insulin concentrations in P and P-HFF dogs were significantly lower than in NP dogs 30 and 60 min after the OGTT. Therefore, mean islet size and area were evaluated in P and NP dogs. These values did not differ between groups, and proliferating endocrine cells were rare in pregnancy. During exposure to high insulin, glucose infusion rate and hindlimb glucose uptake were ∼30% greater (P < 0.05) and net hepatic glucose output was more suppressed (-5.5 ± 6.1 vs. 7.8 ± 2.8 mg·100 g liver(-1)·min(-1), P < 0.05) in P than in P-HFF dogs. In conclusion, in the 2nd trimester the canine pancreas does not exhibit islet hypertrophy, hyperplasia, or neogenesis. Combined with the lack of pancreatic adaptation, a HFF diet during late pregnancy produces a canine model of IGT and GDM without hyperinsulinemia but exhibiting liver and muscle insulin resistance.

Bronchopulmonary dysplasia (BPD), the most common chronic lung disease in infants, is associated with long-term morbidities, including pulmonary hypertension (PH). Importantly, hyperoxia causes BPD and PH; however, the underlying mechanisms remain unclear. Herein, we performed high-throughput transcriptomic and proteomic studies using a clinically relevant murine model of BPD with PH. Neonatal wild-type C57BL6J mice were exposed to 21% oxygen (normoxia) or 70% oxygen (hyperoxia) during postnatal days (PNDs) 1-7. Lung tissues were collected for proteomic and genomic analyses on PND 7, and selected genes and proteins were validated by RT-qPCR and immunoblotting analysis, respectively. Hyperoxia exposure dysregulated the expression of 344 genes and 21 proteins. Interestingly, hyperoxia down-regulated genes involved in neuronal development and maturation in lung tissues. Gene set enrichment and gene ontology analyses identified apoptosis, oxidoreductase activity, plasma membrane integrity, organ development, angiogenesis, cell proliferation, and mitophagy as the predominant processes affected by hyperoxia. Further, selected deregulated proteins strongly correlated with the expression of specific genes. Collectively, our results identified several potential therapeutic targets for hyperoxia-mediated BPD and PH in infants.

Hyperoxia contributes to the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of infants that is characterized by interrupted alveologenesis. Disrupted angiogenesis inhibits alveologenesis, but the mechanisms of disrupted angiogenesis in the developing lungs are poorly understood. In pre-clinical BPD models, hyperoxia increases the expression of extracellular signal-regulated kinases (ERK) 1/2; however, its effects on the lung endothelial ERK1/2 signaling are unclear. Further, whether ERK1/2 activation promotes lung angiogenesis in infants is unknown. Hence, we tested the following hypotheses: (1) hyperoxia exposure will increase lung endothelial ERK1/2 signaling in neonatal C57BL/6J (WT) mice and in fetal human pulmonary artery endothelial cells (HPAECs); (2) ERK1/2 inhibition will disrupt angiogenesis in vitro by repressing cell cycle progression. In mice, hyperoxia exposure transiently increased lung endothelial ERK1/2 activation at one week of life, before inhibiting it at two weeks of life. Interestingly, hyperoxia-mediated decrease in ERK1/2 activation in mice was associated with decreased angiogenesis and increased endothelial cell apoptosis. Hyperoxia also transiently activated ERK1/2 in HPAECs. ERK1/2 inhibition disrupted angiogenesis in vitro, and these effects were associated with altered levels of proteins that modulate cell cycle progression. Collectively, these findings support our hypotheses, emphasizing that the ERK1/2 pathway is a potential therapeutic target for BPD infants with decreased lung vascularization.

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease of human infants that is characterized by disrupted lung angiogenesis. Adrenomedullin (AM) is a multifunctional peptide with angiogenic and vasoprotective properties. AM signals via its cognate receptors, calcitonin receptor-like receptor (Calcrl) and receptor activity-modifying protein 2 (RAMP2). Whether hyperoxia affects the pulmonary AM signaling pathway in neonatal mice and whether AM promotes lung angiogenesis in human infants are unknown. Therefore, we tested the following hypotheses: (1) hyperoxia exposure will disrupt AM signaling during the lung development period in neonatal mice; and (2) AM will promote angiogenesis in fetal human pulmonary artery endothelial cells (HPAECs) via extracellular signal-regulated kinases (ERK) 1/2 activation. We initially determined AM, Calcrl, and RAMP2 mRNA levels in mouse lungs on postnatal days (PND) 3, 7, 14, and 28. Next we determined the mRNA expression of these genes in neonatal mice exposed to hyperoxia (70% O2) for up to 14 d. Finally, using HPAECs, we evaluated if AM activates ERK1/2 and promotes tubule formation and cell migration. Lung AM, Calcrl, and RAMP2 mRNA expression increased from PND 3 and peaked at PND 14, a time period during which lung development occurs in mice. Interestingly, hyperoxia exposure blunted this peak expression in neonatal mice. In HPAECs, AM activated ERK1/2 and promoted tubule formation and cell migration. These findings support our hypotheses, emphasizing that AM signaling axis is a potential therapeutic target for human infants with BPD.

Bronchopulmonary dysplasia (BPD)-associated pulmonary hypertension (PH) is a significant lung morbidity of infants, and disrupted lung angiogenesis is a hallmark of this disease. We observed that extracellular signal-regulated kinases (ERK) 1/2 support angiogenesis in vitro, and hyperoxia activates ERK1/2 in fetal human pulmonary microvascular endothelial cells (HPMECs) and in neonatal murine lungs; however, their role in experimental BPD and PH is unknown. Therefore, we hypothesized that Tie2 Cre-mediated deficiency of ERK2 in the endothelial cells of neonatal murine lungs would potentiate hyperoxia-induced BPD and PH. We initially determined the role of ERK2 in in vitro angiogenesis using fetal HPMECs. To disrupt endothelial ERK2 signaling in the lungs, we decreased ERK2 expression by breeding ERK2flox/flox mice with Tie-Cre mice. One-day-old endothelial ERK2-sufficient (eERK2+/+) or –deficient (eERK2+/−) mice were exposed to normoxia or hyperoxia (FiO2 70%) for 14 d. We then performed lung morphometry, gene and protein expression studies, and echocardiography to determine the extent of inflammation, oxidative stress, and development of lungs and PH. The knockdown of ERK2 in HPMECs decreased in vitro angiogenesis. Hyperoxia increased lung inflammation and oxidative stress, decreased lung angiogenesis and alveolarization, and induced PH in neonatal mice; however, these effects were augmented in the presence of Tie2-Cre mediated endothelial ERK2 deficiency. Therefore, we conclude that endothelial ERK2 signaling is necessary to mitigate hyperoxia-induced experimental BPD and PH in neonatal mice. Our results indicate that endothelial ERK2 is a potential therapeutic target for the management of BPD and PH in infants.