Fetal pulmonary hypertension: dysregulated microRNA-34c-Notch1 axis contributes to impaired angiogenesis in an ovine model.

BACKGROUND: Persistent pulmonary hypertension of the newborn (PPHN) occurs when pulmonary vascular resistance (PVR) fails to decrease at birth. Decreased angiogenesis in the lung contributes to the persistence of high PVR at birth. MicroRNAs (miRNAs) regulate gene expression through transcript binding and degradation. They were implicated in dysregulated angiogenesis in cancer and cardiovascular disease. METHODS: We investigated whether altered miRNA levels contribute to impaired angiogenesis in PPHN. We used a fetal lamb model of PPHN induced by prenatal ductus arteriosus constriction and sham ligation as controls. We performed RNA sequencing of pulmonary artery endothelial cells (PAECs) isolated from control and PPHN lambs. RESULTS: We observed a differentially expressed miRNA profile in PPHN for organ development, cell-cell signaling, and cardiovascular function. MiR-34c was upregulated in PPHN PAECs compared to controls. Exogenous miR34c mimics decreased angiogenesis by control PAEC and anti-miR34c improved angiogenesis of PPHN PAEC in vitro. Notch1, a predicted target for miR-34c by bioinformatics, was decreased in PPHN PAECs, along with Notch1 downstream targets, Hey1 and Hes1. Exogenous miR-34c decreased Notch1 expression in control PAECs and anti-miR-34c restored Notch1 and Hes1 expression in PPHN PAECs. CONCLUSION: We conclude that increased miR-34c in PPHN contributes to impaired angiogenesis by decreasing Notch1 expression in PAECs. IMPACT: Adds a novel mechanism for the regulation of angiogenesis in persistent pulmonary hypertension of the newborn. Identifies non-coding RNAs that are involved in the altered angiogenesis in PPHN and thus the potential for future studies to identify links between known pathways regulating angiogenesis. Provides preliminary data to conduct studies targeting miR34c expression in vivo in animal models of pulmonary hypertension to identify the mechanistic role of miR34c in angiogenesis in the lung vasculature.

Acute and chronic changes in the control of breathing in a rat model of bronchopulmonary dysplasia.

Infants born very prematurely (<28 wk gestation) have immature lungs and often require supplemental oxygen. However, long-term hyperoxia exposure can arrest lung development, leading to bronchopulmonary dysplasia (BPD), which increases acute and long-term respiratory morbidity and mortality. The neural mechanisms controlling breathing are highly plastic during development. Whether the ventilatory control system adapts to pulmonary disease associated with hyperoxia exposure in infancy remains unclear. Here, we assessed potential age-dependent adaptations in the control of breathing in an established rat model of BPD associated with hyperoxia. Hyperoxia exposure ( FIO2 ; 0.9 from 0 to 10 days of life) led to a BPD-like lung phenotype, including sustained reductions in alveolar surface area and counts, and modest increases in airway resistance. Hyperoxia exposure also led to chronic increases in room air and acute hypoxic minute ventilation (V̇e) and age-dependent changes in breath-to-breath variability. Hyperoxia-exposed rats had normal oxygen saturation ( SpO2 ) in room air but greater reductions in SpO2 during acute hypoxia (12% O2) that were likely due to lung injury. Moreover, acute ventilatory sensitivity was reduced at P12 to P14. Perinatal hyperoxia led to greater glial fibrillary acidic protein expression and an increase in neuron counts within six of eight or one of eight key brainstem regions, respectively, controlling breathing, suggesting astrocytic expansion. In conclusion, perinatal hyperoxia in rats induced a BPD-like phenotype and age-dependent adaptations in V̇e that may be mediated through changes to the neural architecture of the ventilatory control system. Our results suggest chronically altered ventilatory control in BPD.

Quantitative optical measurement of mitochondrial superoxide dynamics in pulmonary artery endothelial cells.

Reactive oxygen species (ROS) play a vital role in cell signaling and redox regulation, but when present in excess, lead to numerous pathologies. Detailed quantitative characterization of mitochondrial superoxide anion ( O2•- ) production in fetal pulmonary artery endothelia cells (PAECs) has never been reported. The aim of this study is to assess mitochondrial O2•- production in cultured PAECs over time using a novel quantitative optical approach. The rate, the sources, and the dynamics of O2•- production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain (ETC) complexes, specifically an uncoupler and inhibitors of the various ETC complexes, and inhibitors of extra-mitochondrial sources of O2•- . After stabilization, the cells were loaded with nanomolar mitochondrial-targeted hydroethidine (Mito-HE, MitoSOX) online during the experiment without washout of the residual dye. Time-lapse fluorescence microscopy was used to monitor the dynamic changes in O2•- fluorescence intensity over time in PAECs. The transient behaviors of the fluorescence time course showed exponential increases in the rate of O2•- production in the presence of the ETC uncoupler or inhibitors. The most dramatic and the fastest increase in O2•- production was observed when the cells were treated with the uncoupling agent, PCP. We also showed that only the complex IV inhibitor, KCN, attenuated the marked surge in O2•- production induced by PCP. The results showed that mitochondrial respiratory complexes I, III and IV are sources of O2•- production in PAECs, and a new observation that ROS production during uncoupling of mitochondrial respiration is mediated in part via complex IV. This novel method can be applied in other studies that examine ROS production under stress condition and during ROS-mediated injuries in vitro.

Impaired cerebral angiogenesis in the fetal lamb model of persistent pulmonary hypertension.

BACKGROUND: Persistent pulmonary hypertension of the newborn (PPHN) is associated with increased risk of neuro-developmental impairments. Whether relative fetal hypoxia during evolution of PPHN renders the fetal brain vulnerable to perinatal brain injury remains unclear. We hypothesized that in utero ductal constriction, which induces PPHN also impairs cerebral angiogenesis. METHODS: Fetal lambs with PPHN induced by prenatal ligation of the ductus arteriosus were compared to gestation matched twin controls. Freshly collected or fixed brain specimens were analyzed by immunohistochemistry, Western blot analysis, and RT-PCR. RESULTS: Cortical capillary density was decreased in PPHN lambs compared to controls (Glut-1, isolectin B-4 and factor VIII, n=6, p<0.05). Hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein levels were decreased in cortical cell lysates of PPHN lambs. PPHN increased angiopoetin-1 (Ang-1) and tyrosine-protein kinase receptor (Tie-2) protein expression while angiopoetin-2 (Ang-2) protein levels were decreased (n=6, p<0.05). PPHN did not change mRNA levels of these proteins significantly (n=6). CONCLUSIONS: PPHN decreased cortical capillary density in fetal lamb brain. PPHN decreased the expression of proteins involved in angiogenesis. These findings suggest that PPHN is associated with impaired cortical angiogenesis.