Pulmonary hypertension (PH) is a severe complication of congenital diaphragmatic hernia (CDH). Transforming growth factor-β (TGFβ) signaling is suggested to be involved in PH development by regulating embryonic angiogenesis, cell proliferation, and cell differentiation. Altered TGFβ signaling has been demonstrated in experimental CDH lungs. Elastin microfibril interfacer 1 (Emilin-1) is an extracellular matrix glycoprotein expressed in endothelial and vascular smooth muscle cells and known to regulate TGFβ processing and arterial diameter. We designed this study to investigate the pulmonary vascular expression of Emilin-1 in nitrofen-induced CDH rats. Following ethical approval (REC913b, REC1103), time-pregnant Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH group and control group. Quantitative real-time polymerase chain reaction (n = 11 each group), Western blot analysis, and confocal microscopy were used to determine the gene and protein expression of Emilin-1. Relative Emilin-1 messenger RNA (ribonucleic acid) levels were significantly downregulated in CDH lung tissue compared with controls (CDH: 0.043 ± 0.003; control: 0.067 ± 0.004; p < 0.001). Western blotting confirmed the decreased pulmonary Emilin-1 protein expression in CDH lungs. Confocal microscopy demonstrated a markedly diminished expression of Emilin-1 in the CDH pulmonary vasculature compared with controls. To our knowledge, this study demonstrates for the first time a decreased Emilin-1 gene and protein expression in the pulmonary vasculature of nitrofen-induced CDH. Emilin-1 deficiency through its interaction with TGFß may result in abnormal vascular remodeling resulting in PH in this model.
Background Caveolin‐1 (Cav‐1) exerts major regulatory functions on intracellular signaling pathways originating at the plasma membrane. Cav‐1 is a key regulator in adverse lung remodeling and the development of pulmonary hypertension (PH) regulating vasomotor tone through its ability to reduce nitric oxide (NO) production. This low‐output endothelial NO synthase (eNOS) derived NO maintains normal pulmonary vascular homeostasis. Cav‐1 deficiency leads to increased bioavailability of NO, which has been linked to increased nitrosative stress. Inhibition of eNOS reduced oxidant production and reversed PH, supporting the concept that Cav‐1 regulation of eNOS activity is crucial to endothelial homeostasis in lungs. We designed this study to investigate the hypothesis that expression of Cav‐1 is downregulated while eNOS expression is upregulated by the pulmonary endothelium in the nitrofen‐induced congenital diaphragmatic hernia (CDH). Methods Pregnant rats were exposed to nitrofen or vehicle on day 9.5 (D9.5). Fetuses were sacrificed on D21 and divided into nitrofen and control groups. Quantitative real‐time polymerase chain reaction, Western blotting, and confocal immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of Cav‐1 and eNOS. Results Pulmonary Cav‐1 gene expression levels were significantly decreased, while eNOS gene expression was significantly increased in nitrofen‐induced CDH(+). Western blotting and confocal microscopy revealed decreased pulmonary Cav‐1 protein expression, while eNOS protein expression was increased in CDH(+) compared to controls. Conclusion The striking evidence of markedly decreased gene and protein expression of Cav‐1 with concurrently increased eNOS gene and protein expression in the pulmonary vasculature suggests that activation of eNOS secondary to Cav‐1 deficiency may play an important role in the pathogenesis of PH in the nitrofen‐induced CDH
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