Suppressed erythropoietin expression in a nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Erythropoietin (EPO), an essential stimulator of erythropoiesis produced by the fetal liver, is important both in vascular remodeling and modulation of the endothelial response in the pulmonary vasculature. In addition, EPO guides alveolar development, along with retinoic acid (RA). EPO is a direct target of RA, and the retinoid pathway is altered in the nitrofen-induced congenital diaphragmatic hernia (CDH) model. In the present study, we tested the hypothesis that the synthesis of EPO is suppressed in a rat model of CDH. MATERIALS AND METHODS: Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D19 and D21 and divided into control and CDH groups. Immunohistochemistry and quantitative real-time polymerase chain reaction (RT-PCR) were performed to determine the expression of EPO in the fetal liver and kidney. We also estimated the expression of EPO receptor in the fetal lung. RESULTS: The relative EPO mRNA expression in the liver on D19 and in the kidney on D21 were significantly lower in the CDH group than in the controls (P = 0.0008 and P = 0.0064, respectively). In addition, the results of immunohistochemistry supported the findings from the RT-PCR analysis. No significant changes were noted in the expression pattern or EPO receptor levels in the fetal lungs of the CDH group compared to the controls. CONCLUSIONS: Our results reveal the suppressed EPO synthesis in the CDH fetus, which may contribute to the pathogenesis of lung hypoplasia and modification of pulmonary vasculature in the CDH rat model. Pediatr Pulmonol. 2017;52:606-615. © 2016 Wiley Periodicals, Inc.

Promotion of muscle regeneration by myoblast transplantation combined with the controlled and sustained release of bFGFcpr.

Although myoblast transplantation is an attractive method for muscle regeneration, its efficiency remains limited. The efficacy of myoblast transplantation in combination with the controlled and sustained delivery of basic fibroblast growth factor (bFGF) was investigated. Defects of thigh muscle in Sprague-Dawley (SD) rats were created, and GFP-positive myoblasts were subsequently transplanted. The rats were divided into three groups. In control group 1 (C1) only myoblasts were transplanted, while in control group 2 (C2) myoblasts were introduced along with empty gelatin hydrogel microspheres. In the experimental group (Ex), myoblasts were transplanted along with bFGF incorporated into gelatin hydrogel microspheres. Four weeks after transplantation, GFP-positive myoblasts were found to be integrated into the recipient muscle and to contribute to muscle fibre regeneration in all groups. A significantly higher expression level of GFP in the Ex group demonstrated that the survival rate of transplanted myoblasts in Ex was remarkably improved compared with that in C1 and C2. Furthermore, myofibre regeneration, characterized by centralization of the nuclei, was markedly accelerated in Ex. The expression level of CD31 in Ex was higher than that in both C1 and C2, but the differences were not statistically significant. A significantly higher expression level of Myogenin and a lower expression level of MyoD1 were both observed in Ex after 4 weeks, suggesting the promotion of differentiation to myotubes. Our findings suggest that the controlled and sustained release of bFGF from gelatin hydrogel microspheres improves the survival rate of transplanted myoblasts and promotes muscle regeneration by facilitating myogenesis rather than angiogenesis.

Increased pulmonary RhoA expression in the nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: Persistent pulmonary hypertension remains a major cause of mortality and morbidity in cases of congenital diaphragmatic hernia (CDH). Recently, RhoA/Rho-kinase-mediated vasoconstriction has been reported to be important in the pathogenesis of pulmonary hypertension (PH). Several recent reports have described that fasudil, a potent Rho-kinase inhibitor and vasodilator, could represent a potential therapeutic option for PH. We designed this study to investigate the hypothesis that the expression level of RhoA is increased in the nitrofen-induced CDH rat model. The expression level of Wnt11, an activator of RhoA, was also evaluated. METHODS: Pregnant rats were treated with or without nitrofen on gestational day 9 (D9). Fetuses were sacrificed on D17, D19 and D21 and were divided into control and CDH groups. Quantitative real-time polymerase chain reaction was performed to determine the pulmonary gene expression levels of both Wnt11 and RhoA. An immunofluorescence study was also performed to evaluate the expression and localization of RhoA. RESULTS: The relative mRNA expression levels of pulmonary Wnt11 and RhoA on D21 were significantly increased in the CDH group compared with the control group (p=0.016 and p=0.008, respectively). The immunofluorescence study confirmed the overexpression of RhoA in the pulmonary vessels of CDH rats on D21. CONCLUSIONS: Our results provide evidence that the RhoA/Rho-kinase-mediated pathway is involved in the pathogenesis of PH in the nitrofen-induced CDH rat model. Our data also suggest that the fasudil, a Rho-kinase inhibitor, could represent a therapeutic option for the treatment of PH in CDH.