A miRNA derived from the 17/92 cluster is a novel player in programming pulmonary hypertension in response to gestational hypoxia via NADPH oxidases and the mTOR pathway

Abstract

Background Gestational hypoxia can lead to intrauterine growth restriction (IUGR) and programming of cardiovascular diseases in adulthood via different, although not completely understood, epigenetic mechanisms. We have previously shown that reactive oxygen species (ROS) derived from NADPH oxidases contribute to hypoxia induced pulmonary hypertension (PH). However, their role in disease programming by gestational hypoxia is not resolved. Purpose We aimed to elucidate the role of NADPH oxidases and redox sensitive miRNAs in the cardiovascular response of adult offspring to transient gestational hypoxia. Methods Wildtype (WT) and nmf333 mice, lacking a functional p22phox dependent NADPH oxidase, were exposed to gestational hypoxia from E10.5 to E11.5. Embryos and adult offspring were analyzed by functional, immunohistochemical and molecular methods including mRNA and miRNA profiling. miRNA mimics or inhibitors were applied in vivo and in vitro using embryoid bodies (EBs). Heart samples from patients with right heart disease associated with congenital heart disease (CHD) were used to validate target miRNA expression. Results Transient gestational hypoxia was sufficient to induce IUGR in embryos and pulmonary hypertension (PH) characterized by right ventricular hypertrophy, increased right ventricular pressure and pulmonary vascular remodeling in adult offspring from WT mice while p22phox-deficient nmf333 mice were protected. Cardiac mRNA and miRNA profiling followed by qPCR validation identified several single miRNAs derived from the 17/92 miRNA cluster as downregulated by gestational hypoxia and NADPH oxidases in embryos and adult offspring hearts. In vitro analyses using EBs validated a single miRNA from this cluster to be sufficient to induce cardiac differentiation in response to hypoxia and p22phox, and target analyses identified the mTOR pathway as dysregulated. Treatment with an inhibitor targeting this miRNA was sufficient to induce PH in adult mice, while treating offspring derived from gestational hypoxia pregnancies with an miRNA mimic prevented the development of PH in the adult and dysregulation of the mTOR pathway. Importantly, expression of this single miRNA from the miRNA 17/92 cluster was negatively correlated to right ventricular pressure in CHD patients. Conclusions In this study, we demonstrate that gestational hypoxia programs PH in adulthood by upregulating NADPH oxidases and downregulating miRNAs derived from the 17/92 cluster subsequently affecting the mTOR pathway. Since dysregulation of a single miRNA from this cluster induced not only PH but was also associated with right ventricular pressure in CHD patients, these findings might open novel options for diagnosis, prevention and treatment of right heart disease. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): DZHK (German Centre for Cardiovascular Research)