Epigenetics Of Endothelial Cells In Development Of Pulmonary Hypertension Following Intrauterine Environmental Changes

There is a wealth of epidemiological evidence that intrauterine growth retardation (IUGR) or lower birthweight is strongly correlated with an increased risk of adult diseases, such as type 2 diabetes mellitus, obesity, metabolic syndrome, hypertension, chronic kidney disease. This phenomenon can be maintained throughout the life or transmitted to the next generation, and also known as the Developmental Origins of Adult Disease Hypothesis or Barker's Hypothesis. Accumulating evidence reveals that epigenetic regulatory mechanisms play an important in the fetal origin of adult disease. Epigenetics refers to all heritable changes in phenotype or in gene expression states that are not involved in the DNA sequence itself, including DNA methylation, histone modification, RNA interference, and genomic imprinting. Bisulfate sequencing and chromatin immunoprecipitation (ChIP) methods are used to detect DNA methylation and histone modification. To date, epigenetic regulatory mechanism in IUGR-ralated adult diseases have been preliminary understood, however, we do not know whether epigenetic regulation is involved in the pathogenesis of pulmonary arterial hypertension (PAH) induced by intrauterine environmental changes. Based on the fact, we hypothesized: 1. Epigenetic modification might participate in the pathogenesis of PAH induced by intrauterine environmental changes.2. IUGR induced by dietary restriction during pregnancy might cause epigenetic modification changes in PAH-related genes, and could be maintained into adulthood. These epigenetic changes would induce IUGR individuals to be highly sensitive to hypoxia, leading to the occurrence of PAH. If the appropriate interventions administered in the early stage of IUGR individuals to prevent or reverse the epigenetic changes in the PAH-related genes would decrease the risk of PAH in later life.Part I Epigenetic Regulation of Endothelial cells in Persistent Pulmonary Hypertension of the Newborn RatObjective:Persistent pulmonary hypertension of the newborn (PPHN) is defined as a continuous increase in pulmonary vascular resistance after birth leading to impedance to the transition from the fetal to the adult-type circulation, and right to left shunt, which would result in severe hypoxemia. Nitric oxide produced by the endothelial nitric oxide synthase (eNOS) in pulmonary vascular endothelial cells (PVEC) plays an important role in the pathogenesis of PPHN, eNOS expression in endothelial cells is controlled by epigenetics. The purpose of the present study is to explore the epigenetic regulatory mechanism of the eNOS gene in PPHN.Methods:1. Rat model of PPHN was co-induced by hypoxia and indomethacin.2. PVEC of the newborn rats were isolated by magnetic-activated cell sorting (MACS). Real-time PCR and western blot were to detect the eNOS expression levels, ChIP and bisulfite sequencing were used for the analysis of the epigenetic regulation of the eNOS gene. Results:1. Assessment of rat PPHN model:The ratio of left ventricular free wall plus septum to right ventricular weight significantly increased in hypoxia plus indomethacin-treated group. The medial thickness percentage of small pulmonary arteries from hypoxia plus indomethacin-treated group was higher than that from control or single treatment group. Vascular elastin area percentage and immunostaining density of eNOS from the combined-treated group were higher than other groups. The relative abundance of a-SMA, elastin, and eNOS, and plasma BNP levels in hypoxia plus indomethacin-treated group also significantly increased compared with other groups.2. The levels of acetylated histone H3 and H4 at the proximal promoter of the eNOS gene in PVEC from PPHN were significantly higher than those from the control group. Total methylation percentage of the eNOS gene promoter in PPHN rat was slightly lower than that of control, but there was no statistically significant difference between the two groups. These changes of epigenetic modifications at the eNOS gene promoter were consistent with increased levels of eNOS mRNA and protein in PPHN.Conclusions:1. Hypoxia plus indomethacin had a synergistic effect on pulmonary vascular remodeling. The rat model of PPHN co-induced by hypoxia and indomethacin may mimic the human PPHN syndrome.2. The increased eNOS protein expression in PPHN was strongly correlated with the increased histone acetylation and possibly DNA hypo-methylation.3. Epigenetic regulation may be one of the pathogenesis of PPHN. Part II Epigenetic Regulation of Endothelial cells in pulmonary arterial hypertension following intrauterine growth ratardationObjective:IUGR induced'by'intrauterine different environmental changes can cause PAH or pulmonary vascular remodeling in later life. However, the underlying mechanism of this phenomenon is riot clear. Considering the role of endothelin-1 (ET-1) in the pathogenesis of PAH, we hypothesized that IUGR might cause epigenetic modification changes of the ET-1 gene. These epigenetic changes could be maintained into adulthood and would induce IUGR individuals to be highly sensitive to hypoxia. If the appropriate interventions in the early stage of IUGR individuals might decrease the risk of PAH in later life.Methods:1. Establishing IUGR rat model, hypoxic model, and the early TSA intervention model.2. Assessment of pulmonary arterial pressure and pulmonary vascular remodeling, isolation of PVEC by MACS. Western blot was to analyze the eNOS protein level; ChIP was used for the analysis of epigenetic changes in the ET-1 promoter, including histone modification and the degree of transcription factor binding.Results:1. Right ventricular systolic pressures (RVSP) in the 6-week control and IUGR rats were 21.6±2.08 mmHg and 22.93±1.60 mmHg respectively, there was no significant statistical difference between the two groups. RVSP was significantly increased in both groups followed by hypoxia; moreover, RVSP in IUGR group was significantly higher than the control group. The percentage of pulmonary artery medial thickness and area was significantly greater than that in the age-matched control hypoxia group. The a-SMA protein expression significantly increased in the two groups, especially in IUGR group. However, there was no significant difference in RVSP between the two groups followed by TSA intervention.2. After hypoxia intervention, the ET-1 protein level in the IUGR group was significantly higher than control group, there was statistically significant difference between the two groups. The histone.acetylation of H3 aiid H3K9/18 of the ET-1 gene core promoter region in the 1-day and 6-week IUGR rats was significantly higher than control group, while no statistically significant changes in H4. However, the H3, H3K9/18 and H4 acetylation levels were significantly increased followed by hypoxia. The degrees of the transcription factor HIF-la binding to the ET-1 gene promoter in IUGR groups were similar to the changes of acetylated H3 in the ET-1 gene promoter.Conclusions:1. Hypoxia can induce IUGR rats to produce significant pulmonary hypertension, pulmonary vascular remodeling, and increased expression of ET-1 protein.2. The response characteristics of IUGR to hypoxia were strongly correlated with the increased histone acetylation and transcription factor HIF-la binding in the ET-1 gene core promoter region. TSA intervention in the early stage of IUGR rats can decrease the risk of the hypoxia-induced PAH and pulmonary vascular remodeling.3. Epigenetic regulation may be one of the pathogenesis of PAH followed by IUGR.