Intrauterine Growth Restriction Programs Intergenerational Transmission Of Pulmonary Arterial Hypertension And Endothelial Dysfunction

Intrauterine growth restriction(IUGR)is defined as two standard deviations of birth weight below the average weight of the same age,or below the 10 th percentile of the average weight of the same gestational age.IUGR is one of the most important complications during pregnancy.Adverse intrauterine environment,such as decreased placental blood flow,maternal malnutrition,drugs,alcohol and long-term stress,can affect fetal development,thus causing IUGR.The incidence of IUGR is about 10% in live-born infants,and the perinatal mortality rate is 4-10 times that of normal newborns.In the 1990 s,David Barker found that the offspring of women who were pregnant during the Holland famine and those who had suffered from intrauterine nutrition deficiency had a significantly higher risk of cardiovascular disease,type 2 diabetes,osteoporosis and obesity in adulthood.This is the doctrine of "the developmental origins of adult diseases hypothesis"(DOHa D),which was subsequently supported by a large number of studies.Epigenetic modifications,which occurs to adapt to the adverse intrauterine environment,play an important role in the development of origins of adult diseases.If this adverse environment is not reversed in time,these adaptive modifications will be changed permanently,resulting in an increased risk of disease in adulthood.Epigenetics,including DNA methylation,histone acetylation,micro RNA and imprinting genes,alters gene expression level without alternation of gene sequence.In addition,increasing evidences show that,not only the fetus itself(the offspring which is exposed to adverse environmental stimuli),but also the embryonic primordial germ cells(which will develop into the offspring in the future)are disturbed by adverse environmental.Therefore,it is possible that both the somatic cells and germ cells of the offspring suffer from such epigenetic modifications simultaneously,which leads to "intergenerational inheritance effect".This effect has been proved in metabolic diseases.However,intergenerational transmission of cardiovascular diseases,especially pulmonary arterial hypertension(PAH),is poorly documented.PAH is a complex pathophysiological disorder involving a variety of clinical manifestations,which eventually leads to right heart failure and death.One of the markers of the pathogenesis and progress of PAH is the impairment of pulmonary vascular endothelial cells(PVECs).The dysfunctional PVECs emerged,labeled as hyper-proliferation,aggressive migration and disordered angiogenesis,finally leading to intimal and medial hypertrophy.Since pulmonary vascular defects are difficult to detect until they reach an advanced stage,hypoxic exposure may be an effective method to overcome this limitation in animal models.We previously found that adults born with IUGR are susceptible to PAH-like pathophysiological developments when exposed to prolonged hypoxia.However,it is unclear whether the PAH risk induced by IUGR is coupled with endothelial dysregulation.Part ? The intergenerational transmission of pulmonary arterialhypertension in IUGR rats Objective:1.To assess the growth and development indexes of both first(F1)and second(F2)generations of intrauterine growth restriction(IUGR)rats.2.To assess the ventricular hypertrophy of IUGR rats.3.To observe the pulmonary vascular remodeling and hemodynamic changes of pulmonary circulation of both first(F1)and second(F2)generations of intrauterine growth restriction(IUGR)rats.4.To verify the expression of genes related with pulmonary arterial hypertension of both first(F1)and second(F2)generations of intrauterine growth restriction(IUGR)rats.Methods:1.The establishment and grouping of IUGR intergenerational inheritance model:After confirmation of pregnancy by vaginal smear plug,females were divided randomly into two groups,with one fed standard chow ad libitum and the other fed 50% of the ad libitum amount until parturition.Pups delivered by fully fed females were labeled as control-F1 group,and pups of food-restricted mothers,with a birth weight below the tenth percentile of the controls,were labeled as IUGR-F1 group.At 9 weeks of age,control-F1 and IUGR-F1 unrelated nonsibling males were mated with external control virgin females to generate second-generation offspring.All the pregnant females were fed with standard chow ad libitum during gestation.Pups of control-F1 were referred to as the control-F2 group,and pups of IUGR-F1 were referred to as the IUGR-F2 group.2.The establishment and grouping of hypoxic pulmonary hypertension rat model:at 9 weeks of age,control-F1,IUGR-F1,control-F2 and IUGR-F2 were placed in room air(normoxia)and the other in a hypoxic chamber with fraction inspired oxygen of 11% for 2 weeks.All the subgroups were fed with standard chow ad libitum.Hemodynamic measurements of all groups were evaluated after 2 weeks.3.Assessment of growth and development parameters: including birth weight and survival rate.4.Assessment of right ventricular hypertrophy: left ventricle and septum were weighed,and RVHI(right ventricular hypertrophy index)was calculated using the formula: RVHI=right ventricular weight/(left ventricular weight+septum weight).5.Assessment of pulmonary vascular remodeling: the smooth muscle layer of pulmonary arterioles was stained with alpha-SMA antibody,and the thickness of pulmonary arterioles was measured using Image-J software.6.Assessment of pulmonary hemodynamics: the mean pulmonary arterial pressure was measured for evaluation.7.Sorting of pulmonary vascular endothelial cells in rats: using a magnetic activated cell sorting method.8.Detection of genes related with pulmonary arterial hypertension: using quantitative PCR and western immune blotting.Results:1.IUGR intergenerational rat model were established successfully.2.The birth weight and survival rate of IUGR-F1 group were significantly lower than that of the control-F1 group.3.There was no significant difference in birth weight and survival rate between IUGR-F2 and control-F2 group.4.Compared with controls,the mean pulmonary arterial pressure and right ventricular heart index showed no significant difference among normoxic groups,but more apparent increases took place in IUGR-F1 after exposure to hypoxia,with the F2 generation showing similar outcomes.There was no difference in mean pulmonary arterial pressure,RVHI between control-F2 and IUGR-F2 in normoxia,but more apparent increases were observed in IUGR-F2 under hypoxic conditions.5.Compared with the control group,there was no significant difference in pulmonary vascular remodeling in the IUGR-F1 rats under normoxia,but was significantly aggravated after hypoxia exposure.Similarly,there was no significant difference in pulmonary vascular remodeling in IUGR-F2 rats under normoxia,but more apparent increases were observed in IUGR-F2 under hypoxic conditions.6.IUGR increased m RNA and protein level of PVECs-derived ET-1 in the first and second generation.Conclusion:1.The growth of the first filial generation of IUGR rats are relatively backward,with survival rate relatively lower.In contrast,there was no significant difference in growth and postnatal survival rate in the second generation.2.IUGR programs intergenerational transmission of pulmonary arterial hypertension pathogenesis,which is related to the increased expression of ET-1 derived from pulmonary vascular endothelial cells.Part ? The intergenerational transmission of pulmonaryvascular endothelial dysfunction in IUGR rats Objective:1.To assess the proliferation of primary pulmonary vascular endothelial cells derived from first and second generations of IUGR rats.2.To assess the migration of primary pulmonary vascular endothelial cells derived from first and second generations of IUGR rats.3.To assess the angiogenesis of primary pulmonary vascular endothelial cells derived from first and second generations of IUGR rats.4.To assess the proliferation,migration and angiogenesis of pulmonary vascular endothelial cells derived from first and second generations of IUGR rats after transfected with ET-1 siRNA.Methods:1.The culture of primary pulmonary vascular endothelial cells(PVECs)derived from IUGR rats: the lungs were perfused via the main pulmonary artery by injection of10 to 15 m L 0.9% cold normal saline into the right ventricle.The perfused(white)lungs were collected for PVECs extraction.2.Identification of cultured cells: flow cytometry,immunofluorescence and observation under microscopy.3.Assess the proliferation of primary pulmonary vascular endothelial cells using CCK-8 kit.4.Assess the migration of primary pulmonary vascular endothelial cells using transwell assay.5.Assess the angiogenesis of primary pulmonary vascular endothelial cells using tube formation assay.6.Assess the proliferation,migration and angiogenesis of pulmonary vascular endothelial cells after transfected with ET-1 siRNA(small interfering RNA).Results:1.Primary pulmonary vascular endothelial cells were cultured successfully.2.IUGR-F1 PVECs showed elevated proliferation and migration compared with control-F1 PVECs in hypoxia,although there was no difference in proliferation or migration between IUGR-F1 and control-F1 in normoxia.Comparison between IUGR-F1 and control-F1 indicated greater angiogenesis in IUGR-F1.3.The proliferation was not significantly different in air condition,but a greater extent increase was found in IUGR-F2 in hypoxic condition.IUGR affected cell migration,with greater migration of IUGR-F2 PVECs compared with controls.IUGR-F2 also showed greater angiogenesis relative to control-F2.4.After transfection with ET-1 small interfering RNA,ET-1 m RNA and protein levels were decreased by ?99.5% and 64%,respectively.5.The proliferation,migration and angiogenesis of pulmonary vascular endothelial cells were significantly decreased after ET-1 siRNA transfected.6.After ET-1 siRNA transfected,there was no significant difference in proliferation,migration and angiogenesis between the IUGR-F1 and the control-F1 group.Similar results were observed in IUGR-F2 and control-F2 group.Conclusion:1.IUGR results in pulmonary vascular endothelial dysfunction in the first and second generations,which is manifested by accelerated proliferation,migration and increased angiogenesis.2.Knockdown of ET-1 inhibited pulmonary vascular endothelial dysfunction in first and second generations of IUGR.Part ? The epigenetic mechanism of intergenerational transmission of pulmonary arterial hypertension and endothelial dysfunctionObjective:1.To explore epigenetic changes of pulmonary vascular endothelial cells in the first generation of IUGR.2.To explore epigenetic changes of pulmonary vascular endothelial cells in the second generation of IUGR.3.To explore epigenetic changes of sperm in the first generation of IUGR.Methods:1.Extraction of mature sperm: free mature sperm was obtained from epididymis tail.2.The separation of pulmonary vascular endothelial cells(PVECs)in rats: the pulmonary vascular endothelial cells were directly separated by MACS.3.DNA extraction: using DNA kit to extract DNA from rat PVECs and sperm cells.4.Assessment of DNA methylation: Bisulfite Genomic DNA Sequencing was used to assess the Cp G island methylation of PVECs and sperm cells.5.Assessment of histone methylation: the histone methylation level of PVECs and sperm cells was evaluated by chromatin immunoprecipitation.Results:1.Methylation at Cp G sites 1,2,5,6,9,13,and 15 of the ET-1 first intron was significantly lower in IUGR-F1 PVECs,relative to their control counterparts.2.Methylation at Cp G sites 3,9,12,and 15 of the ET-1 first intron was significantly lower in IUGR-F1 sperm.3.Methylation at the Cp G sites 2,7,11,15,17,and 20 was significantly lower in IUGR-F2 PVECs compared with control-F2 PVECs.4.Three sites of H3K4me3 in the ET-1 first intron region were further investigated and indicated that levels in the A1,A2,and A3 regions of IUGR-F1 PVECs were all higher than those in control-F1.5.For sperm of IUGR-F1,the A2 region was higher in comparison with control-F1.6.In PVECs of IUGR-F2,there was no significant difference in any region,but the level of H3K4me3 showed an increasing tendency.Conclusion:1.DNA methylation in the ET-1 first intron was decreased in pulmonary vascular endothelial cells(PVECs)of intrauterine growth restriction(IUGR)-first generation(F1)and IUGR–second generation(F2),and in IUGR-F1 sperm.2.DNA methylation in the ET-1 first intron was decreased in sperm of IUGR-F1.3.Tri-methylation of lysine 4 on histone H3(H3K4me3)in the ET-1 first intron was enriched in PVECs and sperm of IUGR-F1.