Notch3 Signaling Activation In Smooth Muscle Cells Promotes Extrauterine Growth Restriction-induced Pulmonary Hypertension

Part I Studies on dysfunction of vascular smooth muscle cells in extrauterine growth restriction induced pulmonary hypertensionObjective:Previous studies have found that extrauterine growth restriction is associated with an increased risk of cardiovascular disease in adulthood,including pulmonary hypertension.Pulmonary hypertension is a serious progressive disease with poor prognosis.The pathological features of pulmonary hypertension include proliferation,migration,anti-apoptosis and cell type conversion of pulmonary artery endothelial cells,pulmonary artery smooth muscle cells and fibroblasts.Since the function of pulmonary vascular smooth muscle cells in extrauterine growth restriction has not been studied,we explore the mechanism of pulmonary hypertension from the perspective of pulmonary arterial smooth muscle cells induced by extrauterine growth restriction.Methods:1.Establishment of EUGR rat model:Pregnant Sprague-Dawley rats maintained standard diet throughout pregnancy.The pups were weighed and randomly assigned to the control group(10 pups/litter)and EUGR group(20 pups/litter)with a 1:1 male-female ratio.On the 21st day after birth,rats were weaned and four male in each cage until the age of 12 weeks.2.Measurement of pulmonary arterial pressure was measured by jugular catheter in adult rats.3.Evaluation of pulmonary vascular remodeling.4.Establishment of primary rat pulmonary artery smooth muscle cells(PASMCs):rat pulmonary artery smooth muscle tissues were isolated and cultured.5.Cell proliferation detection.6.Apoptosis detection:apoptosis was detected by Annexin V-FITC/PI double staining cells and TUNEL assay.7.Cell migration ability test:cell scratch test and transwell cell migration test were performed.8.Relative quantitative PCR was used to detect the expression level of the whole lung of the gene pathway related to pulmonary hypertension.Results:1.The mean pulmonary artery pressure and right heart index of EUGR rats at 12 weeks of adult were significantly higher than those of the Control group.2.Pulmonary vascular remodeling in EUGR rats was increased compared with that in the Control group.3.The proliferation and migration of EUGR rat pulmonary artery smooth muscle cells were enhanced.4.The mRNA expression levels of various genes in EUGR lung tissues were different from the Control group.Conclusion:1.There was significant increase in pulmonary artery pressure,right heart hypertrophy and pulmonary vascular remodeling at adult period of EUGR.2.The proliferation and migration abilities of pulmonary artery smooth muscle cells of EUGR were increased,and the mRNA expression levels of various genes in the lung tissues were changed.Part II Activation of Notch3 signal pathway in pulmonary artery smooth muscle cells promotes extra uterine growth restriction induced pulmonary hypertensionObjective:Extrauterine growth restriction is associated with an increased risk of cardiovascular disease including pulmonary hypertension in adulthood.There is increasing evidence that the Notch3 pathway is activated during pulmonary hypertension.We investigated the mechanism of pulmonary hypertension from the perspective of smooth muscle cells induced by extrauterine growth restriction.Methods:1.Relative quantitative PCR was used to detect the mRNA expression.2.Western blot was used to detect the protein expression.3.Immunofluorescence staining was used to detect the expression of Notch3 in the whole lung.4.Establishment of primary rat pulmonary artery smooth muscle cells(PASMCs)by enzyme digestion.5.Treatment of PASMCs with Notch pathway y-secretase inhibitor DAPT to detect the effect of the inhibiton on EUGR PASMCs.6.Lentivirus short hairpin RNA(shRNA)knockdown Notch3 to detect the role of the Notch3 on EUGR PASMCs7.Rats were injected with the Notch pathway γ-secretase inhibitor DAPT or a carrier of the same volume(DMSO).8.Cell proliferation detection:cell proliferation was assessed by BrdU colorimetric cell proliferation kit and cell count.9.Cell migration ability test:cell scratch test and transwell cell migration test were performed.Results:1.Expressions levels of mRNA and protein of Notch3 and target gene Hey1 were upregulated in EUGR PASMCs and immunofluorescence staining was enhanced in the lung tissue of EUGR.2.Notch signaling pathway inhibitor DAPT reduces the expression of Notch3 and target gene Hey1 in EUGR PASMCs,and reduces the cell proliferation and migration of EUGR smooth muscle cells.3.Knockdown Notch3 in EUGR PASMCs reduces the expression of Notch3 and target gene Hey1,and reduces the cell proliferation and migration of EUGR PASMCs.4.DAPT inhibition of Notch3 in rats reversed the phenotype of pulmonary hypertension in vivo.Conclusion:1.The high expression level of Notch3 is related to the occurrence and development of EUGR induced PAH.2.In vitro DAPT inhibition and shRNA knockdown of Notch3 reduced PASMCs proliferation and migration.3.EUGR rats treated with Notch inhibitor DAPT had a reduced mean pulmonary pressure.