| Part Ⅰ Pyruvate Kinase M2 Promotes CTEPH Vascular Remodeling by Modulating Glycolysis and Extracellular Matrix ExpressionBackground and ObjectivePulmonary vascular remodeling is an important pathophysiological mechanism of CTEPH,which is manifested by intimal thickening and abnormal deposition of extracellular matrix.Pyruvate kinase muscle M2(PKM2)is an important rate-limiting enzyme in the glycolysis process.It is usually overexpressed in proliferating cells and tumor cells to regulate the glycolysis process and the Warburg effect.Studies have shown that abnormal glucose metabolism plays an important role in pulmonary arterial hypertension,but there is still a lack of relevant research in CTEPH,and the pattern of glucose metabolism in pulmonary artery cells of CTEPH patients is not clear.This study intends to investigate the role of PKM2 in the glycolysis and extracellular matrix deposition of pulmonary arterial smooth muscle cells(PASMCs)in patients with CTEPH.Method1.Collected pulmonary endarterectomy(PEA)tissue specimens of patients with CTEPH.Masson staining to observe whether the collagen was abnormally distributed;RT-qPCR method was used to detect the extracellular matrix biomarkers in PEA tissues:tissue inhibitor of metalloproteinase 1(TIMP1),matrix metalloproteinase 2(MMP2),and MMP9.Immunohistochemical method was used to semi-quantitatively detect TIMP1,MMP2,and MMP9 in PEA tissue specimens.2.RT-qPCR method was used to detect the expression of glycolysis related genes in PEA tissues.Immunohistochemistry method was used to semi-quantitate the protein expression of PKM1 and PKM2 in PEA tissues.3.Isolation,culture and identification of PASMCs from CTEPH patients.4.The seahorse energy metabolism assay was used to detect the glycolysis level and energy metabolism of PASMCs.5.Detected the expression levels of PKM2,TIMP1,MMP2,Collagenl in PASMCs by using RT-qPCR method and Western blot method.6.Used siRNA interference method to knock down the PKM2 expression of PASMCs,and detected the glycolysis level of PASMCs after knocking down PKM2.7.Used siRNA interference and PKM2 activator TEPP46 to stimulate PASMCs.Used CCK8 method to detect proliferation ability,and used scratch healing test to detect migration ability.8.Used siRNA to interfere with patient PASMCs,and used RT-qPCR and Western blot to detect the expression of extracellular matrix biomarkers.Results1.The PEA tissues of CTEPH patients have abnormal collagen deposition.The expression of TIMP1 is higher than that in the pulmonary artery tissue of lung transplantation donors.There is no significant difference in the changes of MMP2 and MMP9.The expression of PKM2,lactate dehydrogenase A(LDHA),and hexokinase 2(HK2)in PEA tissues increased,but there was no significant difference of PKM1.2.The glycolysis level of PASMCs in CTEPH patients is higher than that of healthy PASMCs.The expression of PKM2,LDHA,monocarboxylate transporter 4(MCT4)and hypoxia inducible factor 1α(HIF1α)of PASMCs in CTEPH patients was higher than that of healthy PASMCs.After PKM2 knockdown,the glycolysis level of PASMCs decreased,the mitochondrial oxidative phosphorylation level increased,and the expression of LDHA and HK2 decreased.After the PKM2 knockdown in healthy PASMCs,there was no change in energy metabolism pattern.3.PKM2 knockdown can inhibit the proliferation and migration of patients PASMCs;stimulation of PKM2 activator TEPP46 can enhance the proliferation and migration of patients PASMCs.4.The extracellular matrix expression of PASMCs in CTEPH patients was abnormal,the expression of TIMP1 and collagen I was increased,and there was no significant difference in MMP2.PKM2 knockdown can inhibit the expression of TIMP1,and downregulate the expression of extracellular matrix collagen I and collagen III.ConclusionsThere are increased glycolysis levels and abnormal extracellular matrix metabolism in the pulmonary arteries of CTEPH patients.The expression of PKM2 and TIMP1 in the pulmonary artery of CTEPH patients increased.PKM2 can regulate the level of glycolysis and the expression of extracellular matrix.Knockdown of PKM2 can inhibit the level of glycolysis and the expression of extracellular matrix in PASMCs of CTEPH patients.This research may provide a potential target for the treatment of CTEPH.Part Ⅱ Preliminary Study on Establishment of Rat Model of Chronic Thromboembolic Pulmonary HypertensionBackground and ObjectiveThe pathophysiological mechanism of chronic thromboembolic pulmonary arterial hypertension(CTEPH)has not been fully elucidated,and the treatment of CTEPH still faces many challenges.However,due to the lack of mature animal models,translational medicine research progresses slowly.Therefore,it is necessary to establish an animal model that is easy to operate,practical,and can objectively simulate the occurrence and development of clinical CTEPH.The purpose of this study is to explore the combination of inhibitor of the vascular endothelial growth factor(VEGF)receptor tyrosine kinase(Sugen5416)and multiple autologous thrombus injections to establish a rat animal model,and provide ideas for establishing small animal models of CTEPH.Method1.Randomly divide male Sprague-Dawley(SD)rats into 5 groups,and divide 3 rats into each group:control group(Ctrl group,injected with equal volume of normal saline),6h injection group,12h injection group,24h injection group and 48h injection group.Blood was collected from orbital vein to prepare autologous thrombi,and the thrombi was injected through the jugular vein.All rats were sacrificed at corresponding time points.Taking the lung tissue forhematoxylin-eosin staining to observe the dissolution of autologous thrombi.2.Randomly divide male Sprague-Dawley(SD)rats into 4 groups,and divide 8-12 rats into each group:control group(Ctrl,injected with equal volume of saline),autologous thrombi injection group(PE),Sugen5416 administration Group(SU),Sugen5416 combined with autologous thrombi injection group(PE+SU).The SU group and PE+SU group received a single dose of Sugen5416(20mg/kg)intraperitoneally one day prior to the first thrombi injection.The PE group and PE+SU group received three autologous thrombi injections through the left jugular vein on the first,third,and fifth days of the experiment.At the same time,tranexamic acid(315mg/kg/d)was injected intraperitoneally every day during the first 2 weeks of modeling.At the end of the 5th week,pulmonary artery pressure,percent media thickness(MT%)and right ventricular hypertrophy index(RVHI)were measured in each group to evaluate the animal model.The expression of α-SMA,PKM1 and PKM2 in the pulmonary arterioles of rats in each group were detected by immunohistochemistry.Results1.Thrombi was dissolving in the rat main pulmonary artery and lobe pulmonary artery at 12 hours after the first injection of thrombi;and thrombi gradually dissolved to 50%of the initial injected thrombi volume after 24 hours;and autologous thrombi was almost completely dissolved after 48 hours.2.Mean pulmonary arterial pressure(mPAP)was elevated in PE+SU group compared to control group,PE group,and SU group[(27.96±4.53)mmHg vs(14.59±2.5 1)mmHg,(17.67±2.99)mmHg,(19.78± 1.68)mmHg,P<0.0001 or P<0.001].3.Percent media thickness(MT%)was increased in PE+SU group compared to control group,PE group,and SU group[(49.04± 10.37)%vs(27.12±5.13)%,(33.52±6.16)%,(37.17±6.90)%,all P<0.0001].It was found that smooth muscle layer was markedly thicken in PE+SU group.4.Right ventricular hypertrophy index(RVHI)in PE+SU group was higher than that in control group,PE group,and SU group[(0.42±0.06)vs(0.26±0.03),(0.29±0.03),(0.34 ± 0.02),P<0.0001 or P<0.001 or P<0.01].5.There was no statistical difference in the expression levels of PKM1 and PKM2 among the groups(P>0.05).ConclusionsIn the present model,we found that the interaction of Sugen5416 and pulmonary arterial mechanical obstruction by multiple autologous thrombi could establish a mild to moderate CTEPH rat model.This new model also suggests the potential connection between the occurrence and development of CTEPH and endothelial injury and thromboembolism. |
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