Study On The New Mechanism Of Pathogenesis Of Congenital Ventricular Septal Defect Associated Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a syndrome resulting from restricted flow through the pulmonary arterial circulation resulting in increased pulmonary vascular resistance and ultimately in right heart failure(ACCF/AHA2009). PAH is a common disease in clinic characterized by increasing of the pulmonay vascular resistance and the progressing of the right heart failure. The pathogenesis of PAH is still unclear. PAH is characterized by excessive pulmonary vasoconstriction and abnormal vascular remodeling processes that usually affect all vessel layers including intima, media, and adventitia, and result in severe loss of cross-sectional area and, therefore, increased right ventricular afterload. Intimal changes include endothelial injury, endothelial cell proliferation, invasion of the intima by (myo)fibroblast-like cells, enhanced matrix deposition with intimal fibrosis and, sometimes, obstruction of the vascular lumen by unique plexiform lesions. Vascular smooth muscle cell (SMC) proliferation is a prominent feature of PAH. Till now, the diagnosis and assessment of PAH rely on the clinical principles, without any molecular biological means including biomarker in serum. Also there is no specific drugs that could cure this disease. Our project aim to screen for some sensitive and specific biomarkers in serum of PAH patients, which may be benefit to the diagnosis and assessment of PAH, then further study the potent mechanism through some new pathways, finally find some therapeutic targets underlying. Patients who were diagnosed with Ventricular Septal Defect(VSD) and PAH were selected randomly from First Affiliated Hospital of Nanjing Medical University. Patients who were diagnosed only with VSD were set as control, with gender and age being matched. The serum was separated and screened to detect the difference of proteins between PAH patients and control group by two-dimensional gel electrophoresis(2-DE), using image analysis software to recognize the different protein spots. Different protein was identified by MALDI-TOF-MS. We searched for the data of the identified proteins to check whether some one could be the biomarker or targets in our next step. On the other side, we enrolled22VSD-PAH patients and25control patients(set as above) and assayed the level of Soluble fms-like tyrosine kinase-1(sFlt-1), Soluble Endoglin(sEng), Monoamine oxidase(MAO), Angiotension II Type-1Receptor Autoantibody(AT1-AA) in their serum using enzyme linked immunosorbent assay(ELISA). After analysing the result of ELISA, we tested the expression of KCNQ1(downstream effector of AT1-AA) on mRNA level in the lung of PAH rat which induced by the injection of Monocrotaline(MCT). In addition, we studied on the movement of isolated mitochondria which isolated from heart tissue of mice, in the mitochondria Isolation-Medium, using the mothod of fluorescence tracking. On the basis of that observations, we assayed the effect of5-hydroxytryptamine(5-HT) on the isolated mitochondria, since5-HT is regarded as a key factor that triggers PAH.With the2-DE and MS, we recognized39different protein spots and identified6of them:Human Zinc-Alpha-2-Glycoprotein, P2X5b, pyruvate kinase isozymes M1, apolipoprotein M, antitrypsin alphal mutant, vitamin D-binding protein. The protein level of MAO and AT1-AA were significantly higher in the PAH group than it in the control group(P<0.05), while protein level of sFlt and sEng didn’t show any statistical difference between the two groups(P>0.05). The mRNA level of KCNQ1was significantly higher in the pulmonay artery and lung tissue of PAH rat than it in the control rat(P<0.01), which suggest that the dysfunction of KCNQ1may induced by over activation of PLC resulting from the increasing of AT1-AA, thus makes the SMCs contraction. The movement of isolated mitochondria could be inhibited by the elevation of concentration of Ca2+in the solution, which is the same as in the cellular circumstance, however, had no relationship with the production of ATP and the activity of mitoKATP and mPTP.5-HT had positive effect on the moving velocity of isolated mitochondria(P<0.05), while it hasn’t been observed in the protoplasts of E.coli(P>0.05). We conclude that5-HT does have effect on mitochondria, which may act via the5-HT receptors localized on the surface mitochondria.