Development Of High-throughput Sequencing-based High Throughput Drug Screening Platform For Hereditary Pulmonary Arterial Hypertension

Pulmonary hypertension is a chronic progressive cardiovascular disease,which is usually characterized by increased pulmonary artery pressure,increased pulmonary vascular resistance(mPAP≥25mmHg,PAWP<15mmHg,PVR≥3WU),and eventually lead to death due to right heart failure.Due to the insidious onset of the disease,the initial stage of the disease has no specific symptoms.Patients are usually diagnosed when the disease has been very serious.The pathogenesis of Pulmonary hypertension is very complex.The main pathological changes include pulmonary vascular remodeling,such as accelerated proliferation of smooth muscle cells in the middle layer of pulmonary arteriole wall,functional damage of endothelial cells,formation of new intima,intima fibrosis,and plexiform lesions.Hereditary pulmonary artery hypertension belongs to the first clinical classification of pulmonary hypertension.At present some mutant genes that are associated with pulmonary hypertension have been found,such as BMPRⅡ BMPR1B,SMAD9,CAV1,KCNK3.Among these genes,BMPRⅡ mutations is the first identified which is related with PAH and has most mutatins.70%～80%of the familial pulmonary artery hypertension patients have BMPRⅡ mutations and 20%-40%patients with idiopathic pulmonary arterial hypertension have BMPRII mutations.Some of severe pulmonary hypertension patients without BMPRⅡ mutations appear decreased BMPRⅡ expression.Compare to patients without BMPRII mutations,Patients with BMPRⅡ mutations have earlier onset time,and the symptom is more serious.Although a series of drugs have been introduced into the treatment of pulmonary hypertension in China since the past decade and the 5-year survival rate has been improved,but the long-term survival rate is still very low.Currently available drugs for the treatment of pulmonary artery hypertension are generally of high price,and patients need to take medicine for life,which brings great burden to the society and individuals.Therefore,innovative drug discovery and new drug screening technology are urgently needed.This thesis take hereditary pulmonary artery hypertension as the research object.The present research knock down BMPRII using Lentivirus mediated shRNA interference in pulmonary artery smooth muscle cells and human pulmonary artery endothelial cells.Then,we respectively carry out whole transcriptome level analysis and phenotypic analysis for pulmonary artery smooth muscle cells and human pulmonary artery endothelial cells with BMPRII gene knockdown to select the appropriate cell model for drug screening.ECM-receptor interaction pathway,Protein digestion and absorption pathway,PI3K-Akt signaling pathway,Focal adhesion pathway were enriched in both pulmonary artery endothelial cells and pulmonary artery smooth muscle cells.And Ras signaling pathway were enriched in pulmonary artery endothelial cells.The results showed that pulmonary artery smooth muscle cells significantly show up declined cell proliferation ability after BMPRII knockdown.And HPAECs have significant transition process of endothelial-to-mesenchymal transition(EndoMT)phenotype when BMPRII knockdown.The pebbles cellular morphology of HPAECs changed to spindle morphology which is similar to mesenchymal cells.The results of RT-qPCR showed that BMPRII gene knockdown lead to smooth muscle cells marker aSMA expression increase significantly on mRNA level in pulmonary artery endothelial cells.Because EndoMT is closely related to pulmonary hypertension vascular remodeling,we chose pulmonary artery endothelial cells with BMPRII knockdown as hereditary pulmonary artery hypertension disease cell model.BMPRII knockdown Pulmonary arterial endothelial cells were used as the model cells of hereditary pulmonary hypertension and HTS2 high throughput drug screening technology based on the gene expression patterns were used as screening method.We screened the compound library containing 2148 small molecular compounds on HPAECs using HTS2 system and found a range of potentially active small molecule drugs.It is worth mentioning that there are four candidates have been reported as potential candidates for pulmonary artery hypertension in other studies among the top 30 candidates.The targets of over 12 small molecule compounds have been reported as potential targets for pulmonary artery hypertension.These targets are mainly in Ras/Raf/MEK signaling pathway,RhoA/ROCK signaling pathway,PI3K/Akt signaling pathway.In addition we found that after K-Ras selective inhibitor 6H05 administration,pulmonary artery endothelial cells with BMPRII knockdown,the expression of aSMA significantly lowered in mRNA level.This indicates that 6H05 has the effect of reversing EndoMT and may be a candidate small molecule for the treatment of hereditary pulmonary hypertension.This indicated that the drug screening system we established is reliable and robust.The results establish the foundation of large-scale drug screening for hereditary pulmonary hypertension,and provide Research ideas for drug discovery of hereditary pulmonary hypertension and other hereditary diseases.