Functional And Mechanistic Characterization Of PDGF-BB-induced Down-regulation Of PTPRD During The Development Of Pulmonary Arterial Hypertension

Background : Pulmonary Arterial Hypertension(PAH)is the most dangerous type of Pulmonary Hypertension,which is another type of malignant and fatal cardiovascular disease in addition to hypertension and coronary syndrome.PAH is known as "malignant tumor of the cardiovascular system" because of atypical clinical symptoms,young onset,and high mortality.Platelet-derived growth factor BB(PDGF-BB),which is the strongest mitogen and chemokine for vascular smooth muscle cells,exerts its biological function through binding to PDGF receptor(PDGFR).Receptor protein tyrosine phosphatase D(PTPRD)is a transmembrane protein receptor,and its main function is to dephosphorylate intracellular phosphorylated tyrosine residues.Signal transducer and activator of transcription 3(STAT3)could be phosphorylated to form a dimer,and then enters the nucleus to transmit stimulation signals and regulate the transcription of target genes.Abnormal activation of STAT3 plays an important role in the development of PAH,and studies have reported that PTPRD can dephosphorylate phosphorylated STAT3 in tumor cells.In order to investigate the pathogenesis of PAH,PTPRD,which was down-regulated by PDGF-BB,was selected as the research in this project to study its effect on Pulmonary artery smooth muscle cells(PASMC)proliferation,migration and cell adhesion and its mechanism of action.The research on the relationship between PTPRD and STAT3 has been widely studied in tumors,but no research has been reported in the field of pulmonary hypertension.Objective: To study the function and intrinsic pathogenesis of PTPRD down-regulated by PDGF-BB during the development of pulmonary vascular remodeling and pulmonary hypertension.Methods and Results:Using deep RNA sequencing technology to select PTPRD in the PTPR family as the research object,which was significantly down-regulated by PDGF-BB.The results of RNA sequencing were firstly validated by q RT-PCR,and then consistently verified in PASMCs of PAH rats induced by Monocrotaline(MCT)and clinical samples of PAH patients.In RPASMCs,PTPRD expression was down-regulated by PDGF-BB in a time-and concentrationdependent manner.Utilization of small molecule inhibitors SH-4-54 or Cryptotanshinone to inhibitSTAT3 activity was found to be able to restore the down-regulation of PTPRD upon PDGF-BB stimulation,which suggested that PDGF-BB may regulate PTPRD expression through the STAT3 signaling pathway.In order to study the biological function of PTPRD,sh RNA mediated inhibition of PTPRD expression in RPASMC,wound healing assay and single cell movement tracing analysis were conducted,and it was found that knocking down PTPRD can significantly promote the overall migration ability of RPASMC and the mobility of single cells.Cellular immunofluorescence staining experiments in RPASMC found that inhibition of PTPRD expression could promote the extension of microtubules and the relocation of microtubule tissue centers during migration,and promoted the formation of cell adhesion junctions.In order to explore the interaction between PTPRD and STAT3 and the molecular mechanism of PTPRD in affecting RPASMC migration,immunoblotting experiments were performed,and it was found that the phosphorylation level of STAT3 could be significantly increased after suppressing the expression of PTPRD.Conclusions: This study found that PDGF-BB could regulate the expression of PTPRD through affecting the activity of the PTPRD promoter,and it might be achieved through the STAT3 signaling pathway.PTPRD could affect the expression of downstream target genes in RPASMC by regulating the phosphorylation level of STAT3,thereby affecting the extension of microtubules,the relocation of MTOC and the formation of cell adhesion connections during cell migration.In summary,the studies of PTPRD in this project could provide a theoretical reference for the internal mechanism of PASMC migration during pulmonary vascular remodeling,and will be helpful for further understanding the process of pulmonary vascular remodeling and the development of PAH.