Transforming Growth Factor-β1 Upregulation Of Insulin-like Growth Factor-Binding Protein Via PI3K-ERK Pathway In Smooth Muscle Cells In Pulmonary Arterial Hypertension Induced By Monocrotaline

Background:Pulmonary arterial hypertension (PAH), Group I PAH according to WHO classification for pulmonary hypertension (PH), is a diverse disease characterized by progressive right ventricular (RV) failure and premature death. In the past decades, a great achievement of medications has significantly improved the clinical outcomes of PAH patients. However, limitations of medical treatment leaves a long list of lung transplantation among PAH patients who were unresponsive to standard pharmacotherapy, mainly driven by the fact that the mechanisms correlated with PAH remain fully unknown. Previous studies have reported the complexity of pathological mechanisms involving in the worsening of PAH, including inflammation of endothelial cells, vasoconstriction, abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs), and subsequent resultant pulmonary arterial (PA) remodeling. Insulin-like growth factor (IGF), as an important mediator, plays a critical role in arterial remodeling by promoting SMC proliferation through binding with its specific receptor, IGF binding proteins (IGFBPs). On the other hand, the activity of IGFBPs is regulated by transforming growth factor-β1 (TGF-β1). However, the underlying mechanism of TGF-β1 in regulating IGFBPs expression in PASMCs is still under complete study.Objective:The present study aimed to analyze the mechanisms through which TGF-(31 regulates IGF-IGFBPs in PAH rat model induced by Monocrotaline (MCT).Methods:Animals were randomly assigned to PAH (intraperitoneal injection of MCT, 60mg/kg) and Control(intraperitoneal injection of saline at same dosage) groups. At four weeks after injection, PASMCs from each animal were harvested, and R-TqPCR was carried out to measure the expression of IGFBPs mRNA. Then, the protein phosphorylation of IGFBPs differentially expressed between two groups were measured by Western blot before and after interventions using TGF-β1 and its antibody, inhibitors of ERK and PI3K in PAH group.Results:Compared with Control group, at four weeks after injection, there were significant elevation in mean pulmonary artery pressure (mPAP,75.69±6.72 mmHg vs. 34.58±2.49 mmHg,p<0.001), right ventricular anterior wall thickness (RVAW, 0.79±0.02 mm vs.0.40±0.07 mm,p=0.04), percentage of RV mass ratio [defined as RV mass/(left ventricle+septal) mass,47.0±10.0 vs.20.0±9.50, p=0.02], and percentage of pulmonary arterial media wall thickness (%MWT,24.0±1.10% vs. 17.0±3.0%,p=0.01) in the PAH group. We also found that the mRNA expression of IGFBP 3 and IGFBP 5 in PASMCs in the PAH significantly increased when compared with those in the Control group, without statistical difference in the mRNA expression of IGFBP 1, IGFBP 2 and IGFBP 4 between two groups. Pre-treatment by TGF-β1 further upregulated the IGFBP 3 and IGFBP 5 proteins phosphorylation in PASMCs by activating Smad 2 and Smad 3 signaling, an effect was inhibited by either anti-TGFβ1 antibody or PD98059 (an inhibitor of ERK signaling). In addition, LY294002, a blockade of PI3K signaling, could increase IGFBP 5 but rather IGBP 3 phosphorylation.Conclusion:PAH-induced by MCT is associated with increased mRNA expression and phosphorylation of IGFBP 3 and IGFBP 5 proteins, regulated by TGF-β1/Smad 2-Smad 3 pathways. ERK and PI3k involved in the regulation of IGFBPs activity in different directions.