Role Of PDGFR-β/PI3K/AKT Signaling Pathway In PDGF-BB Induced Rat Cardiac Fibroblasts Caused Of Myocardial Fibrosis

Backgro and Objective : Myocardial fibrosis is a pathological feature at the end stage of some cardiovascular diseases and characterized by the proliferation of cardiac fibroblasts in the myocardial interstitium,the transformation of these fibroblasts into myofibroblasts and the excessive deposition of extracellular matrix(ECM). Collagen accounts for about 80% of ECM and is a major component of ECM. Collagen I(Col I) and collagen III(Col III) are the dominant types of collagen in the myocardial fibrosis [1]. Fibroblasts and myofibroblasts are the major cells which synthesize Col I and Col II during the myocardial fibrosis. Platelet derived growth factor-BB(PDGF-BB) is an important mitogenic factor and can promote the proliferation of fibroblasts and secretion of collagens, playing an important role in the myocardial fibrosis [2]. However, how the PDGF-BB acts to activate fibroblasts and mediate the myocardial fibrosis is still poorly understood. Our previous study showed DOCA induced fibrosis in rats had significantly increased expression of PDGF and its receptors(PDGFRs) which were mainly localized in fibroblasts and myofibroblasts. In addition, PDGFR-β plays a key role in the proliferation, transformation and collagen secretion of fibroblasts [3, 4]. PI3K/Akt signaling pathway is an important regulatory pathway and its activation has been found to be involved in the regulation of cell pr- oliferation, migration, differentiation and angiogenesis [5]. In the present study, cardiac fibroblasts(CFs) were separated and purified, and then treated with PDGF-BB. The proliferation, transformation, collagen secretion, PDGFR and factors in the PDGFR-β/PI3K/Akt signaling pathway were determined in these CFs, aiming to explore the role of PDGF-BB in the pathogenesis of myocardial fibrosis and its down-stream mechanisms. METHODS: Purified CFs were obtained by the methods of digestive enzyme and differential adhesion.Use Appropriate concentration of PDGF- BB to induce cardiac fibroblasts Simulation the environment in body;Imatinib as PDGFR phosphorylase inhibitor can inhibit the PDGFR-βphosphorylated activated, thereby inhibiting the activation of downstream channel;LY294002 for PI3 K phosphorylation inhibitor, can block PI3K/AKT signaling pathway in the role of a series of physiological mechanism.Around PDGFR-β/PI3K/AKT signaling pathway set the following four groups: blank control group, PDGF- BB model groups(20ng/ml), the treatment group 1(PDGF-BB20ng/ml +IMA 100 u M), the treatment group 2(PDGF-BB20ng/ ml +LY294002 13 u M).Trying to clarify the role of the channel in myocardial fibrosis;Application determined by MTT, RT-PCRand Western Blotting method to detect myocardial fibroblasts proliferation and PDGFR-β, Col Ⅰ, Col Ⅲ, PI3 K, and Akt expression in cells.And given some correlation analysis these results. RESULTS: Immunofluorescence staining showed about 90% of cells were positive for vimentin and 10% for α-SMA. After incubation for 7 days, fluorescence microscopy revealed more than 90% of cells were positive for α-SMA, which was significantly higher than that in CON group(P < 0.01), but markedly lower than that in IMA group and LY group(P < 0.01). Group training determined by MTT method after 48 hours measured PDGF-BB group cells compared with blank control group increased significantly(P < 0.01), cells in PDGF-BB + IMA group and PDGF-BB + LY294002 decreased significantly than PDGF-BB group, the difference was statistically significant(P < 0.01). The m RNA and protein expression of PDGFR-β, Col I, Col III, PI3 K and Akt increased dramatically at 48 h after PDGF-BB treatment when compared with CON group(P < 0.01). However, IMA and LY294002 significantly inhibited the expression of PDGFR-β and p-PI3K(P < 0.05). In addition, the m RNA expression of PDGFR-β, PI3 K and Akt in IMA group and LY group was also markedly lower than those in P group(P <0.01), and the m RNA and protein expression of Col I and Col III reduced remarkably when compared with P group(P< 0.01). Of note, the m RNA expression of PDGFR-α was comparable among 4 groups, and PDGFR-β expression after PDGF-BB treatment increased significantly when compared with PDGFR-α expression(P < 0.01). CONCLUSION: PDGF-BB induces the proliferation and transformation of CFs into myofibroblasts, leading to increase the synthesis of collagen. This is closely associated with PDGFR- β, but not PDGFR-α. PDGFR- β/PI3K/Akt signaling pathway is involved in the PDGF-BB induced transformation of cardiac fibroblasts into myofibroblasts.