The Role Of CTGF-integrin α_vβ₅ Signal Pathway In Proliferation, Migration And Extracellular Matrix Deposition Of PASMCs

Objective 1.To explore the effects of integrinαvβ5 and the extracellular regulated protein kinase (ERK1/2) on the proliferation, migration, change of cytoskeleton and extracellular matrix deposition of pulmonary artery smooth muscle cells (PASMC) induced by connective tissue growth factor (CTGF) in vitro.2.To investigate the mechanisms of CTGF-integrinαvβ5 signal pathway in pulmonary vascular remodeling in pulmonary hypertension. Methods 1. PASMCs of SD rat was cultured in vitro, and cell purity was evaluated by a-SMA immunohistochemical staining. The experiment was divided into control group, CTGF (50ng/ml) group, CTGF (50ng/ml)+integrinαvβ5 antibody (P1F6) group.2.WST-1 assay was used to detect different concentrations of P1F6 for cell's proliferation, and then determined their optimal intervention time piont and optimal intervention levels.3.Transwell chambers were used to observe the effects of P1F6 on CTGF-induced migration.4.The impact of CTGF, CTGF+P1F6 on the changes of cytoskeleton was determined by Coomassie brilliant blue R250 staining. The formulation of FAK of PASMCs was detected by using laser confocal microscopy techniques.5.Real-time fluorescent quantitative RT-PCR was used to detect the role of CTGF and P1F6 in mRNA expression of CollagenⅠ-α1, CollagenⅢ-α1 and Fibronectin-1 genes.6.The expression of CollagenⅢ, ERK1/2 and p-ERK1/2 protein of PASMC induced by CTGF was detected by western blot and Immunohistochemistry assay. Results 1. The cultured cells were determined to be PASMCs by a-SMA immunohistoc-hemical staining.2.PASMCs were exposed to P1F6(0,5,10,15μg/mL) and treated with CTGF (50ng/ml) at the same time for Oh,24h,48h,72h and 96h. Compared with control, WST-1 assay showed that there was significant difference between each group.The inhibition rate of P1F6 (10μg/mL) for 72h was 25.6%(P<0.01). The inhibition rate of P1F6 (15μg/mL) for 72h was 35% (P<0.01).3.Transwell chambers assay indicated that the number of PASMC migration induced by CTGF was decreased with P1F6 (10μg/ml) at 24h(P<0.001).4. Coomassie brilliant blue R250 staining assay indicated that the cytoskeletal rearrangement of PASMCs were not changed significantly between each groups at 24h; compared with the control, CTGF could change cytoskeletal rearrangement of PASMCs briefly at 12h, while P1F6 inhibited this phenomenon. The impact of CTGF, CTGF+P1F6 on the fomulation of FAK of PASMC was assayed by using laser confocal microscopy techniques. Compared with control, the formulation of FAK of PASMC in CTGF group decreased significantly at 12h and 24h, especially at 24h. when PIF6 was added in medium, the formulation of FAK of PASMC treated with CTGF increased, compared with control.5. Compared with the control, RT-PCR assay indicated that the mRNA expression of Collagen typeⅠ-α1, Collagen typeⅢ-α1, Fibronectin-1 were significantly increased in PASMC treated with CTGF (50ng/ml) for 72h (P<0.05), while P1F6 inhibited these extracellular matrix genes mRNA expression (P<0.05).6.Western blot and Immunohistochemistry assay indicated that CTGF (50ng/ml) promoted significantly the expression of CollagenⅢprotein, while P1F6 inhibited the expression of CollagenⅢprotein, but the CollagenⅢprotein expression were still more than control group. The expression of p-ERK1/2 protein was increased for CTGF group by using western blot and Immunohistochemistry assayed, but P1F6 inhibited the expression of p-ERK1/2 protein, p-ERK1/2 and ERK1/2 ratio decrease (P< 0.05). Conclusions 1. Integrinαvβ5 mediates proliferation, migration, cytoskeletal rearrangement of PASMCs induced by CTGF.2. ERK1/2 signaling pathway may mediate the effect of CTGF-integrinαvβ5 signaling pathway on proliferation, migration, cytoskeleton changes and ECM deposition of PASMCs, which may play an important role in the pulmonary vascular remodeling of pulmonary hypertension, and maybe the new target to prevent the occurrence and development of pulmonary vascular remodeling.