The Role Of CTGF-integrin β3 Signal Pathway In Proliferation, Migration And Extracellular Matrix Deposition Of PASMCs

Abstract:Objective 1. To explore the effects of integrinβ3 on CTGF-induced proliferation, migration, change of cytoskeleton and extracellular matrix deposition of PASMCs.2. To investigate the effects of CTGF-integrinβ3 signal pathway on pulmonary vascular remodeling in pulmonary hypertension. Methods 1. Pulmonary artery smooth muscle cells of SD Rats were cultured in vitro and 3-7 passages of cultured PASMCs were used in the experiments.2. The PASMCs were cultured with CTGF and different concentrations of anti-integrinβ3 antibody for 24h,48h,72h and 96h, WST-1 assay was used to detect the effects of anti-integrinβ3 antibody on CTGF-induced proliferation.3. The PASMCs were cultured with CTGF and different concentrations of anti-integrinβ3 antibody for 24h, Transwell chambers were used to observe the effects of anti-integrinβ3 antibody on CTGF-induced migration.4. The cultured PASMCs were divided into control group, CTGF group and CTGF+anti-integrinβ3 antibody group, the cytoskeletal rearrangement was observed with coomassie brilliant blue R250 staining at Oh,12h and 24h.5. The cultured PASMCs were divided into control group, CTGF group and CTGF+anti-integrinβ3 antibody group, RT-PCR assayed the mRNA expressions of CollagenⅠ-α1, CollagenⅢ-α1 and Fibronectin-1 at 24h and 48h.6. The cultured PASMCs were divided into control group, CTGF group and CTGF+anti-integrinβ3 antibody group, Western blot and Immunohistochemistry assayed the effect of anti-integrinβ3 antibody on CTGF-induced expression of Fibronectin-1 protein at 24h. Results 1. PASMCs were exposed to anti-integrinβ3 antibody (5,10,15,20 mg/L) and treated with 50ng/ml CTGF at the same time for 24h,48h,72h and 96h. Between each groups compared with control (Omg/L), WST-1 assay showed that there was significant difference (P<0.01) when the antibody concentration meet or exceed 10mg/L. Inhibition rate of PASMC proliferation was the highest in 10mg/L at 48h. The data indicated that integrinβ3 involved in PASMCs proliferation stimulated by CTGF.2. Transwell chambers assay indicated that the number of PASMC migration induced by CTGF was decreased with increasing anti-integrinβ3 antibody concentration; there was concentration-dependent; it was obvious that 20 mg/L antibody inhibited the migration of PASMCs. This result suggested that integrinβ3 may play an important role in migration of PASMCs stimulated by CTGF.3. Coomassie brilliant blue R250 staining assay indicated that the cytoskeletal rearrangement of PASMCs were not changed significantly between each groups at Oh and 24h; compared with the control, CTGF could change cytoskeletal rearrangement of PASMCs briefly at 12h, while anti-integrinβ3 antibody inhibited this phenomenon; the results showed that integrin β3 mediates the short-term change of cytoskeletal rearrangement induced by CTGF in PASMC cultured for 24h.4. compared with the control, RT-PCR assay indicated that the expression of Collagen typeⅠ-α1, Collagen typeⅢ-α1, Fibronectin-1 mRNA were significantly increased in group of 50ng/ml CTGF (P<0.01), while compared with the group of 50ng/ml CTGF, anti-integrinβ3 antibody inhibited these extracellular matrix genes mRNA expression (P<0.05), but the these genes mRNA expression were still more than control group (P<0.05). This result implied that integrinβ3 involved partly in the mRNA expression of Collagen typeⅠ-α1, Collagen typeⅢ-α1 and Fibronectin-1 promoted by CTGF.5. Compared with the control, both of Western blot and Immunohistochemistry assay indicated that the group of 50ng/ml CTGF promoted significantly the expression of Fibronectin-1 protein, while anti-integrinβ3 antibody inhibited the expression of Fibronectin protein, but the Fibronectin-1 protein expression were still more than control group. This result implied that integrin (33 involved partly in the expression of Fibronectin-1 protein in PASMCs promoted by CTGF.Conclusions 1. Integrinβ3 mediates proliferation, migration, cytoskeletal rearrangement of PASMCs induced by CTGF.2. CTGF could promote the expression of Collagen typeⅠ-α1, Collagen typeⅢ-α1 , Fibronectin-1 mRNA and protein of Fibronectin-1.3. Integrinβ3 may mediate the expression of Collagen typeⅠ-α1 , Collagen typeⅢ-α1 , Fibronectin-1 mRNA and Fibronectin-1 protein in PASMCs induced by CTGF. 4. The CTGF-integrinβ3 signal pathway may play a key role in proliferation, migration, cytoskeletal rearrangement and extracellular matrix deposition of PASMCs, and this signal pathway may be a new target to intervent the pulmonary vascular remodeling in PAH.