The Role Of CTGF-integrin β₁ Signal Pathway In Proliferation, Migration And Extracellualr Matrix Deposition Of PASMCs

Objective 1. To explore the mechanisms of integrinβ1 on CTGF-induced proliferation, migration, change of cytoskeleton and extracellular matrix deposition of PASMCs in vitro.2. To investigate the effects of CTGF-integrinβ1 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-integrinβ1 antibody for 24h,48h,72h,96h and 120h, WST-1 assay was used to detect the effects of anti-integrinβ1 antibody on CTGF-induced proliferation.3. The cultured PASMCs were divided into control group, CTGF group and CTGF+anti-integrinβ1 antibody group.Transwell chambers were used to observe the effects of anti-integrin Pi antibody on CTGF-induced migration at 24h.4. The cytoskeletal rearrangement was observed with coomassie brilliant blue R25o staining and Confocal Lasar Scanning Microscopy(CLSM) at 12h and 24h.5.RT-PCR was used to assay the mRNA expression of CollagenⅠ-α1, CollagenⅢ-α1 and Fibronectin-1 at 72h. In addition, the expression of Collagen III protein and the phosphorylation of ERK1/2 were evaluated by Western blot analysis at 72h. Results 1. PASMCs treated with CTGF (50ng/ml)were exposed to anti-integrinβ1 antibody (0,5,10,15 mg/L) for 24h,48h,72h, 96h and 120h. WST-1 assay showed a pattent of concentration-dependent, and there was significant difference (P<0.01) when the concentration of anti-integrinβ1 antibody meet 15mg/L, and inhibition rate of PASMC proliferation was the highest at 72h.2.The migration of PASMCs treated with anti-integrinβ1 antibody (15mg/L) was inhibited by Transwell chambers assay at 24h.3. 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 anti-integrinβ1 antibody inhibited this phenomenon; Meanwhile, Changes of intracellular FAK was observed by CLSM. The cell morphology and green fluorescence (FAK) of cytoplasmic were not channged significantly between each group at 12h. Compared with the control group, CTGF could reduce cell morphology and decrease green fluorescence of cytoplasmic of PASMCs at 24h, while anti-integrinβ1 antibody inhibited this phenomenon.4. compared with the control groups, RT-PCR assay indicated that the expression of Collagen typeⅠ-α1, Collagen typeⅢ-α1, Fibronectin-1 mRNA of PASMCs treated with CTGF (50ng/ml) were significantly increased at 72h (P<0.01), anti-integrinβ1 antibody inhibited these extracellular matrix genes mRNA expression (P<0.05).5. Compared with the control, Western blot assay indicated that CTGF(50ng/ml) promoted significantly the expression of CollagenⅢprotein, while anti-integrinβ1 antibody inhibited the expression of CollagenⅢprotein, but the CollagenⅢprotein expression were still more than control group. Meanwhile, anti-integrinβ1 antibody also obviously reduced phosphorylation of ERK1/2 induced by CTGF. Conclusions 1. Integrinβ1 mediates proliferation, migration, cytoskeletal rearrangement of PASMCs induced by CTGF.2. CTGF could promote the expression of CollagenⅠ-α1, CollagenⅢ-α1, Fibronectin-1 mRNA and protein of CollagenⅢ.3. Integrinβ1 may mediate the expression of CollagenⅠ-α1, CollagenⅢ-α1, Fibronectin-1 mRNA and CollagenⅢprotein in PASMCs induced by CTGF, which may be related to the phosphorylation of ERK1/2 signal pathway.4.The CTGF-integrinβ1 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 interven the pulmonary vascular remodeling in PAH.