[Integrin ß3 pathway mediated connective tissue growth factor-induced proliferation, migration and extracellular matrix deposition of pulmonary arterial smooth muscle cells].

ABSTRACT

OBJECTIVE: To explore the effects of integrin ß3 pathway on the proliferation, migration and extracellular matrix deposition of pulmonary arterial smooth muscle cells (PASMCs) induced by connective tissue growth factor (CTGF). METHODS: PASMCs of SD Rats were cultured in M199 culture system in vitro and the 3rd-7th passages of PASMCs were used in the experiments. The cells were randomly divided into three groups (1) CONTROL GROUP culture system contained no any stimulation factor; (2) CTGF group culture system was added into 50 ng/ml CTGF; (3) CTGF+ anti-integrin ß3 antibody groupculture system was added with 50 ng/ml CTGF and 10 mg/L anti-integrin ß3 antibody. The PASMCs were cultured with 50 ng/ml CTGF and anti-integrin ß3 antibody (0, 5, 10, 15, 20 mg/L) for 24, 48, 72 and 96 h, the proliferation of PASMCs was detected by WST-1 Cell Proliferation Assay Kit. The migration of PASMCs was observed by Transwell cell test under the phase contrast microscope. RT-PCR assay was applied to detect the mRNA expression of collagenI-α1, collagen III-α1 and fibronectin-1 gene of PASMCs. The expression of fibronectin protein was examined by Western blotting and immunohistochemistry. RESULTS: The results of WST-1 test showed that the anti-integrin ß3 antibody inhibited significantly the proliferation of PASMCs induced by CTGF (P < 0.05), among which the inhibition rate of anti-integrin ß3 antibody (10 mg/L) was the most significant. Transwell test results showed that CTGF group of PASMCs migration numbers (25 ± 1.57) were higher than that of the control group (11 ± 2.08, P < 0.01); PASMCs migration numbers of CTGF+ integrin ß3 antibody group (17 ± 4.16) were less than that of the CTGF group (P < 0.05). Compared with the control group, the mRNA expression of collagen typeI-α1 (4.28 ± 0.33), collagen typeIII-α1 (4.41 ± 0.35), fibronectin-1 (4.05 ± 0.33) of PASMCs was increased in CTGF group, with a time-dependence (P < 0.01); Compared with the CTGF group, the mRNA expression of collagen typeI-α1 (3.38 ± 0.30), collagen typeIII-α1 (3.40 ± 0.30), fibronectin-1 (3.12 ± 0.29) of PASMCs was reduced in CTGF+ anti-integrin ß3 antibody group (P < 0.05), which was higher than that of the control group (P < 0.05); Western blot and immunohistochemical tests showed that compared with the control group, CTGF group could stimulate the expression of fibronectin protein of PASMCs (P < 0.01); the anti-integrin ß3 antibody could inhibit the expression of fibronectin protein induced by CTGF(P < 0.01), which was more remarkable than that in the control group (P < 0.01). CONCLUSION: Integrin ß3 pathway can mediate CTGF-induced proliferation, migration and extracellular matrix deposition of PASMCs, CTGF-integrin ß3 signaling pathway may play an important role in pulmonary vascular remodeling.