[Integrin β3 pathway mediated connective tissue growth factor-induced proliferation, migration and extracellular matrix deposition of pulmonary arterial smooth muscle cells].

Objective To explore the effects of integrin β3 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 β3 antibody group: culture system was added with 50 ng/ml CTGF and 10 mg/L anti-integrin β3 antibody. The PASMCs were cultured with 50 ng/ml CTGF and anti-integrin β3 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 β3 antibody inhibited significantly the proliferation of PASMCs induced by CTGF(P<0.05), among which the inhibition rate of anti-integrin β3 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 β3 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 β3 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 β3 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 β3 pathway can mediate CTGF-induced proliferation, migration and extracellular matrix deposition of PASMCs, CTGF-integrin β3 signaling pathway may play an important role in pulmonary vascular remodeling. Key words: Integrin beta3; Connective tissue growth factor; Extracellular matrix; Hypertension, pulmonary; Cell proliferation; Cell migration