RESUMO
Abstract Purpose: To investigate the mechanisms by which PD98059 and LY294002 interfere with the abnormal deposition of extracellular matrix regulated by connective tissue growth factor (CTGF) of rat pulmonary artery smooth muscle cells (PASMCs). Methods: Rat PASMCs were cultured and separated into a control group. Real-time fluorescence quantitative PCR was performed to detect the expression of collagen III and fibronectin mRNA. Immunohistochemistry and western blot analyses were performed to detect the expression of collagen III protein. Results: The expression of collagen III and fibronectin mRNA was greater in PASMCs stimulated with CTGF for 48 h, than in the control group. After 72h of stimulation, the expression of collagen III protein in the PASMCs was greater than in the control. The equivalent gene and protein expression of the CPL group were much more significant. Conclusions: CTGF can stimulate the gene expression of collagen III and fibronectin in PASMCs, which may be one of the factors that promote pulmonary vascular remodeling (PVR) under the conditions of pulmonary arterial hypertension (PAH). PD98059 and LY294002 can inhibit the ERK1/2 and PI3K/PKB signaling pathways, respectively, thus interfering with the biological effects of CTGF. This may be a new way to reduce PAH-PVR.
Assuntos
Animais , Masculino , Flavonoides/farmacologia , Cromonas/farmacologia , Fibronectinas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Colágeno Tipo III/metabolismo , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Artéria Pulmonar/citologia , Expressão Gênica/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica , Fibronectinas/genética , Ratos Sprague-Dawley , Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais , Colágeno Tipo III/genética , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismoRESUMO
Connective tissue growth factor (CCN2/CTGF) is a matricellular-secreted protein involved in extracellular matrix remodeling. The P19 cell line is an embryonic carcinoma line widely used as a cellular model for differentiation and migration studies. In the present study, we employed an exogenous source of CCN2 and small interference RNA to address the role of CCN2 in the P19 cell aggregation phenomenon. Our data showed that increasing CCN2 protein concentrations from 0.1 to 20 nM decreased the number of cell clusters and dramatically increased cluster size without changing proliferation or cell survival, suggesting that CCN2 induced aggregation. In addition, CCN2 specific silencing inhibited typical P19 cell aggregation, which could be partially rescued by 20 nM CCN2. The present study demonstrates that CCN2 is a key molecule for cell aggregation of embryonic P19 cells.