The Role Of Connective Tissue Growth Factor In Cigarette Smoke-induced Pulmonary Vascular Remodeling In Rats And Its Relationship With Cyclin D1

Objective:To investigate the expression variations of connective tissue growth factor(CTGF) and cyclinDl in pulmonary vasculature in rats exposed to cigarette smoke and their roles in pulmonary vascular remodeling Methods:Twenty-four male Wistar rats were randomly divided into four groups:control group(C group), smoke exposure groups (S2w, S4w, S8w group). Arterial partial pressure of oxygen was measured. Pulmonary artery remodeling was observed by Hematoxylin-Eosin staining and the percentage of muscularised small pulmonary arteries. Immunohistochemistry methods were performed to observe CTGF and cyclinD1 expressions in pulmonary artery smooth muscle. Real time quantitative RT-PCR and Western Blotting analysis were used for detection of mRNA and protein expressions in pulmonary artery smooth muscle. Results:There was no significant difference in arterial partial pressure of oxygen among all groups. The percentage of muscularised small vessels and %WT were significantly increased in S2w,S4w and S8w group compared to control group (P<0.05). Compared to control group, significant increases of CTGF and cyclinDl expressions in smoke exposure groups were observed (P<0.05). The expressions of CTGF and cyclinDl were significantly positively correlated with the severity of pulmonary vascular muscularization, and there was statistically significant positive correlation between the expression of CTGF and cyclinD1. Conclusion:CTGF and cyclinDl expressions significantly upregulated in pulmonary arteries from rats exposed to cigarette smoke(2w～8w) and there was significant positive correlation between their expressions. Their expression variations may be associated with abnormal proliferation of pulmonary artery smooth muscle cells induced by cigarette smoke. Objective:Cigarette smoke has been demonstrated to induce pulmonary vascular remodeling, which is characteristic of increased medial thickening of the pulmonary arteries mainly resulting from the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). However, the molecular mechanism underlying this process is still unclear. In the present work, we investigated whether connective tissue growth factor (CTGF) regulates rat pulmonary artery smooth muscle cells (rPASMCs) proliferation induced by cigarette smoke extract (CSE) by cyclin D1 upregulation in vitro. Methods: Primary cultured rPASMCs were exposed to CSE. CTGFsiRNA or cyclin D1siRNA were transfected to rPASMCs. Cell proliferation was determined by cell counting and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Real time RT-PCR was employed to examine relative expression levels of mRNA. Protein expression was analyzed by western blotting and immunofluorescence staining. Cell cycle distribution was measured using flow cytometry. Results:The expression of CTGF was significantly increased in rPASMCs at both mRNA and protein levels when rPASMCs were treated with 2% CSE, which significantly promoted the proliferation of rPASMCs. CTGFsiRNA could inhibit the proliferation induced by CSE. Furthermore, CTGFsiRNA could markedly suppress the mRNA and protein expression of cyclin D1 in rPASMCs and led to cell cycle arrest in G0/G1 phase and thus reduced rPASMCs proliferation. Conclusion:These findings suggest that CTGF contributes to the proliferation of rPASMCs induced by CSE at least in part by upregulating cyclin Dl expression. Objective:Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell proliferation. Connective tissue growth factor (CTGF) is a cysteine-rich peptide implicated in several biological processes such as cell proliferation, survival, and migration. This study investigated the potential role of CTGF in pulmonary vascular remodeling. Methods:We constructed a plasmid-based short hairpin RNA (shRNA) to knock down the expression of CTGF in primary cultured rat pulmonary artery smooth muscle cells (rPASMCs) and in rat lung vessels. Rat PASMCs were challenged with cigarette smoke extract (CSE). Rats were exposed to cigarette smoke for three months in the absence or in the presence of plasmid-based short hairpin RNA against CTGF which was administrated by tail vein injection. Results:CTGFshRNA significantly prevented CTGF and cyclin D1 expression, arrested cell cycle at G0/G1 phase and suppressed cell proliferation in rPASMCs exposed to CSE. CTGFshRNA administration ameliorated pulmonary vascular remodeling, inhibited cigarette smoke-induced CTGF elevation and reversed the cyclin D1 increase in pulmonary vessels in rats. Conclusion:Collectively, our data demonstrated that plasmid-based shRNA against CTGF attenuated pulmonary vascular remodeling in cigarette smoke-exposed rats.