The Relationship Of Connective Tissue Growth Factor With Extracellular Signal-regulated Kinase Pathway And Protein Kinase C Pathway In Cigarette Smoke-induced Pulmonary Vascular Remodeling In Rats

Part one Changes of connective tissue growth factor, PKCa and ERKl/2expression in pulmonary artery smooth muscles of cigarette smoke exposed rats and their relationship with pulmonary vascular remodelingObjective:To investigate the expression changes of CTGF, PKCa and ERK1/2in pulmonary artery smooth muscles of cigarette smoke exposed rats and their relationship with pulmonary vascular remodeling.Materials and methods:24male wistar rats in8-week were randomly divided into4groups:C group (control group), S-1M, S-3M and S-6M (cigarette smoke exposure respectively for1,3,6months groups). Their arterial partial pressures of oxygen were measured. Hematoxylin-Eosin staining was performed to observe the pulmonary artery wall thickness (%WT) and anti-a-smooth muscle actin antibody staining to determine the percentage of muscularized arteries to represent the pulmonary vascular remodeling. Immunohistochemistry methods were carried out to determine the expression levels of CTGF and ERKl/2in pulmonary vessels. Western blotting analysis was executed to detect the protein expressions of CTGF, PKCa and ERKl/2in pulmonary artery smooth muscles of rats.Results:The arterial partial pressures of oxygen have no significant difference among all groups. The pulmonary arterial wall thickness (%WT) and percentage of muscularized arteries were significantly increased parallel with the time of cigarette smoke exposure. The same changes were also detected in protein expressions of CTGF, PKCα and activated ERK1/2in pulmonary arterial smooth muscles. And the protein expressions of them were positively correlated with the severity of pulmonary artery remodeling.Conclusions:The severity of pulmonary artery remodeling was positively correlated with the time of cigarette smoke exposure in cigarette smoke exposed rats, and also were the protein expressions of CTGF, PKCα and activated ERK1/2in pulmonary arterial smooth muscles. It is suggested that their expressions may be associated with pulmonary artery remodeling that induced by cigarette smoke exposure in rats. Part two Role of connective tissue growth factor participating in PKCa and ERK1/2signaling pathways in cigarette smoke extract induced proliferation of rat pulmonary artery smooth muscle cells and their relationshipObjective:To investigate whether connective tissue growth factor regulating proliferation of rat pulmonary artery smooth muscle cells induced by cigarette smoke extract via PKCa or ERK1/2signaling pathwayMaterials and methods:Primary cultured rPAMSCs were stimulated by cigarette smoke extract (CSE) in different concentration for24h. The proper concentration of CSE in promoting proliferation of rPAMSCs was determined by cell counting and5-bromo-2’-deoxyuridine (BrdU) incorporation assay. A small interfering RNA in knock down the expression of CTGF had been validated and was used to down-regulate the expression of CTGF in CSE-induced proliferation of rPAMSCs. PD98059was used to down-regulate the activation of ERK1/2in CSE-induced proliferation of rPAMSCs. The proliferation of rPAMSCs was evaluated by cell counting and5-bromo-2’-deoxyuridine (BrdU) incorporation assay. Real-time PCR and Western blotting were performed to determine the mRNA and protein expression of CTGF, PKCa and ERK1/2.Results:When CSE stimulated at20μg/ml for24h, the proliferation of rPAMSCs reached its peak. The mRNA and protein expression of CTGF, PKCa and activated ERK1/2were up-regulated in rPAMSCs under CSE stimulation at20μg/ml, accompanied with proliferation of rPAMSCs. CTGFsiRNA did not only down-regulated CTGF expression in CSE-induced proliferation of rPAMSCs, but also suppressed the activation of ERK1/2and up-regulation of PKCa. PD98059only suppressed the activation of ERK1/2and the proliferation in CSE stimulated rPAMSCs, and it did not shown any influence in CTGF expression.Conclusion:Connective tissue growth factor promotes the proliferation of rPAMSCs induced by cigarette smoke extract not only via ERK1/2signaling pathway, but also PKCa signaling pathway. Part three Knockdown of connective tissue growth factor by lentivirus prevented pulmonary vascular remodeling in cigarette smoke exposed rats and relationship with PKCa and ERK1/2signaling pathwaysObjective:To investigate whether connective tissue growth factor regulating pulmonary vascular remodeling induced by cigarette smoke via PKCa or ERK1/2signaling pathway.Materials and methods:A lentivirus-mediated small interfering RNA in knock down the expression of CTGF had been validated and was used to down-regulate the expression of CTGF in CSE-induced proliferation of rPAMSCs and pulmonary vascular remodeling induced by cigarette smoke in rats.24male wistar rats in8-week were randomly divided into4groups:controls group, smoking group, smoking and negative lentivirus infected group and smoking and positive lentivirus infected group. Cigarette smoke was exposured to rats for6weeks in somking groups. Their arterial partial pressures of oxygen were measured. Hematoxylin-Eosin staining was performed to observe the pulmonary artery wall thickness (%WT) and anti-a-smooth muscle actin antibody staining to determine the percentage of muscularized arteries to represent the pulmonary vascular remodeling. Real-time PCR and Western blotting were performed to determine the mRNA and protein expression of CTGF, PKCa and ERK1/2.Results:The lentivirus-mediated CTGFsiRNA could not only down-regulated mRNA and protein expression of CTGF in CSE-induced proliferation of rPAMSCs, but also in cigarette somke exposed rats. The mRNA and protein expression of CTGF, PKCa and activated ERK1/2were aslo down-regulated in cigarette somke exposed rats under infection of lentivirus-mediated CTGFsiRNA, accompanied with supression of pulmonary vascular remodeling.Conclusion:Connective tissue growth factor promotes the pulmonary vascular remodeling in cigarette smoke exopsed rats not only via ERK1/2signaling pathway, but also PKCa signaling pathway.