The Role Of Extracellular Signal-regulated Kinase Pathway In Rat Pulmonary Vascular Remodeling Induced By Cigarette Smoke And Its Relationship With CyclinE1

Part1Changes of ERK1/2and cyclinEl expressions in pulmonary vascular remodeling induced by cigarette smoke in ratsObjective:To investigate the variations of ERK1/2and cyclinEl expressions in pulmonary vasculature in rats exposed to cigarette smoke and the correlations between pulmonary vascular remodeling and them.Materials and methods:24male wistar rats were randomly divided into4groups:C group (control group), S-1M, S-3M and S-6M groups (smoke exposure for1,3,6months respectively). Hematoxylin-Eosin staining was performed to observe the pulmonary artery wall thickness (WT), and percentage of muscularized arteries was determined by anti-a-smooth muscle actin antibody staining. Immunohistochemistry methods were performed to evaluate ERK1/2and cyclinEl expression levels in pulmonary vessels. Real time RT-PCR and Western blotting analysis were used for detection of mRNA and protein variations in pulmonary vascular muscle.Results:Pulmonary vessel wall thickness and percentage of muscularized vessels were significantly increased in S-1M, S-3M and S-6M group compared to those in control group. Compared to control group, significant increases of activated ERK1/2and cyclinEl expressions in smoke exposure groups were observed. Expressions of ERK1/2and cyclinEl were positively correlated with the severity of pulmonary vascular muscularization, and there was statistically positive correlation between the expressions of ERK1/2and cyclinEl.Conclusion:Smoke exposure led to pulmonary vascular remodeling of rats. Meanwhile, activated ERK1/2and cyclinEl expression levels were significantly upregulated in pulmonary arteries from rats exposed to cigarette smoke (1-6m). It is suggested their expression variations might be associated with pulmonary vascular remodeling induced by cigarette smoke. Part2Role of ERK1/2and cyclinE1in proliferation of rPASMCs induced by cigarette smoke extract and the relationship between themObjective:We are aimed to investigate whether cigarette smoke extract (CSE) could lead to abnormal proliferation of rat pulmonary arterial smooth muscle cells (rPASMCs) through upregulating cyclinE1mediated by activated ERK1/2signal pathway.Materials and methods:We constructed a small interfering RNA (siRNA) to knock down the expression of cyclinEl and gave PD98059to inhibit the activation of ERK1/2in primary rPASMCs which were challenged with2%cigarette smoke extract. Cell proliferation was assessed by cell counting and5-bromo-2-deoxyuridine (BrdU) incorporation. Western blotting and immunofluorescence technique were use to detect expression and location of ERK1/2and cyclinE1. And cell cycle was analyzed by flow cytometry.Results:Expression of cyclinE1and p-ERK1/2were upregulated by2%CSE, accompanying with proliferation of rPASMCs. CyclinE1siRNA significantly prevented cyclinE1expression, and suppressed proliferation of rPASMCs exposed to CSE. PD98059suppressed phosphorylation of ERK1/2and downregulated expression of cyclinE1of rPASMCs challenged by CSE. And then, cell cycle was also arrested at G0/G1phase and proliferation of rPASMCs was partly inhibited by PD98059.Conclusion:Activated ERK1/2played a vital role in abnormal proliferation of rPASMCs induced by CSE, which might be partly as a result of upregulating expression of cyclinE1. Part3Small interfering RNA against ERKl/2alleviates pulmonary vascular remodeling of cigarette smoke exposed rats via down regulating cyclinEl expressionObjective:Cigarette smoke may contribute to pulmonary vascular remodeling that resulted by pulmonary artery smooth muscle cell proliferation before pulmonary hypertension in chronic obstructive pulmonary disease. Activated extracellular signal-regulated kinases1and2(ERK1/2) is considered to participate in this procession. This study is investigated the potential role of ERK1/2in rat pulmonary artery smooth muscle cells (rPASMCs) proliferation and pulmonary vascular remodeling induced by cigarette smoke.Materials and methods:We constructed a small interfering RNA (siRNA) to knock down the expression of ERK1/2in rPASMCs and in rat lung vessels. Rat PASMCs were challenged with cigarette smoke extract (CSE). Rats were exposed to cigarette smoke for3months in the presence of ERK1/2siRNA or negative control siRNA which was administrated intratracheally.Results:ERK1/2siRNA significantly prevented ERK1/2and cyclinEl expression, arrested cell cycle at GO/G1phase and suppressed proliferation of rPASMCs exposed to CSE. ERK1/2siRNA administration ameliorated pulmonary vascular remodeling, inhibited cigarette smoke-induced ERK1/2elevation and reversed the cyclinEl increase in pulmonary vessels in rats.Conclusion:Our data demonstrated that siRNA against ERK1/2attenuated pulmonary vascular remodeling in cigarette smoke-exposed rats.