Smoking Induced Rat Pulmonary Vascular Remodeling Via PKCα-mediated CyclinD1 Up-regulation

It is well known that smoking is a major risk factor for the development of chronic obstructive pulmonary disease (COPD) and pulmonary hypertension. The structural change of pulmonary vessels, called pulmonary vascular remodeling, plays an important role in the development and maintenance of pulmonary hypertension. Accumulating evidences from animals and humans have demonstrated that smoking might induce pulmonary vascular remodeling in patients with mild-to-moderate COPD as well as smokers with normal lung fuction. hese findings suggested that smoking might directly result in pulmonary vascular remodeling at the initial stage of COPD.As a critical feature of pulmonary vascular remodeling, the hypertrophy and hyperplasia of pulmonary artery smooth muscle cells (PASMCs) contributed greatly to the sustained elevation of pulmonary vascular resistance and pulmonary artery pressure. Smoking could induce a persisting production of some mediators such as endothelin and endothelial nitric oxide synthase et al, some of which might play important role in vascular musclarization and contraction. Our previous studies have demonstrated that smoking could induce rat pulmonary artery smooth muscle cells (rPASMCs) proliferation in vitro. However, the molecular mechanism underlying this process remains not clear.Functioning as a collaborative oncogene, the cyclin D1 gene is frequent amplified and overexpressed in human malignancy. Accumulating data in recent years indicated that cyclin D1 also regulated proliferation of many other mammalian cells including hepatocytes and cardiomyocytes. As a key regulator of G1 cell cycle progression in mammalian cells, cyclin D1 promotes cell cycle progression via encoding the rate-limiting step in transitions from the G1 to S phase. However, some researches demonstrated that overexpression of cyclin D1 either inhibit cells proliferation or lead to cell apoptosis and death. Previous studies and our recent research indicated that cyclin D1 might induce the proliferation of rat airway smooth muscle cells. To date, the influence of cigarette smoke on the expression of cyclin D1 and its regulatory role in rat pulmonary smooth muscle cells (rPASMCs) is unknown. The protein kinase C (PKC) family is an improtant intracellular mediator in signal transduction, which was responsible for a multitude of cellular processes including proliferation, differentiation, apoptosis, migration and secretion. As a member of the conventional PKC subgroup, PKCa also plays a critical role in the regulation of smooth muscle cells proliferation.In the past decade, increasing evidence manifested PKC as a key regulator of cell cycle progression. Nevertheless, the precise role of PKC in the regulation of cyclin D1 expression remains controversial. PKC might suppress or up-regulated the expression of cyclin D1 even in the same type of cells, such as smooth muscle cells. So far, the precise role of PKCa in regulating cyclin D1 expression in rPASMCs remains unclear.So, the present study focused on the influence of cigarette smoke on the expression of cyclin D1 in smoking rats and explore whether PKCa regulates the cell cycle and proliferation via cyclin D1 up-regulation in vivo and in vitro.Methods1. Wistar rats were exposed to air or the whole smoke of cigarettes for three months. The mRNA and protein levels of cyclin D1 and PKCa were determined by RT-PCR or Western-blot respectively. The pulmonary vascular remodeling was evaluated via the wall thickness of pulmonary vessels and the percentage of muscularized vessels.2. To explore the effect of cigarette smoke extract (CSE) on cells proliferation, CSE were prepared with different concentrations and were applied to the culture of the rat pulmonary smooth muscle cells (rPASMCs). Moreover, the rPASMCs were transfected with cyclin D1 siRNA or negative control siRNA and then stimulated with CSE. The mRNA and protein levels of cyclin D1 were determined by RT-PCR or Western-blot respectively. Cell proliferation was measured by cell count and BrdU incorporation. The cell cycle was determined by flow cytometry.3. The rPASMCs were transfected with PKCa siRNA or negative control siRNA and then stimulated with CSE. The mRNA and protein levels of PKCa and cyclin D1 were determined by RT-PCR or Western-blot respectively. Cell proliferation was measured by cell count and BrdU incorporation. The cell cycle was determined by flow cytometry.4. A plasmid-based short hairpin RNA (shRNA) against rat cyclin D1 was constructed and then applied to transfect rats. Rats were exposed to air or the whole smoke of cigarette for three months. The mRNA and protein levels of cyclin D1 in pulmonary vessels were determined by RT-PCR or Western-blot respectively. The pulmonary vascular remodeling was evaluated via the wall thickness of pulmonary vessels and the percentage of muscularized vessels.Results1. Exposure to cigarette smoke induced the pulmonary vascular remodeling in rats and significantly increased the mRNA and protein levels of cyclinD1 in pulmonary vessels. The protein levels of cyclinDl in pulmonary vessels were positively correlalted with the wall thickness of pulmonary vessels and the protein levels of PKCa.2. CSE at different concentrations played dual role in the proliferation of rPASMCs. CSE at low concentration (1% to 5%) significantly increased the cell number and BrdU incorporation of rPASMCs. However, CSE at high concentration (20%) significantly decreased the cell number and BrdU incorporation of rPASMCs.2% CSE significantly improved the mRNA and protein levels of cyclinDl in rPASMCs as compared with control. However, the cyclin D1-specific siRNA significantly prevented the expression of cyclin D1 at mRNA and protein levels. The cyclin Dl-specific siRNA also induced a G0/G1 arrest in rPASMCs as compared with control. Furthermore, CSE-induced cell proliferation was markedly inhibited by cyclin D1-specific siRNA.3.2%CSE significantly improved the mRNA and protein levels of PKCa and cyclinDl in rPASMCs as compared with control. The PKCa specific siRNA significantly prevented the expression of PKCa at mRNA and protein levels. Moreover, it also decreased the mRNA and protein levels of cyclin D1 in CSE-treated rPASMCs. The PKCa-specific siRNA also induced a G0/G1 arrest in rPASMCs as compared with control. Furthermore, CSE-induced cell proliferation was markedly inhibited by PKCa specific siRNA.4. The plasmid-based shRNA against cyclin D1 could efficiently transfect pulmonary vessels in vivo via tail vein injection. The transfection of plasmid-based shRNA also significantly decreased the mRNA and protein levels of cyclin D1 in pulmonary vessels in smoking rats. Moreover, it also decreased the wall thickness of pulmonary vessels and the percentage of muscularized vessels. Conclusions1. Cigarette skome exposure up-regulated the expression of cyclin D1 in pulmonary vessels and directly resulted in pulmonary vascular remodling in rats.2. The cyclin D1 specific siRNA successfully prevented the upregulation of cyclin D1 and cells proliferation in CSE-treated rPASMCs. Cyclin D1 is required for CSE-induced rPASMCs proliferation.3. PKCa specific siRNA blocked the expression of cyclin D1 and subsequently inhibited the proliferation of rPASMCs in CSE-treated rPASMCs. PKCa mediated up-regulation of cyclin D1 was involved in cigarette smoke-induced proliferation of rPASMCs in vitro.4. The plasmid-based shRNA against cyclin D1 gene attenuated pulmonary vascular remodeling in smoking rats. Cyclin D1 might play a critical role in cigarette smoke-induced pulmonary vascular remodeling via regulating rPASMCs proliferation.In summary, all these findings indicated the important role of cyclinD1 in the pulmonary vascular remodeling in smoking rats. PKCa mediated up-regulation of cyclin D1 was involved in cigarette smoke-induced proliferation of rPASMCs in vitro.The results might be helpful to explore a new molecule machmism in smoking-induced pulmonary hypertension and to find a new approach to the treatment.