Study On The Roles Of Protein Kinase C And Basic Fibroblast Growth Factor In Cigarette Smoke-induced Proliferation Of Pulmonary Artery Smooth Muscle Cells

BackgroundCigarette smoking is not only the major cause of chronic bronchitis and emphysema, but also can lead to pulmonary hypertension and cor pulmonale. The presence of pulmonary hypertension is associated with worse clinical outcomes and less survival rates. At the present time, there is no specific and effective treatment for pulmonary hypertension in chronic obstructive pulmonary disease (COPD). In the past, most of the studies on the pathogenesis of COPD-related pulmonary hypertension were focused on hypoxic pulmonary vasoconstriction and hypoxic pulmonary remodeling. However, recent studies suggest that cigarette smoke has a direct effect on the pulmonary vasculature and this effect may lead eventually to pulmonary vascular remodeling and pulmonary hypertension. Smoke-induced proliferation of pulmonary artery smooth muscle cells (PASMC) plays a vital role in the process, but the underlying molecular mechanisms remain largely unclear. Activation of several intracellular pathways and production of numerous growth factors may be involved in the growth-stimulating effect of cigarette smoke on PASMC.PKC is a crucial family of serine-threonine kinases in the intracellular signal transduction pathway. It has been implicated in a variety of cellular functions including proliferation, apoptosis, differentiation of vascular smooth muscle cells. Previous studies in our laboratory showed that activation of PKC and upregulation of PKC-αisozyme play an important role in hypoxia-induced proliferation of PASMC. Recent work within our group also found that PKC-αmRNA and protein expression in small intrapulmonary arteries were markedly increased either in the smoke-exposed animals or in COPD patients and non-COPD smokers, suggesting a possible role for PKC-αin smoke-induced pulmonary vascular remodeling. However, the role of PKC and PKC-αin smoke-induced PASMC proliferation is yet to be investigated in vitro.As an important growth factor and mitogen, bFGF has been reported to stimulate the proliferation of pulmonary artery endothelial cells and adventitial fibroblasts, enable the medial smooth muscle cells to migrate, proliferate and synthesize extracellular matrix such as collagen and fibronectin. It contributes to pulmonary vascular remodeling in rats with hypoxia-induced or monocrotaline-induced pulmonary hypertension. Studies in smokers with COPD showed an unequivocal increase in bFGF expression in vascular smooth muscle of small pulmonary vessels with a luminal diameter under 200μm. The data suggest a role of bFGF in the pathogenesis of COPD-associated vascular remodeling. However, to our knowledge, no studies have examined the involvement of bFGF in smoke-induced PASMC proliferation in animal models and in PASMC cell culture model.In the present study, we investigated the roles of PKC and bFGF in the proliferation of PASMC and pulmonary vascular remodeling caused by cigarette smoke.PartⅠEffect of Cigarette Smoke Extract on Proliferation of Pulmonary Artery Smooth Muscle Cells and the Relevant Roles of Protein Kinase CObjective To investigate the effects of cigarette smoke extract on proliferation of rat and human PASMC and to evaluate the relevant roles of protein kinase C (PKC).Methods Cultured rat and human PASMC were studied in the following conditions: (1) PASMC were exposed to different concentrations of CSE for 24h, then MTT colorimetric assay was used for detection of cell proliferation. Cell viability was assessed by trypan blue exclusion. (2) PASMC were pre-incubated with PMA for 24h or Ro31-8220 for 30min before exposure to 5% CSE for 24h. Cell proliferation was examined by MTT colorimetric assay, cell cycle analysis and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. (3) PASMC were exposed to 5% CSE for 24h, then PKC-αmRNA expression was detected by Reverse transcription-polymerase chain reaction(RT- PCR)and protein expression by Western Blot. In rat PASMC, intracellular distribution of PKC-αwas also observed by immunofluorescence staining and confocal microscopy. (4) Rat PASMC were transfected with specific antisense oligodeoxynucleotides against PKC-α6h before exposure to 5% CSE for 24h. PKC-αprotein expression and cell proliferation were detected by methods described previously.Results (1) At low concentrations (5%) CSE increased proliferation of PASMC, whereas high concentrations (20%, 30%) were inhibitory as a result of cytotoxicity. (2) The absorbance (A) value, proliferation index (PI), S-phase cell fraction (SPF ) and average optical density of PCNA staining in PASMC from 5% CSE exposure group were significantly increased compared to those of control group (P<0.05). PKC down-regulation by PMA pretreatment or PKC inhibition by Ro31-8220 pre-incubation abolished the effect of 5% CSE on PASMC proliferation. (3) After exposure to 5% CSE for 24h, PKC-αmRNA and protein expression in PASMC (1.054±0.078, 1.185±0.041, respectively) were much higher than in untreated cells (0.573±0.054, 0.671±0.055, respectively) (P<0.01). Moreover, in rat PASMC, 5% CSE induced a translocation of PKC-αfrom cytoplasm toward the perinuclear area and into the nucleus. (4) Specific antisense oligodeoxynucleotides against PKC-αreduced 5% CSE-induced expression of PKC-αprotein (0.713±0.047 vs 1.180±0.056), also abolished the effect of 5% CSE on rat PASMC proliferation significantly.Conclusions CSE can be cytotoxic at high concentrations. But at low concentrations, it makes a mitogenic effect on cultured rat and human PASMC. PKC, especially its alpha isozyme, seems to play an important role in CSE-induced proliferation of both rat PASMC and human PASMC. PartⅡSection 1 Basic fibroblast growth factor expression and pulmonary vascular remodeling after cigarette smoke exposure in the ratObjective To investigate basic fibroblast growth factor expression and pulmonary vascular remodeling in rats exposed to cigarette smoke.Methods Thirty male Wistar rats were randomly divided into five groups: control group(C group), smoke exposure groups (S2w, S4w, S8w, S12w group). Mean pulmonary artery pressure (mPAP) was measured by right cardiac catheterization. The percentage of muscularised small pulmonary arteries adjacent to alveolar ducts was determined byα-SM-actin staining and vascular cell proliferation by proliferating cell nuclear antigen staining. RT- PCR and immunohistochemistry staining analysis were used for the detection of bFGF mRNA and protein expression in pulmonary arteries.Results Compared to control group, artery oxygen partial pressure and mPAP were not altered in smoke exposure groups. The percentage of completely muscularised small vessels adjacent to alveolar ducts was significantly increased in S4w, S8w and S12w group. Cigarette smoke caused marked proliferation of pulmonary vascular cells. The expressions of bFGF mRNA (0.327±0.076, 0.483±0.131, 0.828±0.207, 0.743±0.159, respectively) and protein (0.184±0.012, 0.230±0.017, 0.292±0.013, 0.325±0.020, respectively) in pulmonary arteries in smoke exposure groups were higher than those of C group (P<0.05). The percentage of muscularised vessels, proliferation index of vascular wall cells correlated strongly with levels of bFGF mRNA expression and the values of average optical density of bFGF protein.Conclusions These data suggest that smoke causes marked production of bFGF in pulmonary arteries. bFGF appears to play an important role in the pathogenesis of smoke-induced vascular remodeling. Section 2 Cigarette smoke extract induces bFGF expression via protein kinase C in rat pulmonary artery smooth muscle cellsObjective To investigate the effect of CSE on bFGF expression in rat PASMC and the possible role of PKC in this process.Methods Cultured PASMC were prepared by an explant method from intrapulmonary artery in normal male Wistar rats. Sub-confluent PASMC were serum-starved to induce quiescence, then were exposed to 5%CSE for different time periods. Some PASMC were pre-incubated for 30min with PKC inhibitor Ro31-8220 before exposure to 5%CSE for 12h. RT-PCR and immuocytochemistry staining were performed for the detection of bFGF mRNA and protein expression in PASMC.Results The control PASMC expressed bFGF mRNA and protein at low levels (0.093±0.034, 0.142±0.013, respectively). PASMC treated with 5%CSE for various times showed a time-dependent increase in bFGF expression. Increased bFGF mRNA and protein expression were evident at 4h after CSE treatment. Maximal increase in bFGF mRNA occurred at 8h (0.436±0.103, P<0.01), while peak expression of bFGF protein was at 12h (0.237±0.031, P<0.01). Then bFGF mRNA and protein expression were slowly declined and were still elevated at 24h. Pretreatment with Ro31-8220 nearly completely inhibited CSE-induced bFGF mRNA and protein expression.Conclusion These results indicate that 5%CSE stimulates bFGF mRNA and protein expression in cultured rat PASMC. The induction of bFGF seems to occur through activation of the PKC pathway.