| Exposure to acute or chronic hypoxia leads to the development of pulmonary artery hypertension ( PAH). The cardinal features of PAH are persistent vaso-constriction and structural remodeling of the pulmonary vessels. Hyperplasia of pulmonary artery smooth muscle cells ( PASMCs) is the main component of pulmonary vascular remodeling which is associated with progressive elevation in pulmonary arterial pressure.Several studies have shown that 5 -hydroxytryptamine (5 - HT) plays an important role in the pathogenesis of PAH. 5 - HT exerts potent mitogenic and co - mitogenic effects on PASMCs, and these effects are associated with celluar internalization of 5 - HT mediated by 5 - HT transporter (5 - HTT). Therefore, inhibition of 5 - HTT might abolish the proliferative response of PASMCs to 5 -HT.Many stimuli resulting in cell growth, differentiation and vascular contraction may activate mitogen - activated protein kinase ( MAP kinase) - dependent signaling pathways. Among this family, the extracellular signal -regulated kina-ses (ERKs) are activated in response to growth and differentiation factors. Previous studies have reported that 5 - HT may induce the activation of ERKl/2 in the rat aortic smooth muscle cells and the rabbit isolated renal artery smooth muscle cells. However, whether the intracelluar signal pathway of 5 - HT in PASMCs is dependent on the activation of ERK is largely unconcerned.In this study, we first observed the effect of fluoxetine, an selective inhibitor of 5 - HT transporter on the proliferation of PASMCs in response to 5 - HT. Then, we hypothesized that 5 - HT induced the proliferation of PASMCs is de-pendent on the activation of ERK. We studied the effect of downregulation of ERK1/2, using antisense oligodeoxynucleotides (ODNs) , on the proliferation of 5 - HT stimulated PASMCs in vitro.MethodsLiposomal transfection was used to introduced ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the fluorecein isothiocynate ( FITC) - laBcled antisense ODN in PASMCs. The effects of fluoxetine and ODNs on the proliferation of PASMC were evaluated by cells number counting and cell cycle analysis, and measured by Microculture tetrazolium (MTT) assay and Flow cytometry (FCM) separately. All the data are presented as means + SD, and assessed by ANOVA and t -test. P < 0.05 was considered significant.Results1. Uptake of ODNs by PASMCsAfter liposomal transfection 24 h and 48 h, FITC - laBcled antisense ODN were observed in the cytoplasm and the nuclei of PASMCs. There were more than 90% of the cells exhibited fluorescence.2. Effect of fluoxetine and ODNs on the proliferation of PASMCs induced by 5-HTMTT assay showed that 5 - HT ( 10-6M) could induce the proliferation of PASMCs. Pretreatment of the cells with fluoxetine (10-5M, 10-6M and 10-7 M) could produce a concentration - dependent reduction in proliferation rates ( PR) . According these results, we chose 10-6M as the concentration of fluoxetine used in other experiments.Pretreatment of PASMCs with ERK1/2 antisense ODN resulted in a significant inhibition of 5 - HT - induced cell proliferation and this effect is stronger than fluoxetine. In contrast, sense ODN and random ODN showed no effect.3. Effect of fluoxetine and antisense ODN on cell cycleFlow cytometric analysis of cell cycle distribution showed that the cells treated with 5 - HT had larger values of S - phase cell fractions ( SPF) and proliferation index ( PI) than control. While pretreatment with fluoxetine or antisense ODN could decrease these values. These results indicated that 5 - HT could promote the PASMCs from the G0/G1 phase of cell cycle into S phase and this effect could be inhibited by fluoxetine and antisense ODN.DiscussionOur result demonstrate that 5 - HTT plays a key role in the mitogenic effect of 5 - HT on PASMCs. Fluoxetine, a highly selective inhibitor of 5 - HT transporter inhibited the proliferation of PASMCs induced by 5 - HT in vitro. Meanwhile , we foun...
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