| Popis: |
Experimental and clinical evidence indicates that secretory phospholipase A2 group IIA (sPLA2-IIA) contributes to atherogenesis. Activity of sPLA2-IIA may be pro-atherogenic both in the circulation leading to altered lipoprotein profile and/or locally in the artery wall where it contributes to lipid accumulation both intra and extra cellularly. Smooth muscle cells (SMC) are the main source for sPLA2-IIA in the artery wall. However, knowledge about the regulation of the enzyme in this cell type is very limited. The general purpose of this thesis was to establish an in vitro model to investigate the signaling pathways involved in the regulation of sPLA2-IIA expression in human arterial SMC (HASMC). We show that sPLA2-IIA is regulated on three different levels in HASMC in vitro. Expression of sPLA2-IIA in HASMC was induced after prolonged culture periods resulting in cell differentiation, measured by the expression of, p21, p27, heavy caldesmon and smoothelin. Basal sPLA2-IIA secretion and/or transcription were further modulated by different pro- and anti-inflammatory cytokines among which IFNg had the most pronounced effect. IFNg induction was accompanied by phosphorylation, nuclear translocation and binding of the transcription factor STAT3 to the sPLA2-IIA promoter, as measured by western blot and electrophoretic mobility shift assay (EMSA) respectively. Binding of STAT3 was inhibited by the PPARa activator fenofibrate, followed by a decrease in sPLA2-IIA expression. Moreover, IFNg activation was accompanied by increased accumulation of diacylglycerol a potent endogenous activator of protein kinase C (PKC). However, based on the results with PKC inhibitors this pathway did not appear to be essential for IFNg induction of sPLA2-IIA in HASMC. Furthermore, IFNg induced sPLA2-IIA secretion was independent on endogenous production of eicosanoids and was not accompanied by increased eicosanoid secretion by HASMC. The third level of sPLA2-IIA expression was IFNg primed cells that were found to be sensitized to further stimulation by AA or prostaglandin E2. This effect was associated with intracellular accumulation of cyclic AMP and nuclear translocation and binding of cJun/CREB2 to the sPLA2-IIA promoter measured by EMSA. Using different inhibitors we found that the upstream signals for cJun/CREB2 involve both PKC and protein kinase A. In conclusion, changes in the balance of pro- and anti-inflammatory cytokines towards a pro-inflammatory state result in accumulation of cytokines including IFNg, IL1b and TNFa that induces sPLA2-IIA secretion by HASMC. This may contribute to the increased amount of extracellular enzyme identified in the arterial intima and/or increased serum levels observed in atherosclerosis. Extracellular sPLA2-IIA in the atherosclerotic plaque may play an important role to sustain chronic inflammation and contribute to the local accumulation of intracellular and extracellular lipoprotein deposits in the artery wall. Moreover increased serum sPLA2-IIA alters the lipoprotein profile rendering it more atherogenic. Furthermore we provide evidence that the regulation of sPLA2-IIA is cell and species specific. This property is extremely important especially when drawing conclusions relevant to human physiology and patophysiology. |
