| Diabetes mellitus is a comman, frequently-occurring disease and its chronic vascular complications such as atherosclerosis are the principal cause of morbidity and mortality in diabetic patients. It has been showed that advanced glycation end products (AGEs) play an important role in the pathogenesis and progression of accelerated atherosclerosis. AGEs are a group of nonreversible and poisonous products formed by nonenzymatic glycation of glucose with protein and lipids under hyperglycemia condition. It not only directly results in the structure and fuction changes of the tissues in which AGEs deposited but also can interact with receptor for AGEs (RAGE), a member of the immunoglobulin superfamily of cell surface molecules which express in range of cells including endothelial cell, smooth muscle cell and mononuclear phagocytes, to result in dysfunction and lesion of the cells and play a key role in initiating vasculopathy.Atherosclerosis is an inflammatory disease process with increased plasma low density lipoprotein-cholesterol level. Monocyte/macrophages contribute to the development of lesions by accumulation of cholesterol esters and the formation of foam cells. As we know, macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor (cholesterol influx), acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1; storage cholesterol ester converted from free cholesterol), and ATP-binding cassette transporter A1 (cholesterol efflux). ACAT catalyzes the formation of cholesteryl esters from cholesterol and long chain fatty acyl coenzyme A. ACAT is believed to play significant roles in lipoprotein assembly and in dietary cholesterol absorption. ACAT-1 and ACAT-2 are two subtypes of ACAT gene family. ACAT-1 protein is present at high levels in macrophages and hormone-producing cells. ACAT-1 gene expression is up-regulated in human monocytes during differentiation and foam cell formation. Under pathological conditions, accumulation of cholesteryl esters produced by ACAT-l can promote foam cell formation in atherosclerotic lesions. However, it's still unknown the molecular mechanism of ACAT-1 gene expression, regulation and its signal transduction induced by AGEs in macrophages.Peroxisome proliferator-activated receptor-γ(PPARγ) is a member of nuclear transcription factors and involes in multiple physiological or pathophysiological process including adipocyte differentiation, inflammation, insulin sensitivity, cell cycle regulation and cancers. It is widely accepted that PPARγis a key regulater during the development of atherosclerosis. The interests have focused on the role of PPARγin modulating macrophage cholesterol and lipid homeostasis.Objectives:In this study, THP-1 macrophages were exposed to AGEs-modified bovine serum albumin (AGE-BSA) at different concentration for 24 hours and at a concentration of 200mg/L for different time to investigate the effects of AGEs on the expression of ACAT-1, PPARγand SR-A, which is in favour of clarifying the mechanism of AGEs promoting macrophage-derived foam cell formation. In additional, we investigate the effects of rosiglitazone, a synthetic PPARγligand, on the expression of ACAT-1 induced by AGEs in THP-1 macrophages. The molecular mechanisms of AGEs accelerating atherosclerosis in diabetic patients were proposed and the protective effects of PPARγactivation during foam formation were discussed.Methods:1. AGE-BSA was prepared by the methods as described previously. Briefly, AGE-BSA was made by incubating BSA with 50 mM D-glucose in 5% CO2/95% air at 37°C for 12 wk in a 10 mM PBS, pH 7.4, in the presence of 100 units/ml penicillin, and 100 mg/ml streptomycin. Unincorporated glucose was removed by dialysis overnight against 1×PBS.2. The human monocytic leukemia cell line, THP-1 cells were cultured in RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. In order to induce phagocytic differentiation, THP-1 cells were cultured in the presence of 0.1μM PMA for 72 h.3. THP-1 macrophages were exposed to AGE-BSA at different concentrations (50, 100, 200 and 400 mg/L) for 24h, and exposed to AGE-BSA at a concentration of 200mg/L for 0,12,24,36 h and 48 h.4. Immunocytochemistry was used to examine the expression levels of proteins for PPARγand SR-A in AGEs-treated macrophages.5. The expression levels of mRNA and proteins for ACAT-1 and PPARγin AGEs-treated macrophages were semi-quantified by RT-PCR and Western blot, respectively. And the transcriptional activity of PPARγin AGEs-treated macrophages was determined by electrophoretic mobility shift assay (EMSA).6. THP-1 macrophages were incubated respectively with PPARγagonist rosiglitazone and anti-PPARγmonoclonal antibody. The expression levels of mRNA and proteins for ACAT-1 and PPARγwere detected by RT-PCR and Western blot.7. THP-1 macrophages were pretreated by rosiglitazone for 4h and then incubated with AGE-BSA at a concentration of 200mg/L for 24h. The expression levels of mRNA and proteins for ACAT-1 were detected by RT-PCR and Western blot.Results:1. AGEs-protein specific fluorescence determinations were performed by measuring emission at 450nm on excitation at 390nm using a Fluorescence spectrophotometer. AGE-BSA contained 180.5 fluorescence U/mg protein while unmodified BSA contained 2.9 fluorescence U/mg protein.2. About 85% of THP-1 cells incubated with PMA (0.1μM) for 72h stopped proliferation and differentiated into macrophages. These macrophages containing granules grew on the wall of culture flask just like ameba cells and extended pseudopods.3. Compared with the control BSA group, AGE-BSA significantly upregulated the levels of mRNA and proteins for ACAT-1 but downregulated those for PPARγin a concentration- and time-dependent manner (P<0.05). Moreover, EMSA showed that AGE-BSA also inhibited the transcription activity of PPARγin a concentration-dependent manner (P <0.05).4. Immunocytochemistry showed that AGE-BSA decreased the expression level of proteins for PPARγbut increased that of SR-A significantly (P <0.05).5. Compared with the control group, the expression of PPARγmRNA increased after PPARγagonist treated but decreased after anti-PPARγmonoclonal antibody treated (P<0.05). Moreover, the expression of ACAT-1 mRNA decreased after PPARγagonist stimulating but increased after anti-PPARγmonoclonal antibody stimulating (P<0.05). The same changes can be observed from Western blot analysis.6. Macrophages were pretreated with rosiglitazone at different concentrations (1μM, 5μM and 10μM) for 4h before exposed to 200mg/L AGE-BSA. We found that rosiglitazone impaired ACAT-1 expression induced by AGEs in a concentration-dependent manner significantly (P <0.05).Conclusions:1. AGE-BSA increased significantly the expression levels of mRNA and proteins for ACAT-1 in a concentration-and time-dependent manner.2. AGE-BSA not only decreased the expression levels of mRNA and proteins for PPARγ, but also inhibited the transcription activity of PPARγin macrophages in a concentration-dependent manner, significantly.3. AGE-BSA could promote macrophage intaking lipids by upregulating the expression of SR-A, which contributes to macrophage-derived foam cell formation.4. The increased expression of PPARγand reduced of ACAT-1 after PPARγligand stimulation suggest that PPARγmay inhibit the expression of ACAT-1.5. Rosiglitazone, a PPARγspecific agonist, could significantly inhibit the expression levels of mRNA and proteins for ACAT-1 induced by AGEs in a concentration-dependent manner, which suggests that activation of PPARγcould regulate ACAT-1 transcriptionally to prevent cholesterol esters accumulate in macrophage, thereby inhibiting the macrophage-derived foam cell formation induced by AGEs.
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