| [Backgrounds and objective]It is well known that cardiovascular disease is the most prominent cause of death and disability in the world. According to the current data, it is estimated that cardiovascular disease caused by atherosclerosis will become the the number 1 cause of death in the world with the improving of living levels and the generalizing of westen life styles in developing countries till 2020.Atherosclerosis is a chronic inflammatory disease, and characterized by chronic inflammation of vessel wall. Monocytes/macrophages are the most important inflammatory cells in the atherosclerotic lesions of vessel wall and the primary components of atherosclerotic palques. Active recruiting of monocytes into the intimal lining of blood vessels is the initial step of atherosclerosis. With the action of chemokines, blood monocytes adhere to the injured endothelial cells, migrate and cross endothelial cells, and then differentiate into macrophages. Macrophages uptake lipid proteins, accumulate lipids, and differentiate into foam cells, and then form atherosclerotic palque. Monocytes/macrophages contribute to the formation of atherosclerosis in various stages, and may be potential biomarkers for the formation of atherosclerotic palque, therefore are regarded as the target cells for treatment of atherosclerosis.Asymmetric dimethylarginine(ADMA) is acknowledged as a new risk factor for cardiovascular disease. ADMA is an intensive endogenous inhibitor of nitric oxide synthase (NOS), can inhibit the synthesis of vasoactive substance NO, and lead to endothelial dysfunction. Endothelial dysfunction is regarded as an early step in the development of atherosclerosis, and is a key step in the formation of atherosclerosis. Studies have reported that the elevation of circulating ADMA levels is correlated with endothelial dysfunction. Plasma ADMA levels of atherosclerotic patients are elevated significantly, and ADMA may be related with the formation of atherosclerosis.ADMA may be one of the common mechanisms of atherosclerosis induced by multiple risk factors and play an important role in the the development of atherosclerosis. Clinical studies reported that plasma ADMA levels were significantly elevated in patients with hypercholesterolemia, hypertriglyceridemia, diabetes, obesity, and hyperhomocysteinemia, and showed correlation with these diseases, suggesting that ADMA may be a common step for atherosclerosis induced by multiple risk factors. Many studies also found that ADMA contributed to multiple pathological processes of atherosclerosis, including the adhesion of monocytes, the expression of proinflammatory cytokines and chemokines, and the accumulation of ox-LDL in macrophages.In the formation of atherosclerosis, the elevation of plasm ADMA levels is related with the metabolic disorder of PRMT/ADMA/DDAH pathway. ADMA is synthesized during the methylation of protein arginine residues by S-adenosylmethionine:protein arginine methyltransferases (PRMT), and most ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) in kidney. We designate the metabolic process of ADMA as PRMT/ADMA/DDAH pathway. PRMT/ADMA/DDAH pathway may be a new anti-atherosclerotic target.In the present study, therefore, we investigated the role of PRMT/ADMA/DDAH pathway in the pathogenesis of atherosclerosis in animal experiments and the effects of ADMA on monocytes/macrophages in the development of atherosclerotic plaques from the perspective of the migration and differentiation of monocytes into macrophages, and the formation of foam cells. The objective of the present study is to clarify the mechanism of ADMA in the development of atherosclerotic plaques and to provide a new target for pharmaceutical intervention against atherosclerosis.[Methods]1. Animal experimentsThirty healthy adult male Wistar rats were randomly divided into 3 groups:control group, model group, and ADMA inhibitor group. Rats in model group were give high lipid diet and one time of peritoneal injection of vitamin D3 to establish rat model of atherosclerosis. Rats in ADMA inhibitor group were given ADMA inhibitor, aspirin(30mg/kg/day), on the basis of high lipid diet. Rats in control group were only given basic diet. Ras in all groups were killed after feeding for 8 weeks.The histopathological changes of aortas were observed under microscope by HE staining. Serum levels of TC, TG, HDL-C, and LDL-C were measured by radio-immunity method. Serum levels of ADMA, IL-8, and TNF-a were determined by ELISA method. The mRNA and protein expression of PRMT1 in aortas, and DDAH1 in kidneys of rats was assayed by RT-PCR and Western blot.2. Cell experimentsHuman monocytic THP-1 cells and human umbilical vein endothelial cells(HUVEC) were cultured in vitro. Cell experiments grouping:negative control group and ADMA groups with different concentration(3.75,7.5,15 and 30μol/L).The adhesion of monocytes to endothelial cells was assayed after incubation with ADMA for different time. The migration of monocytic THP-1 cells was assayed by Transwell. The proliferation of monocytic THP-1 cells were detected by MTT method. The cell cycle of monocytic THP-1 cells was assayed by flow cytometry. Monocytic THP-1 cells were induced to differentiated into macrophages by 160nmol/L PMA. Immunohistochemisty was used to identify THP-1-derived macrophges. Macrophges were induced to differentiated into foam cells by 1 OOmg/L ox-LDL. Oil red O staining was used to identify THP-1-derived foam cells. The content of cholesterol in THP-1-derived macrophages and foam cells was measured by enzyme-linked colorimetric method. ACAT-1 and MIF mRNA and protein expression in THP-1-derived macrophages was assayed by RT-PCR and Western blot. The secretion of TNF-a and IL-8 in THP-1-derived macrophages was determined by ELISA method.[Results]1. Animal experiments(1) Histopathological changes:For rats in model group, the structure of arterial wall of aorta was unclear, the tunica intima was thicker, smooth muscle cells proliferated significantly in the tunica intima, foam cells formed, and smooth muscle cells arranged irregularly.(2) Serum lipids levels:Serum levels of TC, TG and LDL-C in rats in model group and ADMA inhibitor group were significantly higher than those in control group(P<0.05). Serum levels of TC, TG and LDL-C in rats in ADMA inhibitor group were significantly lower than those in model group(P<0.05).(3) Serum ADMA levels:Serum levels of ADMA in rats in model group were significantly higher than those in control group(P<0.05). Serum levels of ADMA in rats in ADMA inhibitor group were significantly lower than those in model group(P<0.05).(4) Serum inflammatory cytokines levels:Serum levels of TNF-a and IL-8 in rats in model group and ADMA inhibitor group were significantly higher than those in control group(P<0.05). Serum levels of TNF-a and IL-8 in rats in ADMA inhibitor group were a little lower than those in model group(P<0.05).(5) PRMT1 expression in aortas:Compared with control group, PRMT1 mRNA and protein expression in aortas of rats in model group was upregulated significantly(P<0.05). PRMT1 mRNA and protein expression in aortas of rats in ADMA inhibitor group was also upregulated(P＜0.05), but the expression levels were significantly lower than those in model gorup(P<0.05).(6) DDAH1 expression in kidney:Compared with control group, DDAH1 mRNA and protein expression in aortas of rats in model group was downregulated significantly(P<0.05). DDAH1 mRNA and protein expression in aortas of rats in ADMA inhibitor group was also downregulated(P<0.05), but the expression levels were significantly higher than those in model gorup(P<0.05).(7) Correlation analysis:Serum lipids levels were positively related with serum levels of ADMA, the correlation coefficients of serum levels of TC, TG, and LDL-C and serum levels of ADMA were 0.859,0.887 and 0.845, respectively. Serum levels of inflammatory cytokines(TNF-a and IL-8) were also positively related with serum levels of ADMA, and the correlation coefficients were 0.924 and 0.876, respectively.2. Cell experiments(1) Effects of ADMA on the adhesion, migration, and proliferation of monocytes:After incubation with monocytic THP-1 cells for different time, ADMA with different concentration (3.75,7.5,15 and 30μmol/L) significantly promoted the adhesion, migration and proliferation of monocytic THP-1 cells(P<0.05), and showed a time-dependent and dose-dependent way.(2) Effects of ADMA on the content of cholesterol in THP-1-derived macrophages and foam cells:Compared with control group, the content of cholesterol in THP-1-derived macrophages and foam cells, which were incubated with different concentration (3.75,7.5,15 and 30μmol/L) of ADMA for different time (6,12 and 24h), were increased significantly (P＜0.05), and showed a time-dependent and dose-dependent way.(3) Effects of ADMA on ACAT-1 mRNA and protein expression in THP-1-derived macrophages:After incubation with different concentration (0,3.75,7.5,15 and 30μmol/L) of ADMA for 24h, ACAT-1 mRNA and protein expression was upregulated significantly in THP-1-derived macrophages with the increase of ADMA concentration(P<0.05), and showed a dose-dependent way. ACAT-1 mRNA and protein expression levels were the highest in THP-1-derived macrophages incubated with 15μmol/L ADMA(P<0.05). After incubation with 15μmol/L ADMA for 0,6,12 and 24h, ACAT-1 mRNA and protein expression was upregulated significantly in THP-1-derived macrophages with the prolongation of time(P<0.05), and showed a time-dependent way.(4) Effects of ADMA on MIF mRNA and protein expression in THP-1-derived macrophages:After incubation with different concentration (0,3.75,7.5,15 and 30μmol/L) of ADMA for 24h, MIF mRNA and protein expression was upregulated significantly in THP-1-derived macrophages with the increase of ADMA concentration, and showed a dose-dependent way. MIF mRNA and protein expression levels were the highest in THP-1-derived macrophages incubated with 15μmol/L ADMA.(5) Effects of ADMA on the secretion of TNF-αand IL-8 in THP-1-derived macrophages:After incubation with different concentration (0,3.75,7.5,15 and 30μmol/L) of ADMA for 24h, the levels of TNF-a and IL-8 in cell-culture supernatants of THP-1-derived macrophages were elevated significantly(P<0.05). The levels of TNF-a and IL-8 were the highest in cell-culture supernatants of THP-1-derived macrophages incubated with 15 umol/L ADMA(P<0.05).[Conclusions]1. Serum levels of ADMA are elevated in atherosclerotic rats, the elevation of ADMA levels may result from high levels of serum lipids.2. PRMT1 mRNA and protein expression in aortas of atherosclerotic rats is upregulated significantly, while DDAH1 mRNA and protein expression in kidneys of atherosclerotic rats is downregulated. The elevation of ADMA levels directly results from the metabolic disorder of PRMT/ADMA/DDAH pathway.3. Serum levels of inflammatory cytokines(TNF-a and IL-8) are elevated in atherosclerotic rats, and are positively related with serum levels of ADMA. Serum levels of TNF-a and IL-8 can be lowered by decreasing serum levels of ADMA, therefore, ADMA may be a new important proinflammatory factor.4. PRMT/ADMA/DDAH pathway plays an important role in the development of atherosclerosis, and may be a potential anti-atherosclerotic target.5. ADMA can promote the adhesion, migration and proliferation of monocytic THP-1 cells.6. ADMA can upregulate ACAT-1 mRNA and protein expression, promote cholesterol accumulation, and increase the content of cholesterol in THP-1-derived macrophages in vitro, which cause the typical morphological changes of THP-1 derived foam cells, and promote the formation of foam cells.7. AMD A can promote THP-1 derived macrophages to uptake lipids and increase cellular cholesterol content, wich is correlated with upregulation of ACAT-1 expression.8. ADMA can upregulate ACAT-1 mRNA and protein expression and promote the secretion of IL-8 and TNF-a in THP-1-derived macrophages in vitro, which promote atherosclerotic plaques development.9. ADMA may be an important factor in promoting the formation of THP-1-derived macrophages and foam cells, intervention of ADMA is a new strategy for stopping the formation of atherosclerotic plaques.
|
PDF Full Text Request