| Background & ObjectiveAtherosclerosis (As) is an early pathological stage of cardiovascular disease, it always undergoes several decades years from early atherogenesis to clinical case. During this long time, people usually ignore the existence of dangers, and the conventional preventives are always not so effective as people expected. In order to promote people's life quanlity, slowing down and reversing the genesis and development of As is an inevitable trend. So, it is necessary and important to understand the mechanism of As, and to get a target of prevention and therapy. On the basis of wide and deep studies on genes, cells and systems, we know that endothelial cells, macrophages and vascular smooth muscle cells (VSMCs) are the major components of fatty streak. Endothelial dysfunction, VSMCs proliferation and lipidosis are the three important steps of lesion formation, the key step is macrophages transforming to foam cells with mechanism not well known yet. Nowadays, researches about As are much more focused on endothelial dysfunction and VSMCs proliferation, and less on the mechanism and regulation of macrophages transforming to foam cells. Recent researches demonstrated that the plasma levels of asymmetric dimethylarginine (ADMA) are increased in cardiovascular diseases, such as coronary heart disease, hypertension, hypercholesterolemia, diabetes mellitus, insulin resistance, hyperhomocysteinemia, heart failure and renal failure. The role and mechanism of ADMA are becoming a focus. ADMA is a competitive inhibitor of endogenous nitric oxidize synthase (NOS), it can lead to endothelial dysfunction and VSMCs proliferation, while it's effect on macrophages transforming to foam cells is unknown. NOS includes neural NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). Different from other two types, iNOS has little expression during physiological state, but increases significantly when cells are stimulated by inflammatory factors and immune factors. Oxidized low-density lipoprotein (oxLDL) is believed to play a key role in accumulation of foam cells in atherogenesis. The cellular uptake of oxLDL is mediated by so-called scavenger receptors. Among them, Lectin-like low density lipoprotein receptor-1 (LOX-1) is the most specific receptor of oxLDL. LOX-1 is present in the endothelium of human coronary arteries, rabbit and rat aorta and bovine endothelial cells. A small amount of LOX-1 has also been identified in macrophages, platelets and VSMCs. Studies showed that ADMA upregulates LOX-1 expression in endothelial cells, VSMCs and macrophages, which suggested that ADMA is related to As closely. In the present study, we used methods of molecular biology to interfere cultured rat celiac macrophages pretreated by oxLDL with ADMA, and then observed the expressions of iNOS and LOX-1, so as to discuss the mechanism of macrophages transforming to foam cells. By measuring rat aorta lipidosis and expression of iNOS and LOX-1 after being fed by ADMA , to probe into the effect and mechanism of ADMA in atherogenesis. We hope to provide a reliable clue for regarding ADMA as a target of cardiovascular disease's prevention and therapy.Methods1. Study in vitroForty-two Wistar rats were enrolled. Celiac macrophages of rats were gathered and cultured. Groups and interference:①Control group: Macrophages were incubated with PBS solution for 48 hours.②oxLDL group: Macrophages were treated by oxLDL (final concentration 50μg/mL) for 48 hours.③ADMA group: Macrophages were pretreated by oxLDL (final concentration 50μg/mL) for 24 hours, then co-incubated with ADMA (final concentration 15μmol/L) for 24 hours.④0+A+L group: Macrophages were pretreated by oxLDL (final concentration 50μg/mL) for 24 hours, then co-incubated with ADMA (final concentration 15μmol/L) and L-Arg (final concentration 1.2mmol/L) for 24 hours. Each group was repeated for 3 times. Immunocytochemistry staining of macrophages were done. Intracellular cholesterol level, NO level and iNOS activity in medium were measured. Total RNA and protein of macrophages were extracted, expression of iNOS, LOX-1 mRNA and protein were analyzed by semi-quantified RT-PCR and Western-blot respectively, ratβ-actin were used as internal standard.2. Study in vivoForty-eight Wistar rats were enrolled and divided into four groups randomly.①Control group (n=8): Rats were fed by normal feedstuff and water.②High fat diet group (n=12): Rats were fed by high fat feedstuff and normal water.③ADMA group (n=14): Rats were fed by high fat feedstuff and normal water, ADMA (0.2mg/Kg/d) was infused into the stomachs once a day.④ADMA +L-Arg group (n=14): Rats were fed by high fat feedstuff, water with 3% L-Arg , ADMA (0.2mg/Kg/d) was infused into the stomachs once a day. Eighteen weeks later, serum levels of cholesterol, triglyceride, NO and iNOS were examined respectively, slices of aorta were inspected. Total RNA and protein of aorta were extracted, expression of iNOS, LOX-1 mRNA and protein were analyzed by semi- quantified RT-PCR and Western-blot respectively, normalized by the internal control geneβ-actin.Results1. Study in vitro①Intracellular cholesterol level in oxLDL group and ADMA group were higher than that in control group (p<0.01, either), it increased significantly in ADMA group compared with oxLDL group (p<0.05), but in O+A+L group it was lower than that in ADMA group (p<0.05).②NO level in ADMA group dropped down markedly compared with control group and oxLDL group (p<0.01 or p<0.05), it was much higher in O+A+L group than that in ADMA group (p<0.05).③INOS activity increased in ADMA group compared with control group and oxLDL group (p<0.05, either), but decreased significantly in O+A+L group compared with ADMA group (p<0.05). Bivariate correlation analysis showed that NO and iNOS was negative related (r=-0.697, p=0.012).④Expression of iNOS mRNA and protein increased in ADMA group compared with control group and oxLDL group (p<0.01, either), but decreased in O+A+L group compared with ADMA group (p<0.01, either).⑤Expression of LOX-1 mRNA and protein increased in ADMA group compared with control group and oxLDL group (p<0.05, either), but decreased in O+A+L group compared with ADMA group (p<0.001 or p<0.05).2. Study in vivo①Aorta slices were stained by sudauⅢ. Inspected by optic microscope, the intima in control group was smooth and integrated, without lipid sediment. There were small amount of lipidosis in high fat diet group. In ADMA group, the intima was broken, with obvious lipidosis, VSMCs proliferation of media was marked and the array was irregular. In ADMA +L-Arg group, the intima was intact, lipidosis and VSMCs proliferation were lower than that in ADMA group.②Serum levels of NO and iNOS in ADMA group elevated compared with high fat diet group and control group (p<0.01or p<0.05), but the levels dropped down in ADMA +L-Arg group compared with ADMA group (p<0.05). Bivariate correlation analysis showed that NO and iNOS was positive related (r=0.512, p=0.003).③Serum levels of TG and TC in high fat diet group, ADMA group and ADMA +L-Arg group were higher than those in control group (p<0.01, either).④INOS mRNA and protein expression of aorta enhanced notably in ADMA group compared with control group and high fat diet group (p<0.05 or p<0.001), but they reduced in ADMA +L-Arg group compared with ADMA group (p<0.01, either).⑤LOX-1 mRNA and protein expression of aorta increased in ADMA group compared with high fat diet group and control group (p<0.01 or p<0.001), but dropped dramatically in ADMA +L-Arg group compared with ADMA group (p<0.01 or p<0.05). Conclusions①ADMA promotes celiac macrophages of rats to intake oxLDL and induce foam cell formation.②ADMA accelerates atherogenesis in high fat diet rat.③Upregulating iNOS and LOX-1 expression might be one of the underlying mechanisms of the process. Supressing the action of ADMA may be an effective target of As prevention.
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