The Effect And Mechanism Of Macrophage Migration Inhibitory Factor In Atherosclerosis

Atherosclerosis is an increasingly important disease worldwide. Atherosclerosis may cause clinical effects symptoms by coronary artery stenosis, producing stable angina or dramatic rupture, producing acute coronary syndrome,such as unstable angina,myocardial infarction or sudden death. Vulnerable atherosclerotic plaques have increased inflammation. Vulnerable plaques usually have a large soft lipid core with a thin fibrous cap. The fibrous cap is formed of collagen and vascular smooth muscle cells and endothelial lining, separating the thrombogenic lipid core contents from the blood in the lumen.The fibrous caps of ruptured atherosclerotic plaques have more macrophages than those of non-ruptured atherosclerotic plaques.Macrophages have numerous mechanisms that may promote atherosclerosis and plaque rupture. Atherosclerotic plaques contain a characteristic inflammatory infiltrate,including monocytes,numerous monocyte-derived macrophages, modified lipid-laden macrophages (foam cells) , and T-lymphocytes. Macrophages are recruited and activated by many signals. Macrophage recruitment by abnormal endothelium overdeveloping atherosclerotic plaques, is aided by endothelial expression of adhesion molecules. MCP-1 may attract macrophage recruitment in atherosclerosis. Macrophages are essential to atherosclerotic in inflammation response.Macrophage activation stimuli is associated with oxidized low density lipoprotein as atherosclerotic risk factors. Thus, inflammation plays an important role to all stages of atherosclerosis. Macrophage migration inhibitory factor may be an important mediator of various inflammatory diseases. The macrophage migratory inhibitory factor (MIF) has been shown to play an important role in inflammatory and immune responses. MIF is strongly expressed in atherosclerotic lesions in humans, and its expression is markedly increased in vascular endothelial cells and smooth muscle cells in atherosclerosis-prone rabbits. MIF exerts its atherogenic properties involved chemokine-like functions.MIF promotes local infiltration of inflammatory cells, including neutrophils and macrophages and their migration to the site of inflammation. MIF also causes cell activation and expression of inflammatory cytokines, including IL-1, and TNF-a, and adhesion molecules,such as ICAM-1 and VCAM-1. The inhibition of MIF using neutralizing antibodies led to plaque regression and reduced monocyte and T-cell content of plaques in mice. MIF controls leukocyte recruitment in atherogenesis.The experiment takes rat to make atherosclerotic model and they were interfere by Fasudil (Rho kinase inhibitor). Detect the expression of MIF and Rho kinase by immunohistochemistry and demonstrate the involvement of MIF in the development of atherosclerotic. It may provide a new therapeutic target for the future treatment of cardiovascular.Methods: 30 healthy male Wistar rats weight 220-250g were divided into 3 groups randomly: 1. normal control group (10), 2. angiosclerosis group, (10) 3. Fasudil group (10). The normal control group was offered normal diet and then undertook fake sacculus proprius damage.Each rat of the other two groups was treated with one intramuscular injection of vitamin D3 (3×105u/kg) at the beginning of the experiment. The rats were bred in the normal diet with addition of 2%cholesterol, 0.5%sodium cholate, 0.2%PTU, vitamin D3 (1.25×106u/kg feed) and 3% lard and then do the factitious sacculus injury opration seven days after the beginning of the esperiment. Weighed every three weeks and given the above-mentioned dose vitamin D3 in right lower linbs by intramuscular injection. Fasudil gropes was given Fasudil 5mg/kg body weight, 2 times a day by intraperitoneal injection. All the rats were weighed before the experiment and every 3 weeks in the experiment, until the ninth week they were detected serum lipid level by collecting blood on fasting serum. Then they were killed and their artery tissues were fixed with 4% ormaldehyde solution for arterial tissues HE staining and immunohistochemical analysis. The process of sacculus-impairment was as follows: The rats were anesthetized with pentobarbitol sodium 30mg/Kg by abdominal cavity. After conventional disinfection, the left skin of neck were cutdown above the breast bone for about 1 cm, and disconnect the left common carotid artery, ligate the distal end of carotid artery, the proximal part blood stream was blocked by operation line. Nitroglycerin were dropped on the arteria carotis communis, cut open the artery anterior wall, the balloon catheter with guide wire were inserted into the proximal part (the balloon inside diameter is 1.5mm, long 20mm). The balloon was posted in the left carotid artery approximately 8cm, then the balloon was infunded 0.2ml Sodium Chloride.This procedure was repeated 3-4cm. When dragging the balloon, we can feel a slight resistance. Then the balloon was posted 8cm after draining. Draining and backswing were repeated 3 times. Then drawed the balloon, the carotid artery was ligated at proximal part. After sterilisating, subcutaneous tissue and skin were suturated. Notho-balloon- operation was cutting down the left skin of neck, and disconnect the left carotid arteries, ligate the proximal part of artery and distal end of carotid artery, shearing artery and did not insert balloon. Sample deal: The rats were anesthetized with pentobarbitol sodium 30mg/Kg by abdominal cavity narcotization after 9 weeks,then they were killed after hemospasia from crotch of arteria kidney. Got out all the artery after cutting the abdomen and breast, slice about 1cm artery organization about 1cm below the aortic arch, then the arteries were fixed by 0.4% formaldehyde solution immediately for HE staining and immunohistochemistry detection. The expression of MIF and Rho kinase in the vascular tissues were detected by immunohistochemistry. And compare fasudil group with Valsartan group of the influence on the production of MFI and Rho kinase by semiquantitative method. Measurement data statistics used SPSS11.5 statistical software and was demonstrated by x±s, the mean for every team were analysised by Student-Newman-Keuls, P<0.05was considered significant.Results:The level of lipids: Compared with the others two groups, TC,TG and LDL-C of the arteriosclerosis group were singnificantly higher (P<0.05), there is no singnificantly difference of TC,TG and LDL-C between fasudil grope and the normal control group(P>0.05). Compared with the others two groups, HDL-C of the arteriosclerosis group were singnificantly lower (P<0.05), there is no singnificantly difference of HDL-C between fasudil grope and the normal control group (P>0.05). HE staining: The intimal, membrane and outer membrane of the normal control grope are clarity; the endothelial cells were integrity;the smooth muscle cells in the middle were normal without proliferation; the outer layer is loose connective tissue. The arteriosclerosis group has the typical plapue formation, the endomembrane was thickening with eccentricity, the plapue were filled with foam cells, lipid deposition, small amounts inflammatory cells and the hyperplatic smooth muscle cell on the surface of endomembrane, the smooth muscle cell of medial were obviously hyperplasy, with punctiform and lamellar calcification; there are no accrementition in endomembrane and the medial in Fusadil group, which have few fatty substance deposition and foam cells forming and inflammatory cell infiltration between the endothelium cell and smooth muscle cell. Semiquantitative method of immunohistochemistry: The expression of MIF in arteriosclerosis group were significantly higher than the normal control group and Fasudil group (P<0.001).The expression of Rho kinase in the arteriosclerosis group were significantly higher than the normal control group and Fasudil group (P<0.05).Conclusions: 1. In the process of atherosclerosis, the expression of MIF and Rho kinase are increased in atherosclerotic lesions. 2. MIF maybe plays a role in atherosclerosis by activating the Rho/Rho kinase pathway. 3. The inhibitor of Rho kinase fasudil inhibited the expression of MIF and Rho kinase in atherosclerotic lesions. 4. The inhibitor of Rho kinase has the role of inhibiting arteriosclerosis.