| Background and ObjectivesThe study of pathogenesis of atherosclerosis (AS) has lasted more than one century. There are four hypotheses: lipid infiltration hypothesis, platelet aggregation (PA) and thrombosis hypothesis, vascular smooth muscle cell (VSMCs) clone hypothesis and response-to-injury hypothesis. In recent years, AS has been identified to resemble some fundamental pathological manifestations of inflammation including alteration, exudation and proliferation. In the development of AS, cell-cell interaction is similar to that in chronic inflammation diseases such as rheumatoid arthritis, chronic pancreatitis, hepaticcirhosis, etc. With the identifying of inflammatory cells and mediators, AS is no longer considered as a simple disease of fatty substance accumulation in the artery wall, but as a progressive inflammatory reaction, which is consistent with the general rule of inflammation. Based on his response-to-injury hypothesis, professor Ross proposed that AS is an inflammation disease. Presently, many inflammatory mediators in AS have been discovered such as PDGF-β,bFGF, TGF-βand VEGF.FIZZ1 (Found in Inflammatory Zone 1) was discovered by EST in 2000 and identified to be a new hypoxia derivation mitgenic factor associated with inflammation. It is a secreting protein, with the molecular weight of 9.4KD, which has the function of enhancing the proliferation of pulmonary artery VSMCs, contracting blood vessel, promoting the neogenesis of blood vessel, and stimulating myofibroblast differentiation. FIZZ1 can be detected in the stromal blood vessel of adipose tissue and alternatively activated macrophages, as well as anoxic pulmonary vessel wall. Because there are Th2 cytokines and generous alternatively activated macrophages in atherosclerotic plaque, and atherosclerotic vessel wall is usually hypoxic, we presume that FIZZ1 may exist in atherosclerotic plaque. In this study, we detected the expression of FIZZ1 in atherosclerotic plague by immunohistochemistry method, explored the effect of FIZZ1 on the proliferation of VSMCs and the expression of VSMCs scavenger receptor A (SR-A), and investigated the effect of Simvastatin on the expression of FIZZ1 in atherosclerotic plaque. MethodsThe first part: sixteen C57BL/6J ApoE-/- mice were fed with high fat diet while 12 C57BL/6J mice were fed with normal diet. Twenty-four week later, all the mice were executed for the collection of aortas which were dissected from aortic root to abdominal aorta. Then the aortic sections were imbedded with paraffin for HE dying and FIZZ1 immunohistochemistry. At the same time, the expression of FIZZ1 mRNA in atherosclerotic plaque was detected by RT-PCR. In vitro, after Th2 cytokine was used to stimulate cultural VSMCs and macrophage, the expression of FIZZ1 mRNA and protein were detected by RT-PCR and laser confocal microscopy, respectively. The second part: The cultural VSMCs were stimulated with FIZZ1 at different final concentrations of 3×10-6mmol/L, 9×10-6mmol/L and 2.7×10-5mmol/L respectively. Then the effect of FIZZ1 on the proliferation of VSMCs was detected by 3H-TdR incorporation and MTT. After the cultural VSMCs were stimulated with ox-LDL and FIZZ1 at different final concentrations of 3×10-6mmol/L, 9×10-6mmol/L, 2.7×10-5mmol/L, respectively. Laser confocal microscopy was applied to ascertain and locate the expression of SR-A. Western-blot and flow cytometry were used to detect the effect of FIZZ1 on the expression of SR-A in VSMCs, and meanwhile, flow cytometry was used to detect the effect of FIZZ1 on the expression of CD36 in macrophages.The third part: The animal models of AS and hyperlipoidemia were established, and then interfered with Simvastatin. 21 weeks later, the effect of Simvastatin on the expression of FIZZ1 was evaluated by RT-PCR and immunohistochemistry. The morphology of atherosclerotic plaque was observed.Results1. After 24 weeks, AS emerged obviously in the aortic root in ApoE-/- mice fed with high fat diet, and the area of plaque was fairly large. The expression of FIZZ1 was detected in atherosclerotic plaque by immunohistochemistry and RT-PCR. In the normal artery of C57BL/6J wild type mice, FIZZ1 was negative. Th2-type cytokine can induce the expression of FIZZ1 in macrophages, but not in VSMCs.2. The proliferation of VSMCs was detected by MTT. Compared to the control group, the A value of FIZZ1 3×10-6 mmol/L group increased obviously (P<0.05). Compared to FIZZ1 3×10-6 mmol/L group, the A value of FIZZ1 9×10-6 mmol/L group increased obviously (P<0.05). Compared to FIZZ1 9×10-6 mmol/L group, the A value of FIZZ1 2.7×10-5 mmol/L group increased obviously (P<0.05). These results indicated FIZZ1 enhance aorta VSMCs proliferation, which was also observed by 3H-TdR incorporation.3. SR-A positive expression was found in VSMCs treated with ox-LDL after 24 hours, which located mainly in cell membrane by laser confocal microscopy. SR-A protein was also detected by Western - blot.4. There were significant differences in SR-A positive rates between FIZZ1 3×10-6 mmol/L group and the control group by flow cytometry (P<0.01). The same results were found between FIZZ1 9×10-6 mmol/L group and FIZZ1 3×10-6 mmol/L group, between FIZZ1 2.7×10-5 mmol/L group and FIZZ1 9×10-6 mmol/L group, too. These results indicate FIZZ1 enhance the expression of SR-A induced by ox-LDL in VSMCs.5. FIZZ1 had no effect on the expression of scavenger receptor CD36 in cultured macrophages, which reveals that it couldn't interfere with the ox-LDL uptake of macrophages and atherosclerosis.6. After 21 weeks, AS emerged obviously in the aortic root in ApoE-/- mice fed with high fat diet, and lipid metabolism in the mice disordered. FIZZ1 in atherosclerotic plaque was positive by immunohistochemistry. Simvastatin could decrease the expression of FIZZ1 mRNA and protein in atherosclerotic plaque (P<0.05).Conclusions1. FIZZ1 can express in the atherosclerotic plaque in C57BL/6J ApoE-/- mice, but not in the normal artery of C57BL/6J wild type mice.2. Th2-type cytokine can enhance the expression of FIZZ1 in mcrophages, but not in VSMCs. Th2-type cytokine is one of the probable factors which induce the expression of FIZZ1 in atherosclerotic plaque.3. FIZZ1 can stimulate the proliferation of VSMCs obviously.4. FIZZ1 had no effect on the expression of scavenger receptor CD36 in cultured macrophages. FIZZ1 can promote the development of AS by the up-regulation of the expression of SR-A and the phagocytose of fatty substance in VSMCs.5. Simvastatin can decrease the area of plaque and the expression of FIZZ1 in atherosclerotic plaque obviously, which may be a mechanism of anti-AS drug.
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