| Chapter one:The expression of MCPIP1on the thoracic aorta of mice with atherosclerosisPurpose:Monocyte chemotactic protein1induced proteins (MCPIP) is a newly discovered CCCH-zinc finger protein family. A large number of studies have shown that MCPIP1may play important regulatory roles in many aspects, including inflammation, immune homeostasis, cell differentiation, autophagy and apoptosis. Atherosclerosis is a chronic inflammatory disease. However, the association of MCPIP1and atherosclerosis has not been explored in the previous stuides. So the aims of the current study were to investigate:1) whether MCPIP1expression is associated with the pathogenesis of atherosclerosis by measuring MCPIP1expression in mouse atherosclerotic lesions.2) localization of MCPIP1in mouse thoracic aorta.Methods:C57BL/6and apoE-/-mice were fed with high cholesterol diet for8weeks to induce atherosclerosis in thoracic aorta. Then the experiments were performed as follows.(1) Aortas were stained with Sudan Ⅲ and H&E to determine the plaque formation in atherosclerotic lesion.(2) MCP1levels in serum were measured by ELISA.(3) Western blot was used to evaluate MCPIP1protein expressions in mouse thoracic aorta.(4) the localization of MCPIP1was determined with immunofluorescence staining. Result:After8weeks of high-cholesterol diet, compared with C57BL/6wild-type mice, apoE-/-mice showed significantly higher serum concentrations of TC and LDL-C. Also, the serum concentrations of HDL-C were significantly higher in apoE-/-mice. Based on Sudan III and H&E staining, thoracic aortic plaque area were significantly higher in high-fat fed apoE-/-mice compared with that of C57BL/6wild-type mice. ELISA analysis showed that serum MCP1levels were higher in apoE-/-mice compared with C57BL/6wild-type mice. Western Blot analysis revealed that MCPIP1protein decreased in the aorta from apoE-/-mice compared with that from C57BL/6wild-type mice. Immunofluorescence staining showed that most of MCPIP1positive cells were smooth muscle cell specific marker SM-a-actin positive. Some CD31positive cells also expressed MCPIP1. However, MCPIP1was mainly co-localized with SM-a-actin positive cells in mouse thoracic aorta. Conlusion:Our results demonstrated that the expression of MCPIP1protein significantly decreased in the aorta from apoE-/-mice, compared with that from C57BL/6wild-type mice. MCPIP1was expressed both in aortic vascular smooth muscle cells and endothelial cells. However, smooth muscle cells were the major population responsible for MCPIP1expression in mouse aorta.Chapter two:The effects of MCPIP1on the expression of vascular smooth muscle cell phenotype markersPurpose:In the previous study, we found that the mice with atherosclerosis has significantly lower expression of MCPIP1protein than the same age wild-type mice. MCPIP1was mainly expressed in the vascular smooth muscle cells. Most of cultured VSMC was thought to be poorly differentiated synthetic phenotype. Our preliminary study also found that only a very small amount of MCPIP1protein expressed in the cultured VSMC. It is well known that vascular smooth muscle cell phenotype transformation is closely related to atherosclerosis. We therefore speculate MCPIP1may regulate SMC phenotypic and thus affect the occurrence and development of atherosclerosis. Therefore, the main objective of this study was to investigate the effects of MCPIP1on the expression of vascular smooth muscle cell phenotype markers and to further explore the possible mechanism.Methods:The rat thoracic aorta vascular smooth muscle cells were evaluated with immunofluorescence. The human and rat smooth muscle cells were treated with different concentrations of MCP1(0,50and100ng/ml) for24hours. Western Blot and qPCR were used to evaluate the protein and mRNA expression of intracellular MCPIP1, myocardin and its downstream target genes SM-a-actin and SM22. hSMCs were transfected with a recombinant adenovirus carrying MCPIP1for48h, Western Blot and qPCR were used to evaluate the protein expression of intracellular MCPIP1, myocardin and its downstream target genes SM-a-actin and SM22.Results:Compared with the control group (0ng/ml MCP1),100ng/ml MCP1treatment induced significantly higher MCPIP1mRNA and protein expression in human and rat vascular smooth muscle cells. Compared with the control group (0ng/ml MCP1),100ng/ml MCP1treatment induced significantly higher mRNA and protein expression of myocardin, SM-a-actin and SM22in human and rat vascular smooth muscle cells. Compared with the control group and the Ad-GFP group, the protein expression of MCPIP1significantly increased in the recombinant adenovirus carrying MCPIP1hSMCs group. In addition, Ad-MCPIP1group showed significantly higher protein expression of SM-a-actin, SM22and myocardin.Conclusion:MCPIP1can promote the expression of VSMC contractile phenotype markers SM22, SM-a-actin. In addition MCPIP1can induce the upregulation of myocardin, which may be involved in mediating MCPIP1on VSMC phenotype regulation.Chapter three:The effect of MCPl on the contractility of cultured arterial ring and it related mechanismsPurpose:We previously showed that MCP1could induce the expression of MCPIP1protein in cultured VSMC, accompanied by the expression of VSMC contractile phenotype markers SM22,SM-α-actin upregulated, suggesting MCPIP1be involved in SMC phenotype modulation. Therefore, the purpose of the current study was to further investigate the effect of MCP1on smooth muscle contractility after serum-free organ culture, and explore its potential mechanisms with focus on the expression of MCPIP1.Methods:Rat thoracic arteries were harvested under a dissection microscope. The aortas were then cut into2mm cylindrical segments and divided into three groups as follows:1) fresh sample;2) incubated in200μl serum-free Dulbecco’s Modified Eagle Medium (DMEM) without MCP1;3) incubated in200μl serum-free Dulbecco’s Modified Eagle Medium (DMEM) with100ng/ml MCP1. Then the contractility of the samples were evaluated with the wired myograph. The expressions of MCPEP1, myocardin, SM-α-actin, SM22were determined both by quantitative real-time PCR and Western Blot analysis.Results:In the tissue myography, compared with fresh sample, there was significantly lower contractility in the cultured samples without MCP1. However, the contractility was partilly rescued by adding MCP1into MEM. Compared with fresh sample, MCPIP1protein and mRNA levels were significantly lower in the cultured samples without MCP1. However, the MCPIP1protein and mRNA levels was partilly rescued by adding MCP1. Myocardin protein and mRNA levels were significantly lower in the cultured samples without MCP1. However, the myocardin protein and mRNA levels was partilly rescued by adding MCP1. The protein and mRNA levels of SM-α-actin and SM22were significantly lower in the cultured samples without MCP1. However, the protein and mRNA levels of SM-α-actin and SM22were partilly rescued by adding MCP1.Conclusions:The expression of MCPIP1decreased during serum-free organ culture of rat thoracic arteries, which may be at least partly responsible for the decreased smooth muscle contractility and perhaps due to the decrease in expression of myocardin and its downstream target genes including SM-α-actin and SM22. |
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