Apolipoprotein E Knockout Rat Model Of Atherosclerosis Induced By Partially Ligating Carotid Arteries And The Correlation Analysis Of MTOR

ObjectiveAt present,the ApoE^-/-mice is successfully established animal model for the research of atherosclerosis.However,the distribution of the atherosclerotic plaques throughout the arterial tree and the cells composition of ApoE^-/-mice plaques differ from the atherosclerosis patients greatly.Additionally,the small size of the mice is not suitable for intravascular angioplasty and stenting for testing the novel drugs and devices.Such as pigs and dogs,the large animals are currently applied in the translational vascular research,but their price is so high.The rat in the study of cardiovascular diseases,especially hypertension and stroke,is excellent and suitable animal model.The physiology of rat is monitored more easily and more similar to the corresponding human condition than mouse.The size of rat is more suitable for performing the intravascular interventional procedures.But so far,unfortunately,there is a lack of rat atherosclerotic model.Although at the early stage,several groups made attempts to use different methods to establish rat model of atherosclerosis,but the results are still uncertain.The rats can be induced to develop hypercholesterolemia,but cannot develop the atherosclerosis plaques by diet.Thus,the rat is considered as to be hypercholesterolemia resistant.The diet-induced hypercholesterolemia is still not high enough to lead to the atherosclerosis,which may be another possible reason.Therefore,we tried to create ApoE^-/-rats by taking advantage of the transcription activator-like effector nuclease?TALEN?-mediated knockout approach and treated them with high-cholesterol diet?HCD?for the purpose of obtain a spontaneous hypercholesterolemia rat.Moreover,we established the rat model of atherosclerosis based on both partial ligation of carotid artery and extremely high cholesterol levels.Mammalian target of rapamycin?mTOR?has a key role in regulating the proliferation of smooth muscle cells,and at recent it is believed that the smooth muscle cells proliferation play important role in atherosclerosis.Our previous study found that the expression of mTOR associated with atherosclerotic plaque load in mice.Therefore,in this study we try to explore further the correlation between the expression level of coronary heart disease patients peripheral blood lymphocytes mTOR and coronary artery lesion degree.MethodsPart?:Establishment of ApoE^-/-rat atherosclerosis model1.Animals and generation of knockout ratsMale Sprague-Dawley?SD?rats were experimented as control group and for genetic background and the transcription activator-like effector nuclease?TALENs?-mediated knockout approach was used to generate the apolipoprotein E?Apo E?-deficient rat.2.High cholesterol diet feedingTwo genotypes ApoE^-/-rats and wildtype?WT?rats?6-8 weeks old?were divided randomly into normal diet or HCD groups.The composition of HCD was 67.8% standard chow,10% lard oil,1.5% cholesterol,0.5% sodium cholate,15% egg yolk powder,0.2% methylthiouracil,and 5% sucrose.3.Physiology measurementAfter diet treatment for 12 weeks,the body weight,random blood glucose levels,the tail-cuff heart rate and blood pressure were examined.4.Analysis of serum lipids and inflammatory factorsSerum triglyceride?TG?level,total cholesterol?TC?level,low-density lipoprotein cholesterol?LDL-c?level,and high-density lipoprotein cholesterol?HDL-c?level were measured.Serum levels of tumor necrosis factor-a?TNF-a?,interleukin?IL?-1b,IL-6 and IL-10 were detected.5.En face oil red o staining of aorta6.Partial ligation of carotid arteryAfter fed with HCD diet for 10 weeks,two genotypes rats were intervened with partial ligation of left carotid artery,and then accepted additional 2 weeks HCD diet.7.Histology of aortic roots and carotid arteries8.Real-time q PCR8.1 Examination of mRNA levels of aortas MCP-1,VCAM-18.2 Examination of mRNA levels of carotid arteries MCP-1,VCAM-1 and mTORPart?:The correlation analysis between peripheral blood lymphocytes mTOR mRNA level and coronary artery lesions1.Collect the all clinical datas of the patients accepted CAG2.Examination of mRNA levels of peripheral blood lymphocytes Mtor3.The correlation analysis between peripheral blood lymphocytes mTOR mRNA level and coronary artery lesionResults1.Successful establishement of ApoE^-/-ratThe ApoE^-/-rat was established in a genetic background of SD rat.The genotype of the rat was determined by gene sequencing.The ApoE^-/-rats demonstrated the lack of a 14-base pair segment.2.Physiology of ApoE^-/-ratsThe body weight was approximate between ApoE^-/-rats and WT rats,but was significantly lower after fed with HCD for 12 weeks.Alike to previous studies,HCD only caused slight elevation in TC and LDL-c in WT rats.Excitingly,HCD extremely increased the TC and LDL-c levels in ApoE^-/-rats.3.ApoE^-/-rat is resistant to hypercholesteremiaWe stained the en face preparations of whole aortas and primary branches with oil red O?ORO?and failed to detect the lipid-rich atherosclerotic plaques in normal-or HCD-fed ApoE^-/-rats and WT rats.Moreover,the H&E-stained aortic root sections can't show the perivalvular lesion as well.HCD diet increased the serum TNF-a,IL-1b,IL-6 and IL-10 levels in WT rats,but not in ApoE^-/-rats.In aortic tissues,the mRNA expressions of MCP-1 and VCAM-1 were increased slightly by HCD feeding in ApoE^-/-rats.4.Partial ligation of carotid arteries induced atherosclerosis in ApoE^-/-ratsTwo weeks after the ligation,similar size plaques were found in the left carotid arteries from WT and ApoE^-/-rats.Masson trichrome stained-sections showed a little collagen deposition was showed in the plaques.ORO staining detected large lipid positive areas in plaques from ApoE^-/-rat,but failed to detect lipid positive area in lesions from WT rats.The apoptotic cells were showed in the lesion from ApoE^-/-rats but none in WT rats.Immunofluorescence staining showed that there were a lot of CD68-positive macrophages in the plaques from ApoE^-/-rats but was absent in the lessions from the WT rats,which contained mainly vascular smooth muscle cells?SMCs?.The neovessels were detected in lesions from ApoE^-/-rats but not in WT rats.The mRNA expressions of MCP-1?VCAM-1and mTOR were extremely elevated in left carotid arteries compared with right carotid arteries.5.The comparison of clinic datasThe age,BMI,TG,TC,LDL-c,and mTOR expression levels in coronary stenosis group were significantly higher than control group?P<0.05?.6.The correlation analysis between peripheral blood lymphocytes mTOR mRNA level and coronary artery lesions.The levels of mTOR?TG?TC and age have positive correlation with the Gensini score?r=0.343,0.254,0.236,0.207,P<0.01?Conclusion:The ApoE^-/-rat is a novel model for hypercholesteremia and could also be used for research model of atherosclerosis by partially ligated carotid arteries.We find that its mTOR expression level rise significantly,and the patients' peripheral blood lymphocytes mTOR expression levels have the positive correlation with the coronary lessions.