Correlation Of AHNAK2 Gene Variation And Atherosclerosis Formation And Validation In Myocardial Infarction Model

Objective:In this study,by establishing a mouse model of AHNAK2 gene knockout,the phenotypic conversion of AHNAK2 gene with glucose and lipid metabolism,carotid intimal injury repair,and vascular smooth muscle cells was investigated from the animal and cell level,so as to investigate the correlation with the pathogenesis of coronary atherosclerotic heart disease,and finally through The Adipose-Derived Stem Cells model of myocardial infarction in the pathogenesis of coronary heart disease.The mechanism of AHNAK2 gene in the pathogenesis of CAD was explored from the cellular level,animal level and molecular mechanism.At the animal experimental level,we establish the AHNAK2^-/-On the basis of carotid injury model,we observed the carotid intimhyperplasia of AHNAK2 knockout mice and MT mice.At the cell level,we selected acetylated LDL,LDL and oxidized LDL by isolating vascular smooth muscle cells,so as to explore the correlation of the formation of smooth muscle cell-derived foam cells by adding H₂O_2.Thus,to simulate the effect of reactive oxygen species on the survival of smooth muscle cells,and further verify its transformation phenomenon.final appeal Adipose-Derived Stem Cells transplantation model of AHNAK2^-/-comprehensively evaluated its role and molecular mechanism in the pathogenesis of coronary heart disease.Methods:Part 1:Choose the AHNAK2 of 3 weeks of age^+/-C57BL/6 JSPF heterozygous mice were identified for breeding and genotype and the AHNAK2^-/-A total of 20 C57BL/6J mice were raised on a high-fat diet to detect lipid metabolism,including triglycerides,total cholesterol,free fatty acids,LDL-cholesterol and glucose.After 4 weeks,a carotid artery injury model was established to measure the area of carotid artery intima and media membrane in each experimental group.Part two:Choose AHNAK2^-/-And carotid tissue from two each of wild-type C57BL/6J mice,The VSMCs cells were obtained and identified by collagenase and elastase digestion,Acylated LDL,LDL and oxidized LDL were subsequently selected for lipophagy experiments,Confirmed its ability to induce the formation of VSMC-derived foam cells,Further evaluation of CD36,SR-A and the mechanism of LOX 1 in foam cell formation by Western blot experiments,Finally,the effects of reactive oxygen free radicals on VSMCs,survival,and cellular transformation phenomena were comprehensively evaluated.Part 3:Isolation and culture of AHNAK2^-/-And adipose stem cells from wild-type C57BL/6J mice,and multidirectional differentiation potential testing,molecular changes in adipose stem cell transplantation after myocardial determined by Western blot through lentiviral transfection of ADSCs carrying Fluc-m RFP-t TK trireporters.Results:Part 1:1)Based on CRISPR/Cas 9 technology,AHNAK2 was obtained by optimizing the breeding conditions of mice^-/-Mice homozygous for the gene deletion and genotyped by PCR;2)compared to WT controls fed RC or HFD,AHNAK2^-/-Mice had significantly reduced weight gain,WT-AHNAK2^-/-Group of mice and the HFD-AHNAK2^-/-Group mice had similar body weight;3)HFD-AHNAK2^-/-The triglyceride levels in the group were significantly lower than HFD-WT mice,statistically significant（P<0.05）;HFD fed Ahnak^-/-Total cholesterol in the group was significantly lower compared with HFD-WT group（P<0.05）;as for LDL-cholesterol,HFD-fed AHNAK2^-/-The level of the group mice was also significantly lower compared with the HFD-WT group,and the difference was statistically significant（P<0.05）,and compared with the HFD-WT mice,HFD-AHNAK2^-/-The group of mice showed lower blood glucose levels under fasting conditions and after glucose administration at 15min,30min,60min,90min and 120min,with statistically significant differences（P<0.05）,but HFD-AHNAK2^-/-The group curve was lower and lower than the HFD-WT group with atypical blood glucose peak,while after insulin injection,insulin versus HFD-AHNAK2^-/-The blood glucose effect of the group was slightly better,only statistically significant at 20min and 120min（P<0.05）;4)and that of the WT group（5447.86±469.51um²）In contrast to the AHNAK2^-/-Group（2155.26±364.40um²）The intima area of the carotid artery decreased with statistically significant differences（P^*<0.01）;The percentage of neointima area/media area（12.19%）was significantly lower compared with the WT group（25.24%）,with a significant difference（P^*<0.01）;AHNAK2^-/-Group medium membrane area（17676.64±2393.82um²）Compared with the WT group（21580.53±3002.87um²）There is a thinning trend.Part II:1)Mouse smooth muscle cells were successfully isolated by tissue block wall adhesion method;2)MT group and AHNAK2^-/-Group after 48h of stimulation for the three lipoproteins,Lipid droplets were present in the cells of VSMCs,However,oil red color increased in VSMCs after ox LDL stimulation,Relatively less,while using LDL,ac LDL,Experiments confirmed that using the same dose at the same time,Strong ability to induce the formation of VSMC-derived foam cells using ox LDL,Following a quantitative analysis of intracellular CE and FC:compared to the Control group,After stimulation with VSMCs 48h at the equivalent dose of 100μg/ml LDL,ac LDL,and ox LDL in the MT group,A significant increase in both the cellular CE and FC content,All were statistically significant（P^*<0.05）,It suggests that VSMCs can indeed absorb various types of lipids to form foam cells.Especially,using ox LDL stimulation,the lipid absorption was significantly increased in AHNAK2^-/-group,Intracellular CE and FC content was also significantly increased compared with the Control group,All were statistically significant（P^*<0.05）;3)Through the correlation experiment of the derived foam cell formation of VSMCs at different doses of ox LDL and ox LDL at different times,The results all confirmed that the induction of VSMC-derived foam cell formation by ox LDL was linear with both the stimulation dose and the stimulation time,And the larger the dose and the longer the time,AHNAK2 The greater the inhibitory effect on the formation of VSMCs derived foam cells;4)The results of the Western blot experiment show that CD36,SR-A,and LOX 1 are both in the MT group and the AHNAK2^-/-Group,the results were positive,and the expression results of LOX 1 protein indicated that its content increased at 48h,meaning that the formation of VSMCs induced foam cells induced by ox LDL may be related to the increased expression of LOX 1,which was statistically significant compared with MT group 0h（P^*<0.05）,whereas the AHNAK2^-/-,However,LOX 1 was significantly reduced at 48h and the expression content at 48 hours from the MT group was statistically significant（P^#<0.05）,which means that the deletion of AHNAK2 gene suppressed the expression of LOX 1,which leads to the formation of foam cells of VSMCs induced by ox LDL;5)to evaluate the effect of reactive oxygen radicals on VSMCs and survival,the results showed that the deletion of AHNAK2 gene may make H₂O₂The reduced possibility,or that the cell lacking the AHNAK2 gene has the ROS clearance ability,may be the result of the smooth muscle cell transition.Superoxide anion fluorescence probe DHE experiment confirmed that AHNAK2 gene deletion can indeed reduce ROS in the environment;6)Western blot test confirmed that smooth muscle cells have a transition after stimulation,while after protein content determination,AHNAK2^-/-The smooth muscle cells of the group did not show excessive transition,suggesting that the absence of AHNAK2 inhibited the transition of smooth muscle cells,while synthetic VSMCs has the ability to synthesize extracellular matrix such as collagen and proliferation,thus participating in the formation of atherosclerotic plaques;Part III:1)The successful isolation of the Adipose-Derived Stem Cells,And performed the validation of the multiple differentiation capacity of adipose stem cells,Differentiated into adipocytes,bone cells and myocardial cells respectively;2);Demonstrated that the transplantation of adipose stem cells into the area of MI still has multiple differentiation potential,And by live-animal tracking technology,We can see that the adipose stem cells are still alive at 28 days after transplantation;3)Calculate and measure the mouse heart-related data by cardiac ultrasound,We found that,when compared to the control group,EDD,ESCs,FS,EF values of the systolic and diastolic function of ADSCs or ADSCs-ko group were statistically significant（P^*<0.05）,It shows that the transplantation of adipose stem cells can improve the cardiac function;4)Microincrease in the infarct region was also similarly confirmed by v WAg immunohistochemical staining in the infarct region,We found that both of these factors,The microvessel density of PBS alone was 63.05±8.8/mm²;The microvascular density in ADSCs group was 159.35±17.34/mm²,Further statistical results indicated that the vessel density was increased in the ADSCs group compared with the PBS group and was statistically significant（P^*<0.05）;5)in the ventricular wall thickness and infarct area data compared with the ADSCs group,AHNAK2^-/-However,the difference was statistically significant;6)TGF-β/Smad signaling pathway and the vascular smooth muscle synthesis phenotype and the expression of TGF-β1,Smad2/3 and OPN in the ADSCs-KO mice,and the expression ofα-SMA and SM22αcompared with the control group.The results were statistically significant（P^*<0.05）,indicating that NF-κB activation could further activate TGF-β/Smad signaling pathway and promote abnormal phenotype conversion in mediated VSMCs.However,the results between ADSCs-KO group and PBS group showed that when the NF-κB activation was inhibited,the ADSCs-KO group showed the opposite trend,and the results were statistically significant（P^#<0.05）.It further showed that the activation of TGF-β/Smad signaling pathway is regulated by NF-κB,and the AHNAK2 gene could inhibit the activation of NF-κB and affect the expression of downstream pathways and proteins,thus inhibiting the transformation of vascular smooth muscle phenotype;7)Western blot in the infarct region showed that the expression levels of VCAM 1 and ICAM 1 in the ADSCs group and ADSCs-KO group,while the cytokine SDF-1 showed similar results.Conclusions:1)The AHNAK2 was found in this study AHNAK2^-/-was also suggested by the mice had body resistance to diet-induced obesity and the reduction of obesity in experimental animals Genes have effects in suppressing obesity,and AHNAK2^-/-mice can show better glucose tolerance and greater insulin responsiveness,which may be involved in the overall regulation of obesity and energy metabolism;2)and finding that AHNAK2^-/-Compared with the WT group,there was a decreasing trend in the percentage of carotid artery intima hyperplasia area and neointima area/media area after injury,It suggests that the AHNAK2gene may aggravate the intimal hyperplasia and hinder the phenotypic transformation of vascular smooth muscle cells;3)It is possible that the AHNAK2 gene has an inhibitory effect on the formation of VSMCs-derived foam cells,Moreover,the formation of VSMC-derived foam cells induced by ox LDL is linear with both the stimulation dose and the stimulation time;4)Loss of the AHNAK2 gene suppressed LOX 1 expression,Thus,ox LDL induced the formation of cell-derived foam cells of VSMCs;5)The AHNAK2gene deletion can reduce the ROS role in the environment;6)The deletion of AHNAK2suppresses the transition of smooth muscle cells,While the synthetic VSMCs has the ability to synthesize extracellular matrix such as collagen and proliferation,Thus participating in the formation of atherosclerotic plaque;7)It is determined that the adipose stem cell transplantation can be differentiated into the myocardial infarction area,into cardiomyocytes,immature myocardial cells,smooth muscle cells and vascular endothelial cells;8)found that AHNAK2 gene deletion was able to inhibit NF-κB nuclear translocation,To inhibit the activation of the NF-κB pathway,And mitigate the activation of the TGF-β/Smad signaling pathway,Reduce the phenotypic conversion of vascular smooth muscle cells;9)Found that AHNAK2 gene deletion may be a key gene in the inhibition of vascular endothelial proliferation.