Research On The Role And Mechanism Of ATF3 In The Progression Of Atherosclerosis

Abstract Objective:Activating transcription factor-3（ATF3）is one of the early response genes activated in response to atherosclerotic stimulation,and may be an important factor in inhibiting the progression of atherosclerosis.In this study,we detected the expression of ATF3 and related factors in human coronary atherosclerotic plaques and knocked down the expression of ATF3 in vivo and in vitro,so as to explore the role of ATF3in atherosclerosis and its relationship with inflammation.Methods:（1）Human specimen experiment:90 samples of coronary artery tissue from autopsy cases were collected from Forensic Judicial Appraisal Center of Guizhou Medical University,which were divided into three groups:32 cases of sudden death with coronary heart disease caused by mechanical injury（CHD group）,36 cases of sudden death of coronary heart disease（SCD group）.And 22 cases were taken as control group（Con group）.The pathological changes of coronary artery were observed by HE staining.And the thickness of coronary intima,fibrous effusion,necrotic focus and degree of vascular stenosis were measured to evaluate the changes of vascular structure.The expression of ATF3 and CD68 in plaque was observed by immunofluorescence double labeling method.Immunohistochemical and Western Blot methods were used to detect the expression and distribution of ATF3,inflammatory factors（CD45,IL-1β,TNF-α）,matrix metalloproteinase 9（MMP-9）and vascular cell adhesion molecule（VCAM1）in coronary artery.Pearson correlation coefficient was used to analyze the correlation between ATF3 protein expression and structure-related indexes,inflammatory factors.To investigate the relationship between the expression of ATF3 and the inflammatory response,structural stability in atherosclerotic plaques.（2）Animal experiment:A total of 32 SPF-grade apolipoprotein gene knockout(Apo E^-/-)mice（8-9 weeks of age）were divided into Con group,AS group,AAV9-e GFP group and AAV9-ATF3 group,with eight mice in each group.After one week of accommodation,the mice in Con group were given normal diet,while those in the other groups were given high-fat diet.After all the mice were fed for 12 weeks,the mice of AAV9-ATF3 group was injected with 5×10¹¹vg/100μl AAV9-ATF3virus through tail vein.The mice of AAV9-e GFP group was injected with 5×10¹¹vg/100μl AAV9-e GFP virus through tail vein,the mice of Con group and AS group were injected with 100μl normal saline through tail vein.The mice of Con group was fed with ordinary feed,and the others were fed with high-fat diet.All the mice were sacrificed after continuous feeding for five weeks under anesthesia.The thoracic aorta near aortic arch of mice was taken for pathological section.Extraction of protein from the remaining vascular tissues of mouse aortic arch,thoracic aorta and abdominal aorta to renal artery bifurcation.Detection of lipid deposition in mouse aortic intima by oil red o staining on the gross and cross sections of mouse aorta.Structural changes of aortic plaques in mice were observed by HE staining,and e GFP transfection carried by AAV9 in mouse aortic plaques was observed by fluorescence microscope.The expression of ATF3,inflammation-related factors（CD45,CD68,IL-1β,TNF-αand VCAM1,MMP-2,MMP-9）,NF-κB signal pathway related factors（IKKβ,p-IKKα/β（Ser176/180）,NF-κB p65 and p-IKK NF-κB p65（Ser536））in mouse aorta were detected by immunohistochemistry and Western Blot.（3）Cell experiment:Target knocking down ATF3 expression in THP-1 cell by siRNA.The THP-1 cells were induced into macrophages by 160nmo1/ml PMA,and further were induced by 80ng/L oxidation of low density lipoprotein（Ox-LDL）to establish foam cell model.Oil red O staining was used to identify lipid deposition in foam cells.The knockdown efficiency of ATF3 gene and the protein expression of NF-κB signaling pathway related factors（IKKβ,p-IKKα/β（Ser176/180）,NF-κB p65and p-NF-κB p65（Ser536））were detected by Western Blot.Results:（1）Human specimen experiment:From the Con group,CHD group to the SCD group,the coronary intima thickness and necrotic lesion thickness gradually increased,and the degree of luminal stenosis increased（P<0.05）.The fibrous cap in the SCD group was thinner than that in the CHD group（P<0.05）.The results of immunohistochemistry showed that there was almost no expression of inflammatory factors（CD45,IL-1βand TNF-α）,VCAM1 and MMP-9 in coronary intima of Con group,but the expression of these proteins was increased in AS plaque of CHD group and SCD group,and the expression of SCD group protein was higher than that in CHD group（P<0.05）,Western Blot also confirmed the above results.The results of immunofluorescence showed that ATF3 and CD68 were partially co-expressed in AS plaque.Immunohistochemistry and Western Blot showed that the expression of ATF3CHD group and SCD group was higher than that in Con group,while the expression of ATF3 protein in SCD group was lower than that in CHD group（P<0.01）.In CHD group and SCD group,the expression of ATF3 protein in AS lesions was negatively correlated with the thickness of coronary intima and necrotic core,and positively correlated with the thickness of fibrous cap,but not with the degree of lumen stenosis.The level of ATF3 protein in AS plaque in the lesion group was negatively correlated with the expression of inflammatory factors（CD45,IL-1βand TNF-α）,MMP-9 and VCAM1 protein,respectively.（2）Animal experiment:e GFP showed uneven and widely distributed green fluorescence expression in aortic plaques,and the transfection rate was about 8%,suggesting that AAV9 was successfully transfected into the aorta of mice.The results of IHC and Western Blot showed that the expression of ATF3 protein was significantly higher than that in AS group and AAV9-e GFP group,while the expression of ATF3 protein in AAV9-ATF3 group was lower than that in AS group and AAV9-e GFP group（P<0.01）.This suggests that ATF3 gene knockdown is effective.The results of oil-red o staining in general combined with frozen sections showed that there was almost no red staining in blood vessels and intima of Con group,but different degrees of lipid red staining could be seen in AS group,AAV9-e GFP group and AAV9-ATF3 group,and the percentage of lipid red staining area in the whole vascular or lumen area was as follows:Con group<AS group,AAV9-e GFP group<AAV9-ATF3 group（P<0 01）.There was no significant difference between AS group and AAV9-e GFP group（P>0.05）.The results of HE staining showed that the intima of aorta was smooth and no AS was formed in Con group,but uneven intimal thickening and lumen stenosis were found in AS group,AAV9-e GFP group and AAV9-ATF3 group.Statistical analysis showed that the plaque area in AAV9-ATF3 group was significantly higher than that in AS group and AAV9-e GFP group,and there was no significant difference between AS group and AAV9-e GFP group（P>0.05）.The results of IHC and Western Blot showed that the expressions of CD45,CD68,IL-1β,TNF-αand VCAM1,MMP-2 and MMP-9proteins in plaques were significantly higher than those in AS group and AAV9-e GFP group（P<0.0l）.After ATF3 gene knockout,the expression of these proteins was further increased compared with AS group and AAV9-e GFP group（P<0.01）,which suggested that the inflammatory reaction in plaque increased after ATF3 gene knockdown.The results of IHC and Western Blot also showed that the expression of p-IKKα/βand p-NF-κB p65 protein in AS group and AAV9-e GFP group was significantly higher than that in Con group（P<0.05）.After ATF3 gene knockdown,the expression of p-IKKα/βand p-NF-κB p65 protein was further increased compared with AS group and AAV9-e GFP group（P<0.01）.IKKα/β,NF-κB p65protein expression had no significant difference between groups（P>0.05）.This suggests that the NF-κB signal pathway is further activated after ATF3 gene knockdown.（3）Cell experiment:The results of oil-red o staining showed that pink or orange granule-like staining could be seen in the cytoplasm of foam cells,which indicated that the foam cells were successfully modeled.The results of Western Blot showed that the expression of ATF3 protein in cells induced by Ox-LDL was significantly higher than that in the group without Ox-LDL induction（P<0.0l）,while the level of ATF3 decreased significantly after siRNA-ATF3 interference（P<0.0l）,which suggested that ATF3 gene knockdown was effective.The results of Western Blot also showed that the expression of p-IKKα/βand p-NF-κB p65 protein in Ox-LDL induced cells was significantly higher than that in non-Ox-LDL induced group（P<0.0l）.After ATF3 knockdown,the expression of p-IKKα/βand NF-κB p65 protein was further increased（P<0.01）,but there was no significant difference in the expression of IKKα/βand NF-κB p65 protein between the two groups（P>0.05）.This suggests that the NF-κB signal pathway is further activated after ATF3 gene knockdown.Conclusion:（1）The occurrence of atherosclerosis can induce the expression of ATF3,and the expression of ATF3 is related to the stability of atherosclerotic plaque.（2）Targeted knockout of ATF3 can promote the expression of many inflammatory genes and promote the process of atherosclerosis,and its mechanism may be through the further activation of NF-κB signal pathway.Therefore,ATF3 may be an important protective regulator of atherosclerosis.（3）The strength of the inflammatory response in atherosclerotic plaques is related to the process of atherosclerosis.The strengthening of the inflammatory response may promote the occurrence of plaque instability.