| Background:Cerebrovascular disease is a common disease in contemporary society,which has the characteristics of high morbidity,disability and mortality in the population.And atherosclerosis is the main cause of the cerebrovascular disease.Atherosclerosis is a chronic inflammatory process,its development process including sedimentation of lipid in blood vessel walls,vascular endothelial cell dysfunction,vascular smooth muscle cell migration and proliferation to the endometrium,aggregation of inflammatory cells in diseased region of the blood vessels,and the formation of foam cells.Atherosclerotic plaques mainly include stable plaques and unstable plaques,also namely vulnerable plaques.Plaque structures include lipid core,fibrous cap,cholesterol crystals,neovascularization,and extracellular matrix.The necrosis of lipid core in plaque,the degradation of extracellular matrix,the thinning and rupture of fibrous cap and neovascularization can all lead to the vulnerability of atherosclerotic plaque,resulting in plaque rupture and triggering cerebral vascular diseases such as thrombosis and stroke.Objective:By studying the negative regulatory effect of TSP-1/CD47/VEGF/VEGFR2signaling pathway on angiogenesis in atherosclerosis（AS）vulnerable plaques,including down-regulation of VEGF expression and downstream phosphorylation of VEGFR2,and further inhibition of angiogenesis.The role of TSP-1/CD47 signaling pathway in atherosclerosis and its regulatory mechanism will be explored.Methods:ApoE-/-male rats are fed with high fat at 8 weeks of age.The abdominal aorta of apoE-/-male rats at different time points（Sham group,4 weeks,8 weeks,10 weeks and12 weeks）were taken to observe the lipid content in oil red staining and the expression of TSP-1,CD47,VEGF and VEGFR2 in western blot.The expression changes of TSP-1,CD47,VEGF and VEGFR2 in western blot after the addition of anti-CD47（B6H12）drug at 6 weeks and 8 weeks of high-fat feeding.In human umbilical vein endothelial cells（HUVECs）,lipopolysaccharide（LPS）was added to form an inflammatory model of HUVECs.After anti-TSP-1（LSKL）,exogenous TSP-1,and anti-CD47（B6H12）drug intervention were added,namely,normal control group（control group）,control+LPS group（LPS group）,LSKL group（A-TSP-1 group）,exogenous TSP-1 group（TSP-1 group）,and anti-CD47 group（A-CD47 group）,the protein expression levels of TSP-1,CD47,VEGF,and VEGFR2 in HUVECs were observed by western blot.Results:1.8 weeks of ApoE-/-male rats aged 8 weeks were fed with high fat,and the abdominal aorta of ApoE-/-male rats at different time points were taken.And the content of lipid in oil red O staining was observed,using the Image Pro Plus 6.0 for the lipid content in the oil red O staining to analysis.According to the results of different experimental group.The lipid content of ApoE-/-in the abdominal aorta of rats reached the peak at 8 weeks,This indicated that the degree of atherosclerosis in the abdominal aorta of mice was the most serious at 8 weeks,and lipid content was significantly increased（P<0.001）.and after adding the anti-CD47 drug intervention,The lipid content in abdominal aorta of rats fed with high fat for 8 weeks was significantly increased（P<0.01）.The lipid content in the abdominal aorta of rats fed with high fat for 8 weeks was also significantly increased compared with 10 and 12 weeks（P<0.01,P<0.001,respectively）.2.ApoE-/-male rats of 8 weeks of age were fed with high fat and divided into experimental groups to obtain the abdominal aorta.After the total protein was extracted,the protein expression levels of HIF-1,TSP-1,CD47,VEGF and VEGFR2 were detected and analyzed by western blot.GAPDH was used as the internal reference protein and its ratio was the relative protein expression level.The results showed that the protein expression of each factor in the ApoE-/-abdominal aorta group was as follows:HIF-1 was the highest expression at 8 weeks with high fat feeding.Compared with the control group,the protein expression level at 8 weeks was significantly increased（P<0.001）.Compared with the high fat feeding for 12 weeks,the protein expression of HIF-1 at 8 weeks was significantly increased（P<0.01）.The highest expression level of TSP-1 was observed after 10 weeks of high fat feeding.Compared with high fat feeding for 4 weeks,the protein expression of TSP-1 was significantly increased at 10 weeks（P<0.001）.Compared with high fat feeding for 12 weeks,the protein expression of TSP-1 was significantly increased at 10 weeks（P<0.05）.The expression of CD47 was the highest in high-fat diet for 8 weeks.Compared with the control group,the protein expression of CD47 was significantly increased at 8 weeks（P<0.05）.Compared with the high fat feeding for 12 weeks,the protein expression of CD47 was also significantly increased at 8 weeks（P<0.05）.The highest expression level of VEGF was observed in high-fat diet for 8 weeks.Compared with the control group,the protein expression of VEGF was significantly increased at 8 weeks（P<0.05）,and the protein expression of CD47 was also significantly increased at 8 weeks of high fat feeding compared with that at 10 and 12 weeks with high fat feeding（P<0.05）.The expression level of VEGFR2 was highest in high-fat diet for 8 weeks.Compared with the control group,VEGFR2 protein expression was significantly increased at 8 weeks（P<0.01）,CD47 protein expression was significantly increased at 8 weeks（P<0.05）compared with the high fat feeding group for 12 weeks.And CD47 protein expression was significantly increased at 8 weeks（P<0.001）compared with the high fat feeding group for 12 weeks.3.The 8-week-old male apoE-/-rats were fed with high fat,and anti-CD47（B6H12）was added at 6 and 8 weeks of high fat feeding,respectively.The abdominal aorta of different groups of rats were obtained after 2 weeks（8 and 10 weeks）.After the total protein was extracted,the protein expression levels of HIF-1,TSP-1,CD47,VEGF and VEGFR2 were detected and analyzed by Western blot.GAPDH was used as the internal reference protein and its ratio is the relative protein expression level.the results showed that:The expression of CD47 in the abdominal aorta of mice is significantly decreased after anti-CD47 supplementation（P<0.05）,and the expression of HIF-1 is also significantly decreased compared with the mice of the same week of age without anti-CD47 supplementation.Compared with the normal group with high-fat feeding for 8weeks,the protein expression of TSP-1 in the high-fat feeding group after anti-CD47intervention decreases significantly at 8 weeks.Compared with the normal group with high-fat feeding for 10 weeks,the protein expression of TSP-1 in the high-fat feeding group for 10 weeks after anti-CD47 intervention is significantly decreased（P<0.01）.Compared with the normal group with high-fat feeding for 8 weeks,the protein expression of VEGF in the high-fat feeding group after anti-CD47 intervention for 8 weeks is significantly decreased（P<0.01）.Compared with the normal group with high-fat feeding for 8 weeks,the protein expression of VEGFR2 after anti-CD47intervention in the high-fat feeding group for 8 weeks was significantly decreased（P<0.05）.4.According to the experimental groups,the total proteins extracted from cultured cells in each group were analyzed by Western blot to analyze the relative expression levels of TSP-1,HIF-1,CD47,VEGF,and VEGFR2 among each group.GAPDH was used as the internal reference protein for control,and the ratio of molecular weight to internal reference of each factor to be measured was taken as the relative expression level.The results showed that the expression of HIF-1,CD47,VEGF and VEGFR2 are increased,while the expression of TSP-1 is decreased after the addition of LPS with HUVECs（P<0.05）.When TSP-1 antagonist（LSKL）,exogenous TSP-1 and anti-CD47（B6H12）drugs were added to HUVECs on the basis of LPS,the expression of HIF-1was not statistically significant,and the expression of TSP-1,CD47,VEGF and VEGFR2 were all affected.Conclusion:（1）ApoE-/-male rats fed with high fat for 8 weeks have the highest lipid content in abdominal aorta,and the degree of atherosclerosis is the most serious.（2）After high fat feeding in ApoE-/-male rats,the expression levels of HIF-1,CD47,VEGF and VEGFR2 reach the peak at 8 weeks,while the expression levels of CD47,VEGF and VEGFR2 all decrease when the expression of TSP-1 increases.（3）The TSP-1/CD47/VEGF/VEGFR2 pathway regulates the progression of atherosclerosis by inhibiting neovascularization.The HIF-1/CD47/VEGF/VEGFR2signaling pathway accelerates atherosclerosis progression by promoting neovascularization.（4）HIF-1 expression is increased in inflammatory response,which affects atherosclerosis through HIF-1/VEGF pathway.（5）Both TSP-1 antagonist and CD47 antagonist can increase the expression of VEGF and VEGFR2 in HUVECs.Exogenous TSP-1 reduces the expression of VEGF and VEGFR2 in HUVECs.（6）TSP-1/CD47 signaling pathway inhibites angiogenesis by affecting the expression of VEGF and VEGFR2. |
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