| At present,cardiovascular diseases(CVDs),as the first fatal disease in the world,seriously threaten human health,and bring heavy social and economic burden to individuals,families and countries.Atherosclerosis(AS)is the most common pathological basis of CVDs.It is very important to study the mechanism of atherosclerosis in order to improve the prevention and treatment of CVDs.Extensive clinical studies have proved that AS lesions are prone to the areas of vascular stenosis,curvature and bifurcation,which are irregular blood flow areas with low and oscillatory shear stress(OSS),and these suggest that the occurrence and development of atherosclerosis are closely related to hemodynamics.Endothelial cells(ECs)locate in the innermost layer of blood vessels,and distinct cellular responses could be induced by different shear stresses in ECs.The change of endothelial function plays an important role in the occurrence of AS.OSS lead to endothelial dysfunction and accelerate the focal distribution of atherosclerotic lesions,while laminar shear stress(LSS)can stabilize endothelial function and inhibit the formation of AS.However,the underlying molecular mechanism that how shear stress regulates AS formation through affecting endothelial function has not been fully elucidated.Stress granules(SGs)are the aggregation of mRNA and protein in eukaryotic cells,which are rapidly formed in response to various stimulation from environment.The formation of SGs is closely related to the development of many diseases,and recent studies have found that SGs persist in atherosclerotic plaques.G3BP2(Ras GTPase activating SH3 domain binding protein)is an important component in the formation of SGs.In this study,both the in vivo partial carotid ligation model and in vitro flow chamber device were used to investigate the role of G3BP2 in OSS induced AS formation,and the mechanism of G3BP2 regulating OSS-induced endothelial dysfunction.The main research contents and conclusions are as follows:1.ApoE–/–mice were used to construct the partial ligation model,and the direction and velocity of left carotid artery blood flow were determined by small animal ultrasonic detection.The results showed that the pattern of blood flow was OSS in the ligation group,and LSS in the control group.The results also showed that G3BP2 was significantly up-regulated.2.ApoE–/–mice were fed a high-fat diet for 12 weeks from 8 to 20-week-old.Then,using a repetitive multiple-sites strategy,mice were immunized with KLH conjugated G3BP2 peptide for 2 weeks from week 16 to week 18.The results showed that the lipid deposition in aorta of mice immunized with G3BP2 was significantly reduced,and the number of pro-inflammatory factors in serum,macrophages in plaques and pro-inflammatory factors were significantly reduced.In vitro studies showed that G3BP2 regulated inflammatory ECs via promoting I?B?retention in cytoplasm by binding with I?B?,and then activating the expression of downstream inflammatory genes of NF-?B signaling pathway.3.The expression of G3BP2 was detected in different regions of human femoral artery,aorta of high-fat diet induced AS model and carotids of ligation model.And we found that G3BP2 was highly expressed in ECs in plaques and disturbed flow regions.In vitro inflammation and flow chamber experiments also showed that G3BP2 was highly expressed in ECs treated with proinflammatory factors or OSS.The results of transcriptomics showed that G3BP2,together with other components of stress granules and pro-inflammatory factors were significantly increased,and anti-inflammatory factors were significantly decreased in the control group treated with OSS.However,that in G3BP2 knockdown ECs was on the contrary.4.ApoE–/–and G3bp2+/–ApoE–/–mice were used to construct high-fat AS model.Pathological analysis of aortic plaque and biochemical analysis of blood were carried out in mice after being fed with high-fat for 8 weeks.ApoE–/–,G3bp2+/–ApoE–/–and G3bp2–/–ApoE–/–mice were used to construct carotid partial ligation model.Pathological analysis of carotid plaque was carried out in mice after being fed a high-fat diet for 4 weeks.And we also examined the percentage of plaque area in descending aorta and lipid profiles in these three different genotypes of mice fed with normal diet for 35 weeks.The results showed that the proportion of plaque,inflammatory cytokines and macrophages in the plaques of G3bp2+/–ApoE–/–and G3bp2–/–ApoE–/–mice were significantly decreased.And there were no significant differences in the levels of lipid profiles,number and proportion of monocytes in the spleen and peripheral blood among three groups.5.The monocytes adhesion experiments demonstrated that the number of adhered monocytes was obviously decreased in G3BP2 knockdown ECs.In vitro and in vivo experiments showed that the permeability of ECs was decreased and the stability of VE cadherin was increased after G3BP2 knockdown.Sequencing data analysis showed that OSS could increased the expression of integrin?3 in control ECs,but cannot increase that in G3BP2 knockdown ECs.Further in vitro and in vivo experiments showed that there was a negative correlation between G3BP2 and YAP phosphorylation.6.Carotid partial ligation ApoE–/–models were immunized with G3BP2 for 2weeks and fed a high-fat diet for 4 weeks.Then the carotid arteries were obtained and pathological analysis was carried out.The results showed that G3BP2 immunization could effectively decrease the percentage of carotid plaque,the level of proinflammatory cytokines and macrophages in plaques.In summary,this study showed that G3BP2 is an important mechanical response protein,which could be induced by OSS,and knockdown G3BP2 in ECs can inhibit the inflammatory response of ECs induced by OSS,and G3bp2+/–ApoE–/–and G3bp2–/–ApoE–/–mice showed less AS plaques in aortas.Further investigation showed that G3BP2 mediated mechanical response induced by OSS via integrin?3-YAP signaling pathway.Therefore,G3BP2 is a potential therapeutic target for the development of AS induced by OSS. |
PDF Full Text Request