The Mechanobiological Mechanisms Of Id1-regulated Lipid Uptake In Atherosclerosis Plaque Formation

Cardiovascular disease is a common disease that seriously threatens human health.The statistic data of theWorldHealth Organization?WHO?showed that cardiovascular disease is the leading cause of death in the world.More than 10 million people die of cardiovascular disease per years,which accounts for 30%of the total number of deaths.China's 2016 cardiovascular disease report pointed out that the number of cardiovascular patients in China showed a sustained upward trend,the mortality rate is higher than other diseases such as cancer.Even if application the most advanced and perfect treatment,there are still more than 50%pepole of heart and cerebrovascular disease survivors cannot completely take care of themselves.The main reason of cardiovascular disease is the arterial obstruction or stenosis caused by atherosclerosis,so it is very important to study of the mechanism of atherosclerosis?AS?formation to prevent cardiovascular disease.Atherosclerosis highly preferred occurs in curves,branches,and bifurcation positions of arteries,these positions are the disturbed flow areas with low and oscillatory shear stress?OSS?,which indicated that the formation and development of atherosclerosis are closely related to hemodynamics and the changes of structure and function in blood vessels caused by hemodynamic factors are important for the formation of AS.Excessive lipid uptake in the vascular endothelial cells can lead to deposition of lipid in the arterial intima and promote the formation of atherosclerotic plaques,so lipid uptake of endothelial cells plays an important role in the formation of AS.Some studies have shown that there was a correlation between lipid deposition and shear stress,so this research take OSS as the main line to study the effect of OSS on lipid uptake and deposition in blood vessels,the related molecular mechanisms.This research would help to partly elucidate the reason of atherosclerosis formation in the low and oscillatory shear stress regions,and it may also provide potential new target for the treatment of AS.In this study,the oscillatory flow model was constructed through partial ligation of Apo E^-/-mouse carotid artery.Blood flow status was detected by small animal ultrasonography.When the model was successfully constructed,the differential protein expression between OSS and laminar shear stress?LSS?was detected by proteomics methods,and the related signal pathways were analyzed by GO and KEGG.Results showed that OSS influenced the formation of AS by affecting lipid digest and uptake,inflammation and so on.As the ligation time increased,the plaque formation,lipid accumulation and lipid uptake of endothelial cells were detected.The role of Id1 in OSS promoted lipid absorption and related molecular mechanisms were also detected in vitro and in vivo.The main results are as follows:1.Partial ligation model of Apo E^-/-mice was constructed,the blood flow and vessel diameter were detected by small animal ultrasonography.Results showed that partially ligated left carotid arteries?LCA?appeared reverse flow in opposite direction with OSS,while the right carotid arteries?RCA?were laminar flow with LSS.Then the total protein of LCA and RCA was extracted respectively.Subsequently,the differentially expressed proteins were detected by iTRAQ.Compared with LSS,OSS regulated 168 proteins expression,including 150 up-regulated proteins and 18down-regulated proteins.Through the GO enrichment analysis and KEGG signaling pathway analysis,these proteins were classified and the involved signal pathways were predicted.The results showed that these differentially expressed proteins were closely related to the process of macromolecule metabolism,transport,localization,fat digestion and absorption and inflammation.It suggests that OSS may influence the formation of AS by regulating these processes.Because abnormal lipid metabolism is closely related to AS,so we will continue to further verify whether OSS can affect lipid deposition and absorption and promote plaque formation in vivo and in vitro.2.OSS model of Apo E^-/-mice was constructed,the blood flow and vessel diameter were detected by small animal ultrasonography.Plasma lipoproteins were examined in the partial ligation and no ligation mice,the results showed that OSS in partial ligation mice did not have a significant effect on the lipoprotein content in the blood.HE staining of the left and right carotid arteries in ligation group and control group showed that the intima of LCA with OSS were thickened with the increase of ligation time.Ten weeks after surgery,the plaques almost blocked the entire blood vessels,while the contralateral RCA and carotid arteries in control group with LSS did not have obvious changes.Carotid arteries were stained with oil red O and results showed that LCA with OSS can promote lipid accumulated in the vessel walls,and with the increase of ligation time,lipid accumulation was more and more serious.Lipid absorption in vascular endothelial cells was detected by CD31 and BODIPY staining in carotid arteries.Results showed that lipid uptake in LCA?OSS?endothelial cells was significantly higher than the contralateral RCA and the carotid arteries?LSS?of control group,and with the increased time,more and more lipids were absorbed.The role of OSS in lipid uptake of human umbilical vein endothelial cells?HUVECs?was also tested by using a shear stress apparatus which can apply OSS and LSS.Results showed that OSS promoted lipid absorption,which was consistent with the results of in vivo experiments.The effect of OSS treatment and subsequent LDL uptake on the adhesion of HUVECs and THP-1 cells was investigated,it was found that OSS treatment and subsequent LDL uptake increased the adhesion.Ki67 immunofluorescence staining showed that OSS treatment and subsequent LDL uptake promoted endothelial cell proliferation.Endothelial cell proliferation and inflammatory induced by OSS and subsequent LDL uptake may be involved in the formation of AS.3.Studies in our laboratory have suggested that the inhibitor of differentiation 1?Id1?is a mechanically sensitive transcription factor and is closely related to lipid metabolism.Therefore,we used Id1 as a research object to analyze whether it participates in the process of lipid absorption regulated by OSS.After ligated the LCA24 hours and 48 hours,the expression of Id1 protein in vascular endothelial cells was examined by en face staining.It was found that OSS could inhibit Id1 expression.HUVECs were applied OSS and LSS,the expression of Id1 protein was detected by Western blot.Results showed that LSS promoted the expression of Id1.As the treating time increased,Id1 expression is more and more and the expression reached its peak at the 12th hour.OSS transiently promoted Id1 expression,but the expression of Id1 was finally inhibited as the treating time prolonged.Then lipid uptake in Id1 overexpressed cells that applied OSS was tested.The results showed that Id1 overexpression inhibited OSS-mediated lipid uptake.4.In order to study the role of Id1 in AS formation,we performed a correlation prediction analysis through IPA software and found that in AS,Id1 regulates the expression of many inflammatory factors,such as NF-?B,TNF,ICAM,etc.In addition,Id1 is also associated with lipid metabolism related proteins and may regulate the expression of low density lipoprotein receptor?LDLR?.Based on this,we investigated whether Id1 affects the process of lipid absorption by regulating LDLR expression in vivo and in vitro.The expression of LDLR protein and lipid uptake was examined in Id1overexpressed and knockdown cells.We found that LDLR was inhibited and the lipid uptake was decreased after Id1 overexpression,while LDLR protein and lipid uptake were significantly increased in Id1 knockdown cells.When Id1 and LDLR were interfered meanwhile,Id1 knockdown caused lipid absorption was inhibited,which indicating that Id1 abnormal expression caused lipid uptake was achieved by regulating LDLR protein.The LDLR expression in OSS and LSS treatment were detected by en face staining and Western blot.LDLR was activated by OSS in vivo.In vitro,LSS can transiently activate LDLR expression,but inhibited its expression as the treating time prolonged.OSS can activate the expression of LDLR continuously.When LDLR was knockdown,lipid uptake induced by OSS was inhibited,which indicated that OSS influenced lipid absorption by regulating the expression of LDLR.LDLR and Id1expression patterns were detected by immunofluorescence and Western blotin vitro.After 24 hours of shear stress treatment,compared with static,LSS activated Id1expression and inhibited LDLR expression,while OSS inhibited the expression of Id1and activated LDLR expression.These results indicated that Id1 and LDLR are both regulated by OSS,and they are negatively correlated.After applied OSS to Id1overexpressed cells,LDLR expression was examined by Western blot.The results showed that Id1 overexpression inhibited OSS-mediated LDLR activation,which indicating Id1 was involved in the LDLR regulation by OSS.Because of Id1 needs to combine with b-HLH proteins to work,we examined the relationship between Id1 and SREBP1?sterol-regulatory element binding proteins1,transcription factors that regulate LDLR expression and belong to b-HLH protein family?.Western blot results showed that Id1 abnormal expression did not significantly influence the expression of SREBP1.Coimmunoprecipitation and immunofluorescence staining results found Id1 can combine with SREBP1 to form a complex.This physical binding may be involved in the process that Id1 regulated LDLR expression.In conclusion,this study used a combination of techniques such as proteomics,biomechanics,pathophysiology,and cell molecular biology to construct a mouse carotid artery ligation model to study the effects of OSS on vascular lipid deposition,lipid uptake,and plaque formation.At the same time,in vitro experiments were performed to study the role of Id1 protein in the process of lipid absorption caused by OSS.Through this research work,we have partly clarified the molecular mechanisms that Id1participate in OSS regulated lipid uptake in vascular endothelial cells.This will provide some new scientific evidences for in-depth understanding why AS usually occurred in OSS regions.