Research For Effect Of Endothelial ILF3 On The Progression Of Atherosclerosis

BackgroundThe incidence and mortality of cardiovascular diseases in our population are increasing year by year.Atherosclerosis(AS)is the most important pathological basis for it and plays a crucial role in the pathogenesis of various cardiovascular events,which seriously threatens the life and health of our population.When lipid levels in the blood are elevated,lipids are deposited on the arterial walls to form plaques that protrude into the lumen causing obstruction to normal blood flow.The process from the beginning to the formation of arterial plaque is irreversible,and once plaque is created,it is difficult to eliminate.The long-term accumulation of arterial plaque can cause various cardiovascular diseases,such as myocardial infarction and stroke.There are many risk factors leading to atherosclerosis,among which hyperlipidemia is considered to be the main risk factor.Due to the increase of lipoprotein level in plasma,cholesterol-based lipids will break the endothelial barrier and enter the subendothelial layer.At the same time,high lipids stimulate endothelial cell damage,and the damaged endothelial cells produce and release large amounts of inflammatory factors and chemokines into the bloodstream,attracting and activating macrophages while causing smooth muscle cells to migrate.The activated macrophages are able to engulf the lipids deposited under the endothelium and turn into foam cells that are difficult to degrade,while the migrating and proliferating smooth muscle cells can make the vessel wall brittle and cause the lumen to thicken and intensify the narrowing of the lumen.In addition,the endothelial infiltration theory suggests that lipids in the blood can be deposited under the endothelium through the transport of endothelial cells,and that lipid deposition in the endothelium can impair endothelial function and aggravate the development of AS.It can be said that the stimulation of endothelial cells by high lipids in the blood is the initiating link in the pathogenesis of AS.Therefore,maintaining lipid homeostasis in endothelial cells is critical for the development of atherosclerosis.Lipid metabolism is an important metabolic process for cells to maintain normal physiological functions,and impaired lipid metabolism is the pathological basis of many metabolic diseases and chronic conditions.When the intracellular lipid content is excessive,intracellular vesicles encapsulate it to form lipid droplets.In many metabolic diseases.such as obesity,fatty liver,cardiovascular disease and neutral fat storage disease,the pathological mechanism is often accompanied by abnormal lipid droplet accumulation.Since peripheral cells are weak in catabolism of cholesterol,lipid droplets deposited in cells are mainly metabolized by lipid transport,in which vesicular transport plays a key role in lipid transport between membranous organelles.Vesicles are membranous structures formed by a single layer of cell membrane,which can transport substances between membranous organelles in the form of endosomes,a process referred to as vesicular transport.The process of vesicle transport is very complex,and several proteins are involved in the regulation,among which Rab proteins play a crucial role in vesicle formation,transport,localization and fusion.Nearly one hundred Rab proteins have been identified,and each type of Rab protein is localized on different organelle membranes.Rab5b is a member of the Rab family of proteins.which is located in the early endosome and acts as a molecular switch for vesicle formation;Rab7a is located in the late endosome and acts as a molecular switch for vesicle formation.Rab7a is located in late endosomes and plays a very important role in the fusion of autophagosomes with lysosomes.Interleukin enhancer binding factor 3(ILF3),considered as an RNA-binding protein,binds to a variety of mRNAs and non-coding RNAs,plays an important role at the transcriptional level,and is widely involved in the translational regulation of many genes.Existing studies have identified that ILF3 is involved in the pathogenesis of multiple cardiovascular diseases.Through genomic association analysis,ILF3 was found to play an important role in regulating sterol metabolism in the body.One study found that ILF3 can regulate the pathological process of plaque calcification.Another study found that ILF3 has a regulatory role in cardiovascular diseases such as pathological cardiac hypertrophy,septic myocarditis,and hypertension,but the role and molecular mechanism of ILF3 in AS are still unclear and need to be further investigated.To explore the possible regulatory role of ILF3 in the development of AS,we proposed the following hypothesis:ILF3 in endothelial cells affects the pathogenesis of AS by regulating lipid deposition.Knockdown of ILF3 affects vesicular transport by regulating gene expression of Rab protein,leading to impaired lipid transport in endothelial cells,triggering accumulation of lipid droplets,resulting in imbalance of endothelial cell homeostasis,and ultimately leading to the development of atherosclerosis.Objectives1.To clarify the effect of ILF3 in endothelial cells on atherosclerosis.2.To investigate the pathways through which ILF3 in endothelial cells affects endothelial cell function.3.To elucidate the molecular mechanism of ILF3 regulation of Rab protein in endothelial cells that leads to the imbalance of endothelial cell homeostasis.Methods1.To clarify the role of ILF3 in endothelial cells on atherosclerosis.1.1 Endothelial cell ILF3-specific knockout mice were constructed,while wildtype mice of the same age were selected as controls,and ILF3EC-KO mice were mated with ApoE-1-mice to obtain ApoE-/-ILF3EC-KO mice.Mice were injected intraperitoneally with Tamoxifen at 6 weeks of age to induce ILF3-TEK-CRE knockout,and fed with high-fat chow for 16 weeks to construct an AS model.At the end of the modeling period,mice were executed and tissues were retained for further experiments.1.1.1 The expression of ILF3 in endothelial cells in the model and control groups was examined by immunofluorescence staining.1.1.2 Measure the lipid profile of mice in the endothelial cell ILF3 knockout group and the control group.1.1.3 Observe and detect the expression of arterial plaques in both groups of mice using oil red O staining method.1.2 Human umbilical vein endothelial cells(ECV304-HUVEC)were cultured and stimulated with oxidized low-density lipoprotein(ox-LDL),and the expression of ILF3 in endothelial cells was detected using Western-blot and PCR techniques.2.To elucidate the mechanism of the role of ILF3 in endothelial cells in the progression of AS lesions.2.1 In vivo experimentsMake sections of the aortic root of control and model mice.2.1.1 Observe the plaque size using HE staining and oil red O staining.2.1.2 Observe the expression of inflammatory factors such as TNF-α,IL1-β,IL-6 and IL-8 in aortic plaques using immunohistochemical chemical staining.2.2 Transcriptome sequencingEndothelial cells were transfected with si-NC and si-ILF3,and RNA was extracted for transcriptomic sequencing.2.2.1 Transcriptomic KEGG database pathway analysis showed that ILF3 knockdown affected endothelial cell lipid metabolism.2.2.2 Transcriptomic GO pathway analysis suggested that ILF3 knockdown affected Golgi-associated vesicle transport.2.2.3 Transcriptomic differential gene analysis showed that ILF3 knockdown decreased cholesterol transporter expression in endothelial cells.2.3 In vitro experimentsHUVEC were treated with the following groupings:si-NC,si-ILF3,si-NC+oxLDL,si-ILF3+ox-LDL.2.3.1 Transfection of si-ILF3 to HUVEC partially silenced ILF3 gene,stimulated with ox-LDL,and cellular oil red O staining was performed to observe the lipid content of endothelial cells.2.3.2 Total cellular proteins were extracted for Western-blot experiments to detect changes in the expression of lipophagy-related proteins LC3-B,P62,Beclin-1,Rab7a and LAMP-2.2.3.3 Extracted cell membrane proteins were subjected to Western-blot assay to observe the changes of endothelial cell membrane lipid receptor LDLR,LOX-1,membrane transporter ABC transporter protein family,and extracted total cell proteins to detect the changes of cholesterol transporter GRAMD1C,STARD3NL expression.3.To investigate the molecular mechanism of ILF3 regulation of lipid transport affecting endothelial function in endothelial cells.HUVEC were grouped and treated as follows:si-NC,si-ILF3,si-NC+ox-LDL,si-ILF3+ox-LDL3.1 The binding sites for the possible action of ILF3 were screened based on the analysis of sequencing results prompted and verified by PCR experiments.3.2 Culture HUVEC and verify the relationship between ILF3 and Rab protein mRNA by RNA binding protein immunoprecipitation assay.3.3 Endothelial cells of si-NC and si-ILF3 were cultured,and the stability of Rab protein mRNA was examined by PCR assay after treating the cells with a time gradient using Actinomycin D.3.4 After overexpression of ILF3 in endothelial cells,cells were treated with Rab5b inhibitor and the protein was extracted for Western-blot assay to detect the changes of cholesterol transporters and lipophagy-related indexes in endothelial cells.Results1.Hyperlipidemia is more likely to activate the expression of ILF3 in endothelial cellsWe cultured HUVEC and added ox-LDL at different concentration gradients and time gradients for stimulation,and showed that the expression of ILF3 in endothelial cells was concentration-and time-dependent with ox-LDL stimulation using Westernblot assay.Subsequently,the endothelial cells were stimulated according to the optimal time and concentration stimulation state,and RNA was extracted for PCR experiments,which confirmed that the mRNA level of ILF3 was similarly elevated under ox-LDL stimulation.Also,we found elevated ILF3 expression in endothelial cells in ApoE-/group mice fed high-fat on C57 background compared to the general diet.2.Deficiency of ILF3 in endothelial cells promotes atherogenesisWe bred ApoE-/-ILF3EC-KO and ApoE-/-ILF3EC-WT mice and found that the plaques were significantly increased in the ILF3EC-KO group by various histological and pathological staining,and the expression of inflammatory factors in the plaque tissue was increased,indicating that ILF3 deficiency in endothelial cells aggravates the development of atherosclerosis.3.ILF3 deficiency in endothelial cells leads to endothelial functional impairmentWe transfected HUVEC with si-ILF3 to partially silence the ILF3 gene,stimulated cells with ox-LDL,and found that ILF3 deficiency in endothelial cells leads to increased lipid droplet content in endothelial cells using oil red O staining,and also found that lipid droplet clearance pathway lipophagy is impaired,and ILF3 deficiency in endothelial cells leads to increased lipid deposition in endothelial cells.The deficiency of ILF3 in endothelial cells leads to increased lipid deposition in endothelial cells,affecting endothelial function and leading to imbalance of endothelial homeostasis.4.Transcriptomic sequencing suggests that ILF3 deficiency in endothelial cells affects cholesterol transportThe transcriptomic sequencing of HUVEC transfected with si-ILF3 small interferers was compared with the NC group,and the analysis revealed that when ILF3 was absent,the cholesterol transporter changed significantly,which led to lipid deposition in endothelial cells.western-blot experiments revealed that the change of ILF3 did not affect the endothelial cell membrane transporter,but the intracellular cholesterol transporters GRAMD1C,STARD3NL changed significantly.STARD3NL changed significantly,indicating that knockdown of ILF3 in endothelial cells interferes with the cholesterol transport process and leads to lipid deposition.5.Endothelial ILF3 interferes with vesicular transport by regulating Rab proteinTranscriptomic sequencing revealed that deletion of ILF3 in endothelial cells leads to "Golgi-associated vesicle transport disorder",while ILF3 deletion was found to decrease Rab protein in differential genes.RIP experiments confirmed that ILF3 binds to Rab5b mRNA and Rab7a mRNA as an RNA-binding protein,and causes vesicular translocation disorder by affecting their mRNA stability.Furthermore,we overexpressed ILF3 in endothelial cells stimulated with Rab5b inhibitor(neandrographolide)and confirmed using oil red O staining and Western-blot experiments that ILF3 affects endothelial cell lipid transport by regulating Rab5b,thus affecting the development of AS.Conclusions1.ILF3 deficiency in endothelial cells promotes the development of atherosclerosis.2.Endothelial cell ILF3 deficiency aggravates endothelial cell lipid deposition,and ILF3 leads to abnormal lipophagy by affecting Rab7a mRNA stability.3.ILF3 in endothelial cells regulates vesicular transport by affecting the stability of Rab5b mRNA in early endosomes,which in turn affects lipid transport and leads to abnormal accumulation of lipid droplets,ultimately leading to imbalance in endothelial cell homeostasis.