The Role Of Endothelial DKK2 Expression Induced By Disturbed Flow In Atherosclerosis

BackgroundCardiovascular disease(CVD)is one of the leading causes of mortality and disability worldwide.Atherosclerosis(AS)is the main cause of CVD,characterized by intimal thickening and atheromatous plaque formation,and is a focal chronic inflammatory vascular disease involving medium and large arteries.Disturbed flow(dflow)is a low-frequency oscillatory shear stress that manifests as slow and disturbed flow,usually occurs at the bend and bifurcation of blood vessels and is one of the main causes of AS lesion formation.Disturbed flow can directly affect the morphology and function of vascular endothelial cells,promotes the occurrence of adverse reactions such as endothelial dysfunction and inflammation,and increases the risk of atherosclerotic disease.However,the mechanism of action of disturbed flow involved in the development of atherosclerosis is still unclear.Our group previously revealed the effect of disturbed flow on the functional heterogeneity of vascular endothelial cells at the single cell level by single-cell RNA sequencing and found that disturbed flow induces the formation of a novel mechanical force-sensitive endothelial cells subpopulation with highly expressed DKK2.However,the molecular mechanisms underlying the formation of this cell subpopulation and its role in atherosclerosis are unknown.In the present study,we found that the formation of DKK2-high-expressing endothelial cell subpopulation may be associated with PIEZO1 channels,and this subpopulation appeared to be endowed with robust cell adhesion function.Importantly,DKK2-high-expressing endothelial cells were also detected in mouse atherosclerotic plaques,and DKK2 deletion obviously reduced the formation of mouse atherosclerotic plaques.This study provided a theoretical basis and experimental clues for the prevention and treatment of atherosclerosis.AimsTo investigate the mechanism of disturbed flow induced DKK2 expression in vascular endothelial cells in the development of atherosclerotic diseases.Methods1.Blood disturbed flow and DKK2 expression of endothelial cells.(1)We constructed a disturbed flow model by partial ligation of the carotid artery in wild-type mice and performed single-cell RNA sequencing of the ligated left carotid artery at continuous postoperative time points(1Day/2Days/5 Days/14 Days/28 Days)to integrate the endothelial cell subpopulations at different time points and analyzed the expression of DKK2 in each subpopulation and the relative ratio changes in DKK2overexpressing cells at different time points.(2)We examined the mRNA and protein expression levels of DKK2 in the left carotid artery exposed to disturbed flow after PCL surgery and in the untreated right carotid artery.DKK2 expression in vascular endothelial cells of the left and right carotid arteries was observed by immunofluorescence staining.(3)We examined the mRNA and protein expression levels of DKK2 by using a modified cone-and-plate shear device and placed HUVECs cultured in vitro in a laminar shear stress environment and an oscillatory shear stress environment to clarify the correlation between disturbed flow and DKK2 expression in vascular endothelial cells.2.Molecular mechanisms of DKK2-high-expressing endothelial cells formation in response to disturbed flow.(1)Single-cell RNA sequencing data of disturbed flow were used to calcul ate the markers in DKK2-high-expressing endothelial cell subpopulation,identifi ed the gene expression of mechanoreceptors,and tested whether the markers w ith mechanoreceptor function were also expressed in other endothelial cell subp opulations.(2)The expression level of DKK2 in endothelial cells was detected by activating mechanoreceptors in vitro using specific agonists,and the changes in the transcript levels of endothelial cell genes were detected using transcriptome sequencing.3.Effect of DKK2 upregulated expression on vascular endothelial cell function.Lenti-hDKK2-GFP lentivirus was infected to HUVECs to construct a DKK2overexpressing HUVEC cell line,and transcriptome sequencing was performed on this cell line and an empty lentivirus-infected HUVEC cell line to explore the potential functional changes in endothelial cells after DKK2 overexpression by functional enrichment analysis.An in vitro cultured DKK2-overexpressing endothelial cell line was used for functional validation.4.Expression of DKK2 in endothelial cells of mouse atherosclerotic plaques.(1)The single-cell RNA sequencing data of mouse atherosclerotic plaques from the GEO database were analyzed,and the endothelial cells were separately reclustered to detect the expression of DKK2 in each subpopulation,while the endothelial cell subpopulations in the plaques were subjected to Spearman correlation analysis with the endothelial cell subpopulations formed by disturbed flow induction.(2)ApoE-/-mice were subjected to PCL surgery and fed a high-fat diet(HFD)for 2 weeks,and the expression of DKK2 in endothelial cells of atherosclerotic plaques was detected by immunohistochemical and immunofluorescence staining.5.Effect of DKK2 knockdown on atherosclerotic plaque formation.(1)After the injection of DKK2-shRNA and null-loaded lentivirus into the left carotid artery site of Ldlr-/-mice by vascular ligation closure,DKK2 expression was knocked down in the left carotid artery of mice,which was partially ligated and fed a high-fat diet for 2 weeks to induce atherosclerotic plaque formation.(2)Mouse carotid arteries were taken for Sudan red IV staining and photographed under a stereo microscope,and the plaque areas at the left carotid artery site were quantified using ImageJ analysis software.Results1.Blood disturbed flow upregulated the DKK2 expression of endothelial cells.(1)The single-cell RNA sequencing data showed that a subpopulation of DKK2-high-expressing endothelial cells appeared after disturbed flow stimulatio n at 1 day.2 days.5 days,7 days,and 14 days,with the highest proportion of DKK2-high-expressing endothelial cells after disturbed flow stimulation at 7 days.(2)The qPCR and Western blot results of the carotid vessel walls showed that DKK2 expression was significantly higher in the left carotid artery after PCL surgery than in the untreated right carotid artery.Immunofluorescence staining showed that DKK2 was mainly expressed in endothelial cells after disturbed flow stimulation,and DKK2 expression was higher in endothelial cells of the left carotid artery after the PCL surgery than in endothelial cells of the right carotid artery.(3)In the modified cone-and-plate shear device,DKK2 expression levels were significantly higher in HUVECs exposed to oscillatory shear stress than in HUVECs exposed to laminar shear stress.2.Endothelial cells upregulated DKK2 expression may via mechanosensitive PIEZO1 in response to disturbed flow stimulation.(1)Six mechanosensitive molecules-Piezo1,Wwtr1,Plxnd1,Nrp1,Tie1,and Gsk3b-were found by analysis of characteristic markers on the surface of the DKK2high-expressing endothelial cell subpopulation,of which Piezo1 was less expressed in the normal endothelial cell subpopulations and more highly expressed in the endothelial cell subpopulations induced by disturbed flow.(2)Activation of vascular endothelial cell PIEZO1 by the specific agonist Yodal significantly increased the level of DKK2 expression.3.Significant enrichment of cell adhesion function in endothelial cells after DKK2 overexpression.Transcriptome sequencing of DKK2-overexpressing endothelial cell lines revealed a total of 224 upregulated and 222 downregulated genes,most of which were associated with cell adhesion function.In vitro coculture experiments confirmed that the adhesion of THP-1 cells to DKK2-overexpressing endothelial cells was significantly increased compared to that of controls.4.DKK2 expression can be detected in vascular endothelial cells of mouse atherosclerotic plaques.(1)Analysis of single-cell RNA sequencing data showed that the DKK2-highexpressing endothelial cell subpopulation was also present in mouse atherosclerotic plaques and showed a high correlation with the DKK2-high-expressing endothelial cell subpopulation induced by disturbed flow.(2)Immunohistochemical and immunofluorescence staining showed that endothelial cells expressed DKK2 in mouse carotid atherosclerotic plaque tissues.5.DKK2 deficiency in mouse carotid arteries reduced atherosclerotic plaque formation.Left carotid atherosclerotic plaque formation was significantly reduced in Ldlr-/mice by injecting DKK2-shRNA lentivirus into Ldlr-/-mice compared with control lentivirus.Conclusions1.Blood disturbed flow induced DKK2 expression in vascular endothelial cells.2.Blood disturbed flow may activate the vascular endothelial cell surface mechanoreceptor PIEZO1 and regulated endothelial cell DKK2 expression.3.DKK2 overexpression in endothelial cell lines significantly enhanced cell adhesion function.4.Endothelial cells in mouse atherosclerotic plaques expressed DKK2,and atherosclerotic plaque formation was inhibited after in situ deletion of DKK2 expression in mouse carotid arteries.