Roles And Mechanisms Of Siglec-5-Mediated OxLDL Transcytosis Across Endothelial Cells In The Development Of Atherosclerosis

Objective:Atherosclerosis（AS）is the main pathological basis for cardiovascular and cerebrovascular diseases and the mechanism is very complex.The deposition and retention of low-density lipoprotein（LDL）,especially oxidized low-density lipoprotein（oxLDL）under the vascular endothelium,is considered to be a key factor in the development of AS.We first discovered sialic acid binding immunoglobulin-like lectin-5（siglec-5）was significantly increased in type 2 diabetes mellitus（T2DM）patients with critical limb ischemia（CLI）.More interestingly,we confirmed siglec-5 could be observed in atherosclerotic plaques.More and more studies have reported that the Sialic acid binding immunoglobulin-like lectins（siglecs）family is closely related to AS,which is a chronic non-controllable inflammatory disease of the vascular wall.However,the specific role and molecular mechanism of siglec-5 in the formation of atherosclerosis has not been illustrated.This section aims to study the role of siglec-5in the occurrence and development of AS from the perspective of oxLDL transcytosis to provide new targets and treatments for the prevention and treatment of atherosclerosis.Methods:（1）The transcytosis characteristics of oxLDL were detected by an in-vitro transcytosis model,and the transcytosis of LDL and oxLDL was compared.（2）The expression of siglec-5 in HUVECs was observed by semi-quantitative reverse transcription-polymerase chain reaction（RT-PCR）and Western blot.（3）Quantitative RT-PCR and Western blot were used to detect the effects of oxLDL on the expression of siglec-5 mRNA and siglec-5 protein in endothelial cells and macrophages.（4）The effects of siglec-5 on the transcytosis and uptake of oxLDL were detected in endothelial cells by an in-vitro transcytosis model and laser confocal microscopy.（5）The effect of siglec-5 on oxLDL endocytosis was detected in macrophages by cell flow cytometry and laser confocal microscopy.（6）Binding of oxLDL to siglec-5 was detected in endothelial cells by laser confocal microscopy and co-immunoprecipitation.（7）After administration of anti-siglec-5 antibody and soluble siglec-5 protein,the transcytosis of oxLDL in endothelial cells and the endocytosis of oxLDL in macrophages were detected.（8）ApoE^-/-mice were overexpressed with exogenous siglec-5 by the adeno-associated virus vector system,and the Oil RedO staining was used to observe lipid deposition in the whole aorta and aortic roots.Results:（1）The in-vitro transcytosis of oxLDL showed that the transcytosis of oxLDL increased with the elevation of oxLDL concentration,indicating that the transcytosis of oxLDL had certain concentration dependence,and oxLDL had a stronger transcytosis effect than LDL.（2）Western blot and laser confocal microscopy showed that siglec-5was expressed on the membrane of endothelial cells.（3）OxLDL could promote the expression of siglec-5 mRNA and siglec-5 protein in endothelial cells and macrophages in a concentration-dependent manner.（4）The knockdown of siglec-5 inhibited the transcytosis of oxLDL,while the overexpression of siglec-5 promoted the transcytosis of oxLDL in endothelial cells.（5）The knockdown of siglec-5 suppressed the endocytosis of oxLDL and the overexpression of siglec-5 promoted the endocytosis of oxLDL in macrophages.（6）Siglec-5 could directly bind to oxLDL and mediate the transcytosis of oxLDL.（7）Anti-siglec-5 antibody and soluble siglec-5 protein not only inhibited the transcytosis of oxLDL in endothelial cells but also attenuated the endocytosis of oxLDL in macrophages.（8）In the ApoE^-/-mouse of atherosclerosis model,overexpression of the siglec-5 gene promoted lipid deposition in the whole aorta and aortic roots,while anti-siglec-5 antibody and soluble siglec-5 protein intervention slowed lipid deposition.Conclusions:The transcytosis of oxLDL has a concentration-dependent characteristic,and oxLDL has a stronger transcytosis effect than LDL,which indicates that oxLDL has a more atherogenic effect from the perspective of transcytosis.Siglec-5 was expressed on the membrane of endothelial cells and found that siglec-5 was a novel oxLDL receptor.Through direct binding to oxLDL,siglec-5 not only mediated oxLDL transcytosis in endothelial cells but also mediated oxLDL endocytosis in macrophages,which promoted the formation of macrophage foam cells,thereby accelerating the occurrence and development of atherosclerosis.