Proatherogenic Effects And Mechanisms Of Glycated Low-Density Lipoprotein Transcytosis Of Endothelial Cells

Atherosclerotic cardiovascular disease（ASCVD）is a major cause of death worldwide and a serious risk to human health.Atherosclerosis（AS）is the underlying pathology of ASCVD,and currently,most studies suggest that lipoprotein retention under the endothelium is the joint link of AS.Diabetes mellitus（DM）is an independent risk factor for AS.Data show that plasma levels of glycated LDL are significantly higher in diabetic patients than in normal controls and that glycated LDL is strongly associated with the development of AS.However,it has not been reported whether glycated LDL can transcytosis across the intact endothelial cell layer.In the present study,we investigated whether glycated LDL could be retained in the endothelium through an in vitro transcytosis model,and then screened the receptors mediating glycated LDL transcytosis,in order to provide a theoretical basis for the prevention and treatment of diabetic AS.Part Ⅰ:G-LDL transcytosis modelingObjective:Lipoprotein transcytosis across the vascular endothelium and retention under the endothelium promotes atherosclerosis（AS）.Glycated low density lipoprotein（G-LDL）is a clear pro-atherosclerosis factor,but it has not been reported whether G-LDL promotes AS through increased transcytosis.Our group has previously demonstrated that by an in vitro transcytosis model,the native LDL（N-LDL）transcytosis was time-dependent and concentration-dependent.The present study was designed to investigate whether G-LDL can transcytosis across vascular endothelial cells and to investigate the characteristics of G-LDL transcytosis.Methods:The successful preparation of G-LDL was verified by color observation and agarose gel electrophoresis to determine electrophoretic mobility and to compare the physicochemical properties of N-LDL and G-LDL.Flow cytometry and confocal microscopic imaging were used to compare the difference in uptake of N-LDL and G-LDL by endothelial cells.The amount of N-LDL and G-LDL transcytosis was compared and characterized by an in vitro cell transcytosis model using fluorescently labeled N-LDL and G-LDL.Changes in the amount of N-LDL and G-LDL transcytosis were observed by adding high concentrations of lipoproteins not labeled with fluorescence.Results:G-LDL showed a brownish-yellow color and the electrophoretic mobility was faster than that of N-LDL,and the chemical reaction detected a reduction in the amount of free lysine in G-LDL,confirming the successful preparation of G-LDL.In vitro transcytosis experiments confirmed that G-LDL could be transported across endothelial cells and that the amount of transcytosis was significantly higher than that of N-LDL.G-LDL transcytosis was concentration-dependent,time-dependent,saturable and competitive.Conclusions:G-LDL is recognized and uptake by endothelial cells in higher amounts than N-LDL,and the transcytosis of G-LDL is time-dependent,concentration-dependent,saturable and competitive.Part Ⅱ:Screening of receptors-mediated G-LDL transcytosis andprobing molecular regulatory mechanismsObjective:Increased G-LDL transcytosis promotes the development of AS.However,it is not known which receptor molecule mediates the G-LDL transcytosis,and the molecular regulation mechanism of G-LDL transcytosis has not been reported.In this study,we aim to screen the receptor molecules that mediate G-LDL transcytosis using an in vitro transcytosis model and further investigate the molecular regulatory mechanisms.Methods:The G-LDL-mediated transcytosis receptor was screened by silencing the expression of eight transcytosis-associated proteins by small interfering RNA（si RNA）to detect changes in the amount of N-LDL and G-LDL transcytosis.After transfection of scavenger receptor A（SR-A）si RNA or overexpression plasmids in endothelial cells,flow cytometry and fluorescence confocal microscopy were used to detect changes in N-LDL and G-LDL uptake by endothelial cells;in vitro lipoprotein transcytosis models was used to detect alterations in N-LDL and G-LDL transcytosis.Direct binding of SR-A and G-LDL was verified by the binding assays.The expression of receptor molecules and protein kinase Cδ（PKCδ）were measured by quantitative real-time polymerase chain reaction（q RT-PCR）and western blot（WB）after N-LDL or G-LDL treatment of endothelial cells.To detect whether N-LDL or G-LDL affects SR-A stability.After transfection of si RNA for PKCδ,changes in SR-A expression after G-LDL treatment of endothelial cells were examined by q RT-PCR and WB and changes in G-LDL uptake and transcytosis was determined.After tail vein injection of Apo E^-/-mice with endothelium-specific knockdown of SR-A by adeno-associated virus and verification of SR-A silencing efficiency in endothelium by q RT-PCR and immunohistochemistry,the two groups of mice were N-LDL and G-LDL were injected and plaque formation and lipid deposition in mouse arteries were detected by gross observation and whole aortic oil red O staining.Peritoneal primary macrophages from control and SR-A knockdown mice were extracted and q RT-PCR was used to compare SR-A expression and to compare whether the uptake capacity was altered.Results:The reduced expression of SR-A in endothelial cells decreased the transcytosis of G-LDL without affecting the transcytosis of N-LDL,while the overexpression of SR-A in endothelial cells significantly increased the transcytosis of G-LDL compared to the control group,while the transcytosis of N-LDL was not significantly affected.The uptake of G-LDL was significantly reduced after silencing of SR-A expression in endothelial cells compared to the control group,while the uptake of G-LDL was significantly increased in the endothelial cells of the SR-A overexpression group.The binding assays confirmed that SR-A directly bound to G-LDL and mediated G-LDL uptake.Endothelial cells in the G-LDL-treated group showed increased expression of both RNA and protein amounts of SR-A and PKCδ,and increased stability of SR-A protein.Decreased PKCδexpression in endothelial cells mitigated the G-LDL-induced increase in SR-A RNA and protein expression.Both G-LDL uptake and transcytosis were reduced in the PKCδknockdown group compared to the control group.In the Apo E^-/-mouse AS model,the G-LDL-injected group had significantly more oil-red O-positive area than the N-LDL-injected group,and exogenously injected N-LDL and G-LDL were deposited mainly in the liver and aorta.Endothelial-specific knockdown of SR-A reduced G-LDL-induced lipid deposition and AS plaque formation without affecting lipid deposition and plaque formation of N-LDL.Endothelial-specific knockdown of SR-A did not affect SR-A expression and the ability to uptake lipoproteins in macrophages.Conclusions:SR-A is the main receptor mediating G-LDL transcytosis across vascular endothelial cells.G-LDL promotes SR-A expression by upregulating PKCδexpression in endothelial cells on the one hand,and inhibits SR-A degradation and increases its stability on the other hand,which together promote G-LDL uptake and transcytosis.Endothelial-specific silencing of SR-A expression may be a new idea for the treatment of diabetic AS.