Effect Of Vaccination Against AGE-LDL On Atherosclerosis In Diabetic Apo-E^-/- Mice And Its Mechanism

Atherosclerosis has been considered as a chronic inflammatory disease concomitantwith dysfunctioned immune system, which is the main cause of morbidity or mortality ofdiabetic patients. Data from animal studies had been demonstrated beneficial effects ofimmunization strategies with different antigens or peptides against atherosclerosis, such asox-LDL, PC, apo-B-100,Hsp65and IL-12. The immune effects of such strategies might beinvolved with different mechanisms, such as induction of immune tolerance or productionof protective antibodies. It has been identified that the association of high level ofadvanced glycation end product of low density lipoprotein(AGE-LDL) with atherosclerosisprogression existed among diabetic patients. We hypothesized that vaccination withAGE-LDL could inhibit the progress of atherosclerosis in diabetic atherosclerosis-pronemice.Objective: In this study, we present data about the effects of vaccination strategy withAGE-LDL against atherosclerosis and investigate the involved mechanism.Methods: LDL was obtained by ultracentrifugation of serum from healthy volunteers andAGE-LDL was obtained by glycosylation reaction for2weeks. Protein modification wasevaluated by measuring pentosidine formation spectrofluorometrically. Four-week-oldmale apo-E deficient mice maintained on a standard diet for2weeks and then changed tohigh fat diet. ApoE-/-mice were divided into three groups as PBS group(250ul), Al(OH)3group(125ul+125ul PBS), and Al(OH)3+AGE-LDL group (125ul+125ul). Male apoE-/-mice at eight week received subcutaneous injection once every two weeks for8weeks.Diabetes was induced by intraperitoneal injections of streptozotocin at a dose of40mg/kgin citrate buffer for5days in sixteen-week-old homozygous apoE-deficient male mice.Two weeks later such mice showed polydipsia, polyuria and polyphagia and diabetes wereidentified by randomly measured blood glucose twice. Mice with blood glucose above than11.1mmol/l were choosed to receive subsequent vaccination. Twenty five-week-old malediabetic apo-E^-/-mice were anesthetized by an intraperitoneal injection of pentobarbitalsodium. Atherosclerotic lesions were analyzed to evaluating the immune effect ofAGE-LDL. The blood obtained by cardiac puncture. Total cholesterol, LDL, HDL andtriglyceride concentrations were measured by autoanalyzer technique. Profiles oflipoproteins were measured by FPLC system. The area of atherosclerosis lesions wereanalyzed by oil red O staining. Both en face of the aorta and aortic root in cryostat sectionswere stained.The aortic root in paraffin sections were stained with MOMA-2antibody, Masson,s trichrome, and É‘-SMC antibody. Masson's trichrome staining was used to assessplaque collagen content. MOMA-2antibody, É‘-SMC antibody, biotinylated secondaryantibody, and DAB detection kit were used to stained. The result of the staining was usedto assess ratio of the macrophages and the smooth muscle cell in plaque,respectively. Inthe end, we did research in mechanism of immunoprotection against atherosiclerosis aboutAGE-LDL. FACS analysis was used to measure intracellular cytokines. One part ofsplenocytes were stained with flurochrome-conjugated antibodies to assess percentage ofTreg cells. The other part of splenocytes were stimulated for4h with50ng/ml PMA,500ng/ml inomomycin, and monensin. Cells were then stained to assess pecentage of Th1Th2T17cells. Total IgG,total IgM, IgG to AGE-LDL,and IgM to AGE-LDL from serumwere analyzed by immunometeric ELISA. ELISA kits were used for determined the levelof IFN-r, IL-4,IL-10,and IL-17in serum or supernatant.Results:(1) Immunization exerted protective effects on atherosclerotic lesions indiabetic apo-E^-/-mice. Compared with single Al(OH)3group and PBS group, subcutaneousimmunization with AGE-LDL+Al(OH)3significantly decrease the atherosclerosis burdennot only from en face of the aorta, but also aortic root. While immunization with Al(OH)3and PBS did not influence the en face of the aorta or aortic root sections plagques.Administration of AGE-LDL+Al(OH)3did not affect body weight, serum cholesterol,profiles of lipoproteins, triglycerides, and composition of plaque,(2) Mechanisms ofimmunization against atherosiclerosis with AGE-LDL+Al(OH)3. Imunization withAGE-LDL+Al(OH)3could upregulate the level of IgG to AGE-LDL and IgM toAGE-LDL as compared with immunization with Al(OH)3or PBS. The level of IgG toAGE-LDL in AGE-LDL+Al(OH)3group was2.5times higher than single Al(OH)3groupor PBS group. The levels of total IgM and IgG did not have shown difference in all threegroups. Imunization with AGE-LDL+Al(OH)3could upregulate the percentage of Tregcells and the level of IL-10from cell supernatant. Imunization with AGE-LDL+Al(OH)3neither alterd the percentages of Th1, Th2, Th17cells from spleen nor the levels of IFN-r,IL-4and IL-17in supernatant from splenocyte culture. There were not detected the contentof IFN-r, IL-4,in the serum.The content of IL-17, IL-10in the serum did not changed.Conclution: AGE-LDL can be obtained by glycosylation reaction with LDL. Diabetic apo-E^-/-mice model can be induced by several intraperitoneal injections withstreptozotocin. Subcutaneous immunization with AGE-LDL+Al(OH)3could inhibit theprogression of atherosclerosis lesions in diabetic apoE-/-mice. Such effects were shown toassociate with up-regulation of IgG and IgM aganist AGE-LDL. In addtion to suchmechanism, the higher percentage of Treg cells and the increased level of IL-10might alsoinvolve in the protective effects.