Human CD59 Mediates Rapid Cellablation After Intermedilysin Administration:Multiple Applications Systemic Application Of 3-Methyladenine MarkedlyInhibited Atherosclerotic Lesion In Apoe^-/- Mice

ObjectiveIn the past over 30 years, biology has greatly benefited from rapid technological advancement. Gene-targeted deletion and gene over expression methods in vivo and in vitro have have developed for studying individual and multiple gene function. A lot of information about gene function has been obtained from the utilization of the elegant gene-targeted and transgenic technologies in mice. At cellular level, conditional and targeted cell ablation is a powerful and widely used approach for studying specific cellular functions, as well as tissue repair and differentiation in vivo.The genetic cell ablation methods that are currently used by researchers include the expression of herpes simplex virus 1 thymidine kinase (HSVtk) and the diphtheria toxin (DT) receptor (DTR) coupled with transgenic strategies. However, these approaches have some limitations. For example, in the model of HSVtk transgenic mice, only dividing cells are eliminated, whereas non-dividing cells are not ablated. Although the DTR cell ablation model has been used in the study of cellular functionalities in vivo, DT administration of only two-to three-fold higher doses than the effective doses required for targeted cell ablation results in significant off-target effects, causing mortality and morbidity independent of DTR. These facts underscore an unmet need to develop a new model that specifically ablates cells in vivo with higher efficiency and fewer off-target effects.Intermedilysin (ILY) is a cholesterol-dependent cytolysin (CDC) that is secreted by Streptococcus intermedius. ILY can specifically lyse human cells by binding to human CD59 (hCD59) and subsequently forming toxin pores; however, ILY does not lyse cells from nine other animal species that were tested because there is no cross-species reactivity of ILY with hCD59. CD59 is a glycosylphosphatidylinositol (GPI)-linked membrane protein that inhibits the formation of the membrane attack complex of complement by binding to complement proteins C8 and C9 and preventing C9 incorporation and polymerization. We previously developed two lines of hCD59 transgenic mice that express hCD59 specifically in erythrocytes or endothelial cells. No obvious adverse phenotypes were observed in these transgenic mice. The injection of ILY causes massive erythrocyte and endothelial damage in erythrocyte-and endothelial-specific hCD59 transgenic mice, respectively, indicating that ILY is able to efficiently and specifically lyse hCD59-expressing cells in mice in vivo. This result suggests that ILY-mediated cell killing might provide an alternative approach to specifically ablate cells in vivo; however, the potential broad application of the ILY-mediated cell ablation model has not been explored.In the current paper, we intend to study from the following two parts:1. We generated a line of Cre-inducible floxed STOP-hCD59 transgenic mice, where specific hCD59 expression occurs following Cre-mediated recombination (ihCD59). By crossing ihCD59 with transgenic mice that express Cre in a cell-specific manner or by delivering an adenovirus expressing Cre, we obtained several lines of mice in which ihCD59 was specifically expressed in a spatially regulated manner on the surface of immune cells, epithelial cells or neural cells.2. ILY injection resulted in conditionally specific cell ablation in various types of cells without any detectable off-target effects on non-targeted cell populations, including the adjacent tissue cells. Moreover, we tested this ablation technique in various disease models and found that this model is valuable for the study of cellular functionalities, tissue injury and regeneration, and neural injury.Methods1. Generation of ILY/ihCD59 mediated cell ablation mice model and abtaincell-specific expression of hCD59 mice1) Generation of transgenic ihCD59 constructpCAG-LSL-hCD59 construct was generated by inserting hCD59 open reading frame to downstream of CAG promoter and LoxP-STOP cassette-LoxP element. hCD59 expression was confirmed in vitro by co-transfection with Cre recombinase in 3T3 cells.2) Generation of Cre-inducible ihCD59+miceThe construct was microinjected into the pronucleus of a mouse zygote which was implanted into CD1 foster mice.. Successful integration from the founder mice was identified by PCR analyses.3) Generation of Cre+ihCD59+mice and characterization of hCD59 expressioni) Generation of hCD59 systemic expression miceihCD59 mice was crossed with systemic Cre+mice, Meox2Cre+, to generate the Meox2Cre+ihCD59+double transgenic mice.ii) Measure hCD59 expression in organshCD59 expression was determined by flow cytometry and Immunohistochemical staining in different organs.4) To facility hCD59 specific expression in micei) Generation of hCD59 specific expression miceihCD59 mice was crossed with various cell specific Cre+mice including LckCre+, CD19Cre+, CD11cCre+, AlbCre+, LyzCre+,Sox9CreERT+ or mGFAP-Cre+ mice to generate the Cre+ihCD59+ double transgenic mice.ii) Measure hCD59 expression in taget cells of different lineshCD59 expression was determined by flow cytometry and Immunohistochemical staining in different cells.2. Characterization of ILY-mediated cell ablation in Cre+ihCD59+mice1) ILY purification and measurement of ILY activityi) ILY purificationHis-tagged recombinant ILY was purified by His-bind purification column.ii) Hemolytic assayThe activity of ILY and heat-inactive ILY was determined by hemolytic assay in vitro.iii) Investigate the pharmacological characteristics of ILYSystemic pharmacokinetics (distribution, elimination half-life, and clearance), the kinetics of tissue distribution, and the toxicity profile of ILY in non-transgenic Wild type mice were characterized.2) Rapid ILY-mediated cell ablation under physiological conditioni) Application of ihCD59 mice model in immune cells ablationDifferent Cre+ihCD59+ were administrated with single or multiple dose of ILY injection, the specific cell ablation in various mice model was inverstigated by flow cytometry and Immunohistochemical/Immunofluorescence staining. The dynamic recovery of each cell population in circulation and tissues after the rapid ablation was also examined. For repopulation of Kupffer cells,7.5X106 GFP+ monocytes with ILY were injected into LyzCre+ihCD59+ mice by i.v injection. Mice were sacrificed 3 d later and Kupffer cells were isolated and analyzed.ii) Application of ihCD59 mice model in organ damage and regenerationTo investigate liver damage and regeneration, AlbCre+ihCD59+, Ad-AlbCre+ihCD59+ and Sox9CreERT+ihCD59+ mice were given a single ILY injection (150 ng/g, i.v.), followed by the measurement of serum ALT and bilirubin, and euthanized 40 h or 48 h post ILY injection. Immunofluorescence staining of BrdU and CK19 was performed in the livers of ILY-treated mice.3) Application of ihCD59 mice for dissecting cellular functionalities in disease modelsi) Immune-related acute diseaseCD11cCre+ihCD59+ mice were given a single PBS or ILY (75 ng/g, i.v.) injection one hour before the injection of 100 ?g/kg (i.v.) a-Galcer. The serum cytokines and ALT were determined 24 h post a-Galcer injection.Lck-CreihCD59+CD19-Cre+ihCD59+,lyz-Cre ihCD59+ and ihCD59+ mice received ILY or PBS by i.v injection,3 h later, Con A (12 mg/kg) was administrated by i.v. injection. Serum was obtained 24 h after ConA injection for ALT and AST measuremen. Liver tissue was fixed in formalin for histological analysis.ii) Immune-mediated chronic diseases:Role of T cell, B cell and monocyte in EAEEAE was induced by subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG) peptide in Lck-Cre+ihCD59+, CD19-Cre+ihCD59+, Lyz-Cre+ihCD59+and ihCD59+mice. Three days after immunization, all the mice received systemic delivery of ILY (100 ng/g/day) via daily subcutaneous injections for 14 consecutive days (until 17 days after immunization). Clinical score was monitored daily after immunization. Histological analyses was performed by myelin basic protein (MBP) and neurofilament stainingiii) Application of ihCD59 mice model for neural cell ablationAfter generation of Brain injury model or Spinal Cord Injury (SCI) model in mGFAP-Cre+ ihCD59+ mice, ILY was injected locally. At day 7, the mice were sacrificed for harvesting the brain and spinal cord. All above tissue was processed GFAP staining.Results1. Generation of ILY/ihCD59 mediated cell ablation mice model and obtain cell-specific expression of hCD59 mice1) Generation of transgenic ihCD59 construct?) Strategy of ihCDS9 constructWe generated ihCD59 transgenic construct first. pCAG-LSL-hCD59 construct was generated by inserting hCD59 open reading frame to downstream of CAG promoter and LoxP-STOP cassette-LoxP element. hCD59 expression was confirmed in vitro by co-transfection with Cre recombinase in NIH 3T3 cells.?) Verificaton of ihCD59 construct in vitroThe construct was verified by in vitro transfection experiments showing that the cells transfected with the construct expressed hCD59 on the surface upon adding Cre-recombinase but did not express hCD59 without Cre expression.2) Generation of ihCD59 transgenic miceBriefly, the construct was introduced into the H11 locus by pronuclear injection to generate ihCD59 knock-in mice at mouse genomic locus H11.3) Test hCD59 expression by crossing ihCD59 mice with germ-line expressing Cre transgenic lineThe ihCD59 mice were crossed with Meox2Cre transgenic mice (a germ-line expressing Cre line), hCD59 protein expression was not detected in any tissues we have tested in Cre negative ihCD59 mice or in naive wild-type C56BL6 mice, but hCD59 protein was highly expressed in all tissues we have tested in Meox2 Cre+ihCD59+ mice. This suggests that hCD59 is constitutively expressed throughout Meox2Cre+ihCD59+ mice.4) Test specific hCD59 expression by crossing ihCD59 mice with cell-specific expressing Cre transgenic lineihCD59 mice was crossed with various cell specific Cre+mice including LckCre+, CD19Cre+, CD11cCre+,AlbCre+, LyzCre+,Sox9CreERT+r mGFAP-Cre+ mice to generate the Cre+ihCD59+ double transgenic mice.ihCD59 transgenic mice were crossed with LckCre+, LyzCre+, or CD11cCre+ mice to generate several lines of double transgenic mice. Flow cytometry analysis confirmed specific hCD59 expression in T cells in LckCre+ihCD59+mice, myeloid cells in LyzCre+ihCD59+ mice, and DCs in CD11cCre+ihCD59+ mice. 2. Characterization of ILY-mediated cell ablation in Cre+ihCD59+ mice 1) To harvest purified ILY and characterize pharmaceutical window of ILYThe acute and chronic toxicity profiles of ILY were examined in Cre negative ihCD59+mice by administrating 1500 ng/g heat-inactivated ILY (no cell lysis activity) or 1500 ng/g active ILY (10-20 times higher than effective doses [75-150 ng/g] used in the ILY-mediated cell ablation model). ILY was given for one dose to assess acute ILY toxicity or daily for 15 d to assess chronic effects of ILY. In ihCD59+ mice treated with one dose or 15 doses (for 15 d) of heat-inactivated or active ILY, various blood cell parameters and serum parameters were not altered. In addition, there were no any abnormalities in major organs examined by histological studies in mice with acute or chronic ILY injection.2) To determine whether ihCD59 mice can be used to ablate various types of cells under physiologic conditioni) Application of ihCD59 mice model in immune cells ablationSplenocytes from LckCre+ihCD59+ mice were isolated and incubated with ILY in vitro. Incubation with ILY lysed almost all of the hCD59+ splenocytes in vitro. Furthermore, an in vitro incubation with ILY killed almost all of the T cells without affecting the B cells of LckCre+ihCD59+ mice.Next, the in vivo administration of a single dose of ILY mediated a specific and rapid depletion of 85% T cells (CD4+or CD8+) and myeloid cells (CD11b+) in the peripheral blood of LckCre+ihCD59+and LyzCre+ihCD59+ mice, respectively. The injection of ILY also rapidly and efficiently depleted DCs and T cells in the spleens of CD11cCre+ihCD59+ and LckCre+ihCD59+ mice, respectively. This specific deletion of T cells in LckCre ihCD59+ mice is also dose dependent. ILY administration also specifically deleted T cells in the spleen but to a lesser extent.The dynamic recovery of each immune cell population in circulation and in tissues after the rapid ablation with a single ILY injection was examined. After a single ILY injection, circulating T cells in LckCre+ihCD59+ mice and circulating monocytes in LckCre+ihCD59+ mice were rapidly depleted by approximately 90% within 0.1 hours (h), but recovered to near normal levels 24 h post injection. Interestingly, in ILY-treated LyzCre+ihCD59+ mice, Ly6C-monocytes recovered much more slowly than did Ly6C+ monocytes. In addition, the injection of ILY also efficiently deleted T cells and DCs in the spleens of LckCre+ihCD59+ and CD11cCre+ ihCD59+ mice, respectively, and recovered to near normal levels 24 h post injection. Multiple ILY injections are able to induce the chronic ablation of immune cells.To determine whether ILY efficiently depletes liver macrophages, immunostaining with F4/80 was performed. ILY injection into LyzCre+ihCD59+ mice markedly reduced the number of F4/80+ cells. Using a BrdU incorporation assay demonstrated that the proliferation of Kupffer cells contributes to the replenishment of Kupffer cells after depletion.To determine whether monocytes also partly contribute to Kupffer cell replenishment, we utilized the adoptive transfer of GFP+ monocytes to mice. FewKupffer cell-like GFP+CD11bloF4/80hi cells were identified after adoptive transfer GFP+monocytes into wild-type C57BL6 mice. Further, GFP+ monocytes were adoptively transferred to ILY-treated LyzCre+ihCD59+ mice in which Kupffer cells were depleted. Interestingly, approximately 1.5% of F4/80hi CDllblo Kupffer cells were GFP+ cells that were contributed from 7.5×106 transferred GFP+monocytes. These results suggest that monocytes can be converted into Kupffer cells after acute cell loss, highlighting that the ihCD59 model is useful to study immune cell differentiation and repopulation.ii) ihCD59 mice mediated highly efficient and selective ablation of liver epithelial cells and liver injuryAlbCre mouse is a widely used Cre line to delete a floxed sequence in both hepatocytes and BECs. In AlbCre+ ihCD59+ mice, hCD59 protein expression was specifically detected in both hepatocytes and BECs in the liver. ILY administration to AlbCre+ihCD59+ mice rapidly induced a dramatic elevation of serum ALT levels (indication of hepatocyte injury) and elevation of bilirubin levels (indication of bile duct injury). Importantly, ILY injection induced liver damage (elevation of serum ALT and AST) in a dose-dependent manner. Second, to exclusively express hCD59 in hepatocytes, we infected ihCD59+ mice with an adenovirus expressing Cre that is controlled by the Alb promoter (Ad-AlbCre) to develop a model in which hCD59 is expressed only in hepatocytes through a viral delivery system (Ad-AlbCre+ihCD59+). The injection of ILY rapidly induced damage to hepatocytes but not damage in BECs, as demonstrated by the elevation of serum ALT but not bilirubin. Finally, to exclusively express hCD59 in BECs, ihCD59+ mice were crossed with tamoxifen-inducible Sox9Cre (Sox9CreERT), a model that is used to express Cre in BECs only in the liver. The treatment of Sox9CreERT+ihCD59+ mice with tamoxifen induced temporal and specific expression of hCD59 on BECs but not on hepatocytes. The injection of ILY into Sox9CreERT+ihCD59+ mice rapidly induced severe bile duct damage but little hepatocyte damage.In the meantime, significant proliferation of both hepatocytes and BECs was observed in AlbCre+ihCD59+ mice, with the peak effect occurring 40 h post ILY injection. In contrast, only hepatocyte proliferation and not BEC proliferation was observed in ILY-treated Ad-AlbCre+ihCD59+ mice. Interestingly, both hepatocyte and BEC proliferation were observed in ILY-treated Sox9CreERT+ihCD59+ mice, despite only BECs being damaged in this model.Significant a-SMA-positive staining was observed in the portal vein area of AlbCre+ihCD59+ mice, suggesting moderate liver fibrosis after multiple ILY injections. Interestingly, we found some oval cells (also called liver progenitor cells [LPCs]) in the a-SMA-positive areas. In contrast, neither liver fibrosis nor LPC expansion was observed in Ad-AlbCre+ihCD59+ or Sox9CreERT+ihCD59+ mice that received multiple injections of ILY.3) Application of ihCD59 mice for dissecting cellular functionalities in disease modelsi) Application of ILY/ihCD59 cell ablation for dissecting cellular functionalities in acute liver injuryFirst, we used a model of a-Galcer-induced natural killer T (NKT)-mediated acute liver injury to evaluate whether DC ablation by ILY affects antigen presentation to NKT cells. Results suggested ILY treatment significantly blocked the a-Galcer-induced cytokine production and serum ALT elevation in CD11cCre+ihCD59+ mice but not in ihCD59+mice.Second, we performed concanavalin A (Con A)-induced acute hepatitis, a widely used model to study immune-mediated liver injury. Pre-treatment with ILY completely abolished Con A-induced liver injury in LckCre+ihCD59+mice and ameliorated the liver injury to a lesser extent in LyzCre+ihCD59+ mice but did not affect the liver injury in CD19Cre+ihCD59+or ihCD59+ mice.ii) Application of ILY/ihCD59 cell ablation for dissecting cellular functionalities in EAEThe ablation of T cells in LckCre+hCD59+ mice completely prevented EAE development, and the ablation of myeloid cells in LyzCre+ ihCD59+ mice also attenuated the EAE scores but to a lesser extent. In contrast, the EAE scores were only modestly reduced in the B cell-ablated CD19Cre+ ihCD59+ mice.iii) Application of ILY/ihCD59 cell ablation for dissecting glial cell functionalities in CNS injuryStereotaxic injection of ILY but not PBS resulted in a dramatic reduction in the number of reactive astrocytes (GFAP+and hCD59+) in the vicinity of the brain injury in mGFAPCre+ ihCD59+ but not ihCD59+ mice. Consistently, i.v. injection of ILY to the mice also mediated the specific ablation of reactive astrocytes, although it was much less efficient than a local injection.In addition, the number of reactive astrocytes was greatly reduced in the mGFAPCre+ihCD59+mice compared to ihCD59+ mice with ILY treatment upon spinal cord injury. ILY-treated mGFAPCre+ ihCD59+mice exhibited slower behavioral recovery than did ILY-treated control miceConclusions1. ihCD59 mice model mediated specific and rapid cell ablation in a dose-dependent manner without any detectable off-target effects.In the current study, we successfully developed ihCD59 transgenic mice, which can be used to ablate a wide variety of cell types in vivo, such as immune cells in peripheral system, epithelial cells in liver and glia cell in CNS, by crossing with various lines of Cre transgenic mice and injecting ILY.2. ihCD59 mediated cell ablation can be used for study cell function in various disease modelsThe unique features of the ILY/ihCD59 model with high specificity and lack of off-target effects make it possible to create a chronic cell ablation model with multiple injections of ILY. This chronic cell ablation feature allowed us to study the functions of various types of immune cells in chronic immune-mediated diseases, such as EAE and liver injury.3. A large pharmaceutical window of ILY-mediated cell ablation modelILY has at least 10-to 20-fold higher pharmacological window than the effective dose required for the targeted cell ablation.Originality and SignificanceIn the current study, we successfully developed ihCD59 transgenic mice, which can be used to ablate a wide variety of cell types in vivo by crossing with various lines of Cre transgenic mice and injecting ILY. Compared to several existing cell ablation models, the ILY/ihCD59 cell ablation method has several attractive features:1. Rapidity.The effective cell ablation occurs within seconds after ILY administration.2. Specificity.ILY-mediated cell ablation is specifically confined to cells expressing hCD59. ILY lyses only cells expressing hCD59 with no cross reactivity in cells expressing CD59 from other species.3. Large pharmaceutical windowILY has at least 10-to 20-fold higher pharmacological window than the effective dose required for the targeted cell ablation.4. ILY mediated cell ablation is dose-dependentLarge pharmacological window in our model system will allow us readily to perform the dose dependent studies to partially or fully ablate the target cells and easily interpret the results.5. Easy to administerILY-mediated cell ablation could be equally achieved by intravenous (i.v.), intraperitoneal (i.p.), intramuscular (i.m.), intracranial or local administration.Limitations of present studyAlthough we have not fully studied the bio-distribution of ILY in mice, we observed that ILY most efficiently ablated circulating leukocytes in peripheral blood, and partially deleted leukocytes in the spleen but barely affect the cells in the thymus and bone marrow, indicating the existence of ILY-privileged compartments.ObjectiveAtherosclerosis is a chronic inflammatory and metabolic disease in the wall of large-and medium-sized arteries. Modified low-density lipoprotein (LDL), such as oxidized LDL (ox-LDL), triggers the disease by deposition at specific sites of the arterial intima, thereby becoming a crucial stimulator of the innate and adaptive immune system. In this way, the uptake of modified lipoproteins by macrophages accompanied by defective cholesterol efflux results in foam-cell formation, which plays an important role in the growth of atherosclerotic plaque and vulnerable plaque.Macroautophagy (hereafter referred to as autophagy) is a highly conserved lysosomal macromolecules (e.g., proteins and lipids) and dysfunctional organelles (e.g., mitochondria and endoplasmic reticulum) are degraded and recycled to maintain cellular homeostasis. Accumulating evidence suggests that autophagy, especially macrophage autophagy, plays an important role in the pathogenesis of atherosclerosis. In advanced atherosclerosis, generation of several atherosclerotic factors, such as ox-LDL,7-ketocholesterol and reactive oxygen species (ROS), may result in dysfunctional autophagy, thereby leading to plaque development and instability. Basal autophagy has an essential role in anti-atherosclerosis. Basic autophagy deficiency in macrophages by specific autophage protein 5 knockout accelerated degradation pathway by which intracellular components, including soluble atherosclerotic plaques in high-fat diet (HFD)-fed ldlr-/- mice via promoting oxidative stress, plaque necrosis or inflammasome hyperactivation. As well, autophagy can enhance the breakdown of lipids in lipid droplets (LDs) and cholesterol efflux from macrophage foam cells and further inhibit atherogenesis.3-methyladenine (3-MA) is a widely used inhibitor of autophagy because of its inhibitory effect on class III phosphatidylinositol 3-kinase (PtdIns3K). However, 3-MA has an autophagic promotion effect under nutrient-rich conditions based on its inhibitory effect on class I PtdIns3K. In vivo,3-MA has had promising theraputic benefit in ameliorating experimental autoimmune neuritis in rat and controlling Enterovirus 71 infection and pathogenesis. Morever,3-MA can suppress tumor metastasis in an autophagy-independent manner. However, the role of 3-MA in atherosclerosis development remains to be investigated.In our experiment, to assess whether 3-MA can affect atherosclerosis, we systemically administered 3-MA to ApoE-/- mice fed a HFD for 8 weeks and analyzed its effect on atherosclerosis. We also tested the effect of 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), another PI3K inhibitor and commonly used as an autophagy inhibitor. Administration of 3-MA significantly inhibited the formation of atherosclerotic lesions and enhanced the stability of plaque; LY294002 had no atheroprotective effects. Our data provide strong evidence for the potential therapeutic benefit of 3-MA in atherosclerosis. We explored the potential mechanism of the role of 3-MA in atheroprotection and found multiple mechanisms, including modulating macrophage autophagy and foam-cell formation and modifying the immune microenvironment.Methods1. To study the effect of 3-MA and LY294002 on development of atherosclerosis1) To set up atherosclerosis model with ApoE-/- mice and perform 3-MA and LY294002 administration.ApoEE-/- mice were fed a high fat diet (HFD) starting from 8 weeks old to 17 weeks old. We performed the first experiment with three groups of randomly divided mice:1) control (PBS, i.p. n=14); 2) 3-MA (30 mg/kg, Sigma Aldrich, n=16; and 3) LY294002 (0.3 mg/kg, Sigma Aldrich, n=6). Each animal received i.p injection twice a week for 8 weeks. Both 3-MA and LY294002 were dissolved in PBS and kept at -20?. We heated the 3-MA solution to 60? immediately each time before injection. One week after the last treatment, mice were killed for further study.2) To detect atherosclerosis development in 3-MA and LY294002 treated mice?) En face staining analysisSix mice from each group were killed and aortas from the heart to the iliac arteries were dissected, soaked overnight in 4% paraformaldehyde for fixation, excised longitudinally and stained with 0.5% Oil-red O for 2 h, then pinned flat on a black surface by using Minutien (Fine Science Tools, Inc).?) Detect the size of plaque and lipid content in aortic rootAfter mice were killed, aortic root vessels were fixed in 4% paraformaldehyde overnight and then embedded in optimal cutting temperature (OCT) compound. Serial cryosections (7 ?m thickness) were cut along the aortic root specimens by using a cryotome. At least three transverse sections (spaced about 50?m) from each aortic root were stained with hematoxylin and eosin (HE) for histopathology analysis. Lipid content in plaque area was evaluated by Oil-red O staining?) Study plaque contents by immunohistochemistry (IHC)Collagen content in plaque area was evaluated by Sirius red staining. To detect the proportion of macrophages and SMCs, corresponding cryosections on separate slides underwent IHC staining with a rat anti-mouse macrophage-specific antibody or a rabbit anti-mouse polyclonal antibody for smooth muscle actin. ?) Apoptotic cells in aortic root sections were detected by using an in situ cell death detection kit (Roche, USA)?) Calculated the vulnerable index of each group of miceWe calculated the vulnerable index, the ratio of plaque area occupied by lipid components (in macrophages+extracellular lipids) and by fibromuscular components (SMCs+collagen fibers).2. To study the potential mechanism by which 3-MA affects atherosclerosis1) To study the effect of 3-MA on autophagy under high fat conditionWe use mouse primary peritoneal macrophage to study 3-MA's function on autophagy during foam cell formation. For foam cell formation assay, mouse primary peritoneal macrophages were pretreated of 5 mM 3-MA (3-MA group) or PBS (control group) in DMEM containing 10% FBS for 30 min at 37?, then 50 ?g/mL oxygenized-LDL (ox-LDL) was added to medium of both 3-MA and control groups to induce foam cell formation. Cells without any treatment were used as a negative control. Cells were extensively washed for total protein extraction 24,48 or 72 h after treatment.For detecting autophagosome in plaque, cryosections were stained for microtubule-associated protein 1A/1B-light chain 3 (LC3) by a rabbit anti-LC3 antibody.2) To study the effect of 3-MA on foam cell formation and viability of macrophagesWe use mouse primary peritoneal macrophage to study 3-MA's function on foam cell formation. For foam cell formation assay, mouse primary peritoneal macrophages were pretreated of 5 mM 3-MA (3-MA group) or PBS (control group) in DMEM containing 10% FBS for 30 min at 37?, then 50 ?g/mL oxygenized-LDL (ox-LDL) was added to medium of both 3-MA and control groups to induce foam cell formation. Cells without any treatment were used as a negative control. Cells were extensively washed for Oil-red O staining 24,48 or 72 h after treatment.Cell viability was tested by cell counting kit-8 (CCK-8) assay. Mouse primary peritoneal macrophages were seeded in 96-well cell culture plates and divided into four groups for treatment:no treatment, ox-LDL treatment only,30 min 3-MA pre-incubation (5 mM) before ox-LDL treatment, and 30 min rapamycin pre-incubation (100 nM) before ox-LDL treatment. Cells were cultured at 37? for 24, 48 and 72 h. At each time, cells were washed. An amount of 10 ?l CCk-8 solution was added to each well for incubation at 37? for 1 h. The absorbance of each well was measured at 450 nm.3) To find out if 3-MA affected cytokine expression in plaque areaReal-time PCR was performed with equal amounts of cDNA in triplicate and Ultra SYBR Mixture to detect IL-17?IFN-??IL-6?TGF-??IL-10?IL-35. Data for relative molecule expression were presented by using the 2??Ct method. Data are reported as fold change in the experimental group normalized to an endogenous reference gene (18S) and relative to the control group.Results1.3-MA markedly inhibited the development of atherosclerotic lesionOil-red O staining of aortas showed that mice treated with 3-MA (n=16) showed significantly less atherosclerotic lesions in the whole aorta (including aortic arch, thoracic and abdominal regions) compared to PBS-treated controls (n=14). Consistently, HE staining and Oil-red O staining of aortic roots also showed significantly decreased atherosclerotic plaques and lipid content in 3-MA-treated mice. To explore whether other autophagy inhibitors have the same effect, we investigated the impact of LY294002 on development of atherosclerosis. LY294002 treatment had no significant effect on atherosclerotic lesions.2.3-MA application improved the stability of plaque3-MA did not affect CD3 proportion in lesions but decreased the number of infiltrated macrophages, presented as elevated MOMA-2 level, as compared with controls. In parallel, the proportion of SMCs was increased as compared with controls. Collagen content within the plaque of 3-MA-treated mice was increased but not significantly.3-MA treatment decreased the number of TUNEL-positive apoptotic cells. We calculated the vulnerable index and found it significantly lower in mice with 3-MA than controls, so 3-MA promoted a stabilized phenotype in the lesion area. However, LY294002 application had no significant effect on plaque contents.3.3-MA affected ox-LDL-induced macrophage autophagy in vitro but decreased the level of autophagy in plaque in vivoCultured primary peritoneal macrophages were stimulated with ox-LDL for 24, 48, and 72 h with and without 3-MA.3-MA significantly increased the protein expression of LC3 and decreased that of sequestosome 1 (SQSTM1, p62) as compared with controls, which suggests that 3-MA improved autophagy flux in ox-LDL-stimulated macrophages. In contrast with in vitro results, plaque of 3-MA-treated mice showed decreased LC3-II expression as compared with controls. We also used immunofluorescence staining to detect the change of LC3-II level in plaque. Moreover, in aortic root of 3-MA-treated mice and controls. Level of pS6 was lower with 3-MA than control treatment although not significantly (p=0.0876).4.3-MA decreased foam-cell formation in vitro and limited the accumulation of LDs within atherosclerotic plaque in vivoThe number of LDs within plaque was markedly lower with 3-MA than control treatment. Furthermore, the number of LDs was reduced in LC3-positive regions but was higher in LC3-negative regions, which supports that autophagy promotes the breakdown of LDs in plaque. Furthermore,3-MA greatly reduced macrophage formation at 24,48 and 72 h after ox-LDL stimulation in vitro.3-MA exposure retained mouse peritoneal macrophages in a round shape with less lipid burden under ox-LDL challenge as compared with the spindle shape and foamy morphology of characteristic foam cells with ox-LDL treatment alone. The lipid burden of macrophages without 3-MA treatment was gradually decreased at 72 h as compared with 24 h. But we did not see this phenomenon in 3-MA-treated macrophages.5.3-MA inhibited the viability of ox-LDL-stimulated macrophages in vitro3-MA treatment significantly reduced macrophage content in plaque lesions. Furthermore,3-MA, as well as rapamycin, the classical mTOR inhibitor, time-dependently inhibited cell viability as compared with controls.6.3-MA improved the anti-inflammatory microenvironment in atherosclerotic plaque3-MA treatment had no effect on the expression of proinflammatory cytokines such as IL-6...