Effect Of Regulatory T Cells On Vulnerable Atherosclerotic Plaque:an Experimental Study

1IntroductionAtherosclerosis is a multifactorial process. In recent years, the role of the immune system in atherosclerosis has received considerable attention. Atherosclerosis is now considered an autoimmune-like disease with both innate and adaptive immune responses contributing to atherosclerosis development and progression. However, the exact immunomodulatory mechanisms remain poorly understood. Histological markers for rupture-prone atherosclerotic plaque include a thin fibrous cap, a large lipid core, enriched activated T cells and macrophages, and depleted extracellular matrix (ECM). As SMCs and collagen-rich extracellular matrix are the major component of the fibrous cap of atherosclerotic plaques, fibrous caps deprived of SMCs and collagen is associated with plaque instability.CD4+CD25+regulatory T cells (Tregs) as a specific subpopulation of T cells are important components of the immune system. Tregs maintain immunological tolerance, terminate an activated immune response and control the progression of autoimmune diseases. Deficiency or dysfunction of Tregs disrupts immune homeostasis and leads to many pathological conditions.Emerging evidence has suggested that Tregs treatment attenuates early atherosclerotic lesions in ApoE-/-mice. However, it is still unclear whether Tregs treatment is able to enhance plaque stability dose-dependently, and if it is, what mechanisms may underlie these therapeutic effects.2Objectives(1) To assess the beneficial effects of Tregs on plaque disruption and plaque composition in apolipoprotein E-knockout mice.(2) To elucidate the inherent molecular mechanisms of Tregs-mediated beneficial effects on vulnerable plaque.3Methods3.1Isolation of TregsThe C57BL/6J mice served as Tregs cell donors. Purified Tregs were isolated from the splenocytes of C57BL/6J mice with use of the CD4+CD25+Regulatory T cell Isolation Kit. The purity of Tregs subsets was>95%as determined by FACS (Fluorescence Activated Cell Sorting) analysis.3.2Animal protocolTwo weeks after high-fat diet feeding, all ApoE-/-mice were anaesthetized with an intraperitoneal injection of pentobarbital sodium (40mg/kg), and a silastic perivascular collar was placed around the right common carotid artery in all mice to induce atherosclerotic lesions as described previously. Eight weeks after surgery, ApoE-/-mice were randomly divided into5groups:control group that received no treatment; PBS group that received injection of200μl PBS via the tail vein; small-dose Tregs group that received intravenous injection of105Tregs; moderate-dose Tregs group that received intravenous injection of3～105Tregs and large-dose Tregs group that received intravenous injection of106Tregs. These treatments were repeated two weeks later. All mice underwent stress stimulation one day after the initial injection of Tregs using our previously reported method with modification. The mice were restrained in a50-ml plastic tube with multiple holes on the wall to ensure sufficient ventilation and sound transmission, and these mice were then put into a noise generator that emits noise every3min at110dB for5sec. The restraint stress and noise stimulation lasted for6h per day for4weeks. At the end of14weeks, all apoE-/-mice were euthanized and the right common carotid arteries and blood from the inferior vena cava were collected for further analysis.3.3Body weight and Blood pressure measurementBody weight was measured at the beginning and the end of the experiment in all ApoE-/-mice. At the end of14weeks, systolic blood pressure (SBP), diastolic blood pressure (DBP) and heat rate (HR) were recorded in ApoE-/-mice by a noninvasive tail-cuff system. Three consecutive measurements were recorded and the values averaged.Mice were starved overnight and blood samples were collected before euthanasia. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined with use of an enzymatic assay.3.4Histopathological analysisSuccessive transverse cryosections of5μm in thickness of the carotid arteries were obtained and selectively stained with hematoxylin and eosin (H&E), picosirus red and Oil-red O staining. Plaque disruption was defined as fibrous cap discontinuation with or without luminal thrombosis, intraplaque hemorrhage or buried fibrous cap. Furthermore, Frozen sections of right common carotid arteries were stained for immunohistochemical analysis, including macrophages (MOMA-2), SMCs (a-smooth muscle actin), monocyte chemoattractant protein-1(MCP-1), tumor necrosis factor-a (TNF-a), interleukin-6(IL-6) interleukin-17(IL-17), matrix metalloproteinase2(MMP-2) and matrix metalloproteinase9(MMP-9). The vulnerability index was calculated as (macrophage stained%+lipid stained%)/(collagen stained%+a-SM actin stained%).3.5Cell separation and cocultureHuman peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and prepared by centrifugation. Tregs and CD4+CD25-T cells were isolated from PBMCs by use of a Human CD4+CD25+Regulatory T cell Isolation Kit according to the manufacturer's instruction.Human aortic smooth muscle cells (HASMCs) were cultured in a serum medium without T cells (control group) or with CD4+CD25-T cells (CD25-group) or with Tregs at3concentrations (1.25×105group,2.5×105group and5×105group, respectively) for40h in the presence of anti-CD3antibody (50ng/ml). Then all cultures were treated with human recombinant TNF-a (100ng/ml) for an additional8h. After floating T cells were discarded, HASMCs were harvested and supernatants were collected. In addition, neutralizing antibodies against TGF-β or IL-10or isotype mAbs were added to the medium at the beginning of the coculture of HASMCs and Tregs to examine the role of TGF-β and IL-10in Tregs-mediated effects. 3.6Quantitative real-time PCRTotal RNA was extracted from the right common carotid arteries of ApoE-/-mice, or HASMCs. In the in vivo experiment, the mRNA expression of Foxp3, TGF-β, IL-10, MMP-2and MMP-9was analyzed. In the in vitro experiment, the mRNA expression of P4Ha1, MCP-1, VCAM-1, ICAM-1, MMP-2and MMP-9in HASMCs was analyzed too.3.7Western blot analysisTotal proteins were extracted from the right common carotid arteries of ApoE-/-mice or HASMCs. The protein expression of Foxp3, TGF-p, IL-10, MMP-2, MMP-9, P4Hal in the right common carotid plaques and P4Hal, MMP-2, MMP-9, Askl, JNK in HASMCs were analyzed by western blot.3.8ELISASoluble type I and III collagen, TGF-β and IL-10levels in culture supernatants was assayed by ELISA.4Results4.1Body weight, blood pressure and serum lipid profilesThere was no significant difference in body weight, SBP, DBP or serum levels of TC, TG, LDL-C and HDL-C among the5groups of ApoE-/-mice.4.2Tregs prevented carotid plaque disruptionHistopathological studies revealed that in ApoE-/-mice, the incidence of plaque disruption was50%(8/16) in the control group,50%(8/16) in the PBS group,43.8%(7/16) in the small-dose Tregs group,12.5%(2/16) in the moderate-dose Tregs group and12.5%(2/16) in the large-dose Tregs group. These results indicated that adoptive transfer of Tregs dose-dependently lowered the incidence of plaque disruption in comparison with the control and the PBS groups in ApoE-/-mice.4.3Tregs changed carotid plaque compositionThe relative contents of macrophages and lipids in the carotid plaque were significantly reduced in the moderate-dose and large-dose Tregs groups versus that in the control, PBS and small-dose Tregs groups. On the other hand, the relative contents of collagen and a-actin of SMCs in the carotid plaque were significantly higher in the moderate-dose and large-dose Tregs groups than those in the control, PBS and small-dose Tregs groups. Consequently, the vulnerability index was remarkably lower in the moderate-dose and large-dose Tregs groups than that in the control, PBS and small-dose Tregs groups.4.4Tregs reduced inflammatory cytokine expression in vivo and in vitroIn ApoE-/-mice, the relative contents of MCP-1, TNF-a, IL-6and IL-17in the carotid plaque assessed by immunohistochemistry was significantly lower in the moderate-dose and large-dose Tregs groups than those in the control, PBS and small-dose Tregs groups. In cultured HASMCs, the mRNA expression levels of MCP-1, VCAM-1and ICAM-1in the three groups of Tregs-treated HASMCs were remarkably lower than in the two groups of HASMCs cultured in a medium without T cells or with CD4+CD25'T cells.4.5Tregs lowered MMP-2and MMP-9expression in vivo and in vitroIn ApoE-/-mice, the mRNA expression of MMP-2and MMP-9in the carotid plaques were lower in the moderate-and large-dose Tregs groups than those in the control, PBS and small-dose Tregs groups. Quantitative measurements of protein expression of MMP-2and MMP-9by immunohistochemistry or western blot analysis revealed similar results, respectively. In cultured HASMCs, the mRNA and protein expression levels of MMP-2and MMP-9in the three groups of Tregs-treated HASMCs were significantly lower than those in the two groups of HASMCs cultured in a medium without T cells or with CD4+CD25-T cells.4.6Tregs modulated P4Hal and ASK1-JNK expressionIn ApoE-/-mice, the protein expression level of P4Hal was significantly increased in the moderate-dose and large-dose Tregs groups in comparison with that in the control, PBS and small-dose Tregs groups. In cultured HASMCs, the mRNA and protein expression level of P4Hal was higher in the three groups of Tregs-treated HASMCs than in the two groups of HASMCs cultured in a medium without T cells or with CD4+CD25-T cells. Moreover, the protein expression levels of type I and III collagen were remarkably higher than in the two groups of HASMCs cultured in a medium without T cells or with CD4+CD25-T cells. TNF-a suppressed P4Hal expression via activating the ASK1-JNK pathway, and we found that the protein expression levels of ASK1and JNK were significantly lower in the three groups of Tregs-treated HASMCs than in the two groups of HASMCs cultured in a medium without T cells or with CD4+CD25-T cells. 4.7Tregs enhanced TGF-β and IL-10expressionThe expression levels of TGF-β and IL-10were significantly increased in the moderate-dose and large-dose Tregs groups than those in the control, PBS and small-dose Tregs groups. Moreover, the levels of TGF-β and IL-10in the three groups of Tregs-treated HASMCs were remarkably higher than in the two groups of HASMCs cultured in a medium without T cells or with CD4+CD25-T cells. These results documented that Tregs increased the levels of TGF-β and IL-10both in vivo and in vitro.4.8TGF-β and IL-10mediated the effects of Tregs on P4Hα1and inflammatory cytokinesTo explore whether TGF-β and IL-10mediated the effects of Tregs on MCP-1, VCAM-1, ICAM-1, MMP-2, MMP-9and P4Hα1expression, neutralizing antibodies against TGF-β or IL-10were applied. The results showed that mRNA expression of MCP-1, VCAM-1and ICAM-1in Tregs-treated HASMCs was increased after treatment with neutralizing antibodies against TGF-β or IL-10. Similarly, mRNA and protein expression of MMP-2and MMP-9in Tregs-treated HASMCs was also markedly increased after treatment with neutralizing antibodies against TGF-β or IL-10. In contrast, mRNA and protein expression of P4Hal in Tregs-treated HASMCs was significantly attenuated when neutralizing antibodies against TGF-β or IL-10were added. These results suggested that Tregs-induced effects on the expression of inflammatory cytokines, MMPs and P4Hal were mediated by TGF-β and IL-10.5Conclusion(1) Adoptive transfer of Tregs dose-dependently entered the carotid plaque, changed plaque composition to a stable plaque phenotype and lowered the incidence of plaque disruption.(2) Tregs have a protective role in the destabilization and rupture of atherosclerotic plaques, and the major mechanisms may involved reduced expression of inflammatory cytokine and MMP-2and MMP-9, increased the secretion of anti-inflammatory cytokine, enhanced expression of P4Hα1and suppressed expression of ASK1-JNK in the carotid plaque of ApoE-/-mice. (3) The beneficial effect of Tregs were partly mediated by TGF-β和IL-10. 1IntroductionThe pathogenesis of atherosclerosis is multifactorial. Much attention has been given to the role of the inflammation in the development of atherosclerosis. There was an association between inflammation and immune system. Many important immune factor played a role in reducing inflammation, while, some inflammatory cell possessed an immune capability. Accelerated atherosclerosis was found in some autoimmune diseases such as systemic lupus erythematosus, systemic sclerosis and rheumatoid arthritis.Statins (HMG-CoA reductase inhibitors) improve endothelial function, reduce inflammation, prevent the progression of atherosclerosis and promote stability in vulnerable atherosclerotic plaque. In addition to their potent lipid-lowering capabilities, statins have beneficial pleiotropic effects. They possess immunomodulatory properties, including inhibiting interferon y (IFN-y)-induced expression of major histocompatibility complex (MHC) class II genes and effector T cell activation, and their role in atherosclerosis depends in part on their immunomodulatory mechanism.CD4+CD25+regulatory T cells (Tregs) are a healthy T cell compartment. We have been documented Tregs were capable of inhibitting the accumulation of inflammatory cell, preventing the produce of proinflammatory factor, reducing the inflammatory response. However, whether statins treatment is able to influence Tregs numbers in atherosclerotic plaques is still unclear.2Objectives (1) To assess the effects of simvastatin treatment on Tregs numbers in atherosclerotic plaques of apolipoprotein E-knockout mice.(2) To investigate the effect of simvastatin treatment on the levels of Tregs and immunosuppressive function in the peripheral circulation of patients with ACS.3Methods3.1Animal protocolMale forty ApoE-/-mice on a C57BL/6background (10weeks old) were obtained. Two weeks after high-fat diet feeding, a silastic perivascular collar was placed around the right common carotid artery in all ApoE-/-mice to induce atherosclerotic lesions. All mice were randomly assigned to2groups (n=20per group):simvastatin group that received intragastric administration of simvastatin (50mg/kg/d) in0.5%methylcellulose; control group that received methylcellulose alone. After6weeks, the mice were euthanized and tissues were harvested for further analysis.3.2Lipid ProfileMice were starved overnight and blood samples were collected before euthanasia. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined with use of an enzymatic assay.3.3Histopathological analysisIn the animals, the right common carotid arteries were removed and consecutive sections (5-μm thickness) were collected from each mouse for immunohistochemic analysis. Frozen sections of right common carotid arteries were performed for immunohistochemical analysis, including natural Tregs (Foxp3), IL-4, IL-1β, IFN-y and IL-17.3.4Quantitative real-time PCRTotal RNA was isolated from the right common carotid arteries from the mice. The mRNA expression of Foxp3, TGF-β, IL-10, IL-4, IL-1β, IFN-y and IL-17was analyzed.3.5Western blot analysisThe right common carotid arteries of apoE-/-mice were isolated and protein was extracted. The protein expression of Foxp3, TGF-β, IL-10were analyzed.3.6ELISA The concentrations of IL-10, IFN-y and IL-17in serum were determined by ELISA.3.7Cell culturePeripheral blood mononuclear cells (PBMCs) were isolated from blood samples of10male ACS patients and prepared at1×106cells/ml in RPMI1640medium containing10%fetal bovine serum (FBS) and1%penicillin/streptomycin without additives (control) or with simvastatin (1or10μM) for96hr. After96-hr incubation, PBMCs in different cultures were harvested for flow cytometry to detect CD4+CD25+Foxp3+TregsIn addition, different ratios of Tregs and CD4+CD25" T cells (1:1,1:2,1:4,1:8) were added to96-well plates with plate-bound anti-CD3monoclonal antibody (1μg/well) and co-cultured in the presence of105CD4-T cells pretreated with mitomycin C for72hr. A total of1μCi [3H]-thymidine was added for the final16hr, and proliferation was assayed with use of a scintillation counter (β-counter). The experiment was repeated in the presence of simvastatin at1or10uM.4. Results4.1Lipid ProfileThere was no significant difference in serum levels of TC, TG, LDL-C and HDL-C between the2groups of ApoE-/-mice.4.2Simvastatin upregulated the expression of Foxp3, IL-10and TGF-β in atherosclerotic plaquesAfter6weeks of simvastatin treatment, immunohistochemical studies showed an increase in the number of Tregs within atherosclerotic plaque. Furthermore, the mRNA and protein expression of Foxp3was markedly increased in the simvastatin group versus those in the control group. These results confirmed that simvastatin might induce an accumulation of Tregs in atherosclerotic plaques.In line with the change in Foxp3expression, mRNA and protein expression of TGF-β and IL-10were significantly increased in the simvastatin group than those in the control group.4.3Simvastatin changed the cytokine production in atherosclerotic plaquesSimvastatin treatment led to a significant increase of IL-4and a decrease of IL-1β, IL-17and IFN-y in mRNA expressions in the carotid plaque of two groups of mice. The protein expression level of cytokine in the carotid plaques was also assessed by immunohistochemistry. As compared with the control group, simvastatin significantly upregulated the relative contents of IL-4and downregulated IL-1β,IL-17and IFN-y in the carotid plaque.4.4Simvastatin influenced circulatory levels of IL-10, IFN-y and IL-17The serum levels of IL-10were greatly increased in simvastatin group than the control group, but the circulatory levels of IFN-y and IL-17were significantly reduced after the simvastatin treatment.4.5Simvastatin improved the proportion and regulatory function of Tregs in PBMCs from ACS patientsPBMCs were cultured with simvastatin at different concentrations, and the proportion of Tregs in PBMCs was analyzed by CD4+/CD25+/Foxp3+staining. Compared with the control group,10μM simvastatin increased the proportion of CD4+CD25+Foxp3+Tregs to total CD4+T cells. Although1μM simvastatin produced a slight increase in the frequency of Tregs relative to control group, the effect was not significant.In addition, the suppression rate with10μM simvastatin was higher than that for the control group at different ratios of Tregs and CD4+CD25-T cells (1:1,1:2,1:4,1:8). Moreover,10μM simvastain increased the extent of suppression relative to1μM simvastain. Simvastain at the low concentration did not have any effect.5. Conclusion(1) Statins treatment resulted in increased expression of Foxp3and accumulation of Tregs in atherosclerotic lesions of ApoE-/-mice, and modulated the Thl/Th2/Thl7balance.(2) Statins treatment increased the number of Tregs and repaired their compromised functional inhibitory properties in patients with ACS(3) The potential beneficial role of statins on atherosclerosis plaque may be in part through Tregs activation. 1IntroductionAbdominal aortic aneurysm (AAA) is a chronic vascular degenerative disease associated with high mortality among elderly people. It is characterized by the deterioration of tissue architecture leading to progressive aortic dilation with a potential for rupture of the vessel wall. Currently, the mechanism and pathogenesis of AAA have not been fully illuminated and the therapeutic methods are limited to surgical repair. However, surgery is not appropriate for all AAA, expecially smy. So, a sall, asymptomatic AAA, or patients with contraindication for surgerafe and effective pharmacological strategy that can prevent aneurysm development and rupture is needed.The extracellular matrix (ECM) of vessels provides a structural support to the arterial wall. Chronic inflammation and excess ECM degradation contribute to aortic expansion and are believed to play an central role in the pathogenesis of AAA. Aneurysmal segments are characterized by an inflammatory response with cell infiltration (primarily macrophages and T lymphocytes) and increased expression of local proinflammatory cytokines in arterial wall tissues. Inflammatory cells produce proinflammatory cytokines and interlace an inflammatory network, which may contribute to tissue destruction that results in a weakened artery and ultimately rupture. Matrix metalloproteinases (MMPs), expecially matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9), the predominant proteinases in the AAA wall, degrade the ECM and are thought to play a key role in AAA formation. Evidence have been supported that defective inflammation and MMP activity may impair aneurysm formation in animal models.Aneurysms have many characteristics of autoimmune diseases, and the role of the immune system in aneurysmal disease has received considerable attention. However, the exact immunomodulatory mechanisms remain poorly understood. CD4+CD25+regulatory T cells (Tregs), a specific subpopulation of T cells, are important components of the immune system. Tregs play a role in maintaining immunological tolerance, and they control the progression of autoimmune diseases. Deficiency or dysfunction of Tregs disrupts immune homeostasis and leads to many pathological conditions.Angiotensin II-mediated progression of AAA and animal models of AAA have been investigated with apolipoprotein E knockout (ApoE-/-) mice. The role of the immune system in AAA formation has received consideration. However, no studies have elucidated the effect of Tregs on the development of AAA.2Objectives(1) To establish the animal models of AAA induced by angiotensin Ⅱ in ApoE-/-mice(2) To assess the beneficial effects of Tregs on angiotensin II-induced AAA in apolipoprotein E knockout mice.(2) To explore the inherent molecular mechanisms of Tregs-mediated beneficial effects on AAA.3Methods3.1Isolation of TregsThe C57BL/6J mice served as Tregs cell donors. Purified Tregs were isolated from the splenocytes of C57BL/6J mice with use of the CD4+CD25+Regulatory T cell Isolation Kit. The purity of Tregs subsets was>95%as determined by FACS (Fluorescence Activated Cell Sorting) analysis.3.2Animal protocolWe fed12-week-old ApoE-/-mice a Western-type diet (0.25%cholesterol and15%cocoa butter) with continuous subcutaneous infusion of angiotensin Ⅱ (1000ng/kg/min) or saline for28days via an osmotic pump.One day before the initial injection of angiotensin Ⅱ infusion, angiotensin II-treated mice were randomly divided into4groups for treatment (n=30each group):control group, tail-vein injection of200μl PBS; small-dose Tregs group, intravenous injection of105Tregs; large-dose Tregs group, intravenous injection of106Tregs; PC group, intravenous injection of106Tregs and together with an intraperitoneal injection of100mg CD25-depleting PC61antibody. These treatments were repeated2weeks later. After28days of infusion, ApoE-/-mice were euthanized and tissues were removed for further analysis.3.3Blood pressure measurementEach week after infusion of angiotensin Ⅱ, systolic blood pressure (SBP) was monitored by a noninvasive tail-cuff system with a specially device designed for ApoE-/-mice. Blood pressure is reported as mean of3consecutive measurements3.4Histology and morphologyThe aorta of ApoE-/-mice were removed and the maximal diameter of the abdominal aorta was measured by computerized morphometry. Aneurysm was defined as50%or more dilation of the external diameter in angiotensin Ⅱ-treated ApoE-/-mice as compared with saline-infused mice. Aneurysm severity was classified too.Corresponding sections on separate slides were visualized by hematoxylin and eosin (H&E) and Verhoeff staining for morphology. Immunohistochemistry involved use of the antibody to assess macrophages (MOMA-2), smooth muscle cells (SMCs), monocyte chemoattractant protein-1(MCP-1), interleukin-6(IL-6), MMP-2, MMP-9and cyclooxygenase2(COX-2).The production of reactive oxygen species (ROS) in aortic tissue was determined by the oxidative fluorescent dye dihydroethidium (DHE). Moreover, Sections of suprarenal aortas underwent terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining.3.5Cell separation and cocultureHuman peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and prepared by centrifugation. Tregs and CD4+CD25-T cells were isolated from PBMCs by use of a Human CD4+CD25+Regulatory T cell Isolation Kit according to the manufacturer's instruction.Human aortic smooth muscle cells (HASMCs) were without T cells (control group) or with CD4+CD25-T cells (CD25-group) or with Tregs at3concentrations (1.25×105group, 2.5×105group and5×105group, respectively) for48h in the presence of anti-CD3antibody (50ng/ml), then an additional24h with angiotensin Ⅱ (1μM). After floating T cells were discarded, HASMCs were harvested and supernatants were collected.3.6Quantitative real-time PCRTotal RNA was extracted from suprarenal aortas of ApoE-/-mice or HASMCs. In the in vivo experiment, the mRNA expression of MCP-1, IL-6, MMP-2and MMP-9was analyzed. In the in vitro experiment, the mRNA expression of MCP-1, IL-6, ICAM-1, MMP-2and MMP-9in HASMCs was analyzed too.3.7Western blot analysis Total proteins were extracted from the suprarenal aortas of ApoE-/-mice or HASMCs. The protein expression of IL-10, ICAM-1, MMP-2, MMP-9in the suprarenal aortas and MCP-1, IL-6, ICAM-1, MMP-2, MMP-9, FasL, COX-2and NF-κB in HASMCs were analyzed by Western blot.3.8ELISAIL-10levels in culture supernatants was assayed by ELISA.4. Results4.1Blood pressure of ApoE-/-miceIn this study we monitored blood pressure of the angiotensin Ⅱ-treated ApoE-/-mice and we found angiotonin Ⅱ infusion significantly enhanced SBP in comparision with baseline level. However, we did not found any effect of Tregs on SBP.4.2Treg transfer attenuated angiotonin Ⅱ-induced AAA in ApoE-/-miceAfter28-day saline or angiotonin Ⅱ infusion, the presence of AAA was examined and the incidence of AAA were shown as follows:aneurysm was absent in the saline control group,80%in the control group,76%in the small-dose Tregs group,27%in the large-dose Tregs group and71%) in the PC group. It is noteworthy that large-dose Tregs markedly reduced the incidence of AAA, with no difference with Tregs and PC61combined.Based on the classification system, we observed that most AAA in control, small-dose and PC groups showed Type Ⅲ or Ⅳ AAA forms; however, in the large-dose Tregs group was mostly Type Ⅰ. The AAA with large-dose Tregs differed from that in other Ang Ⅱ-infused groups. Furthermore, the maximal diameter of abdominal aorta were significantly lower in large-dose Tregs group than control, small-dose and PC groups. Therefore, transfer of Tregs dose-dependently reduced the incidence and severity of angiotonin Ⅱ-induced AAA formation in ApoE-/-mice.4.3Histological and morphological analysisAngiotonin II treatment led to a thickened aortic wall, destroyed media and adventitia, thrombus formation, and breakage and discontinuity of elastin fibers in aortic wall. H&E and Verhoff staining revealed that large-dose Treg treatment partly reversed the histological changes.Macrophages content in aortic aneurysmal tissue was observed by immunohistochemical staining. With large-dose Treg treatment, the infiltration of macrophages was greatly reduced as compared with than those in the control, small-dose and PC groups. Moreover, we found a marked increase in medial SMC content in large-dose Tregs group than those in the control, small-dose and PC groups.4.4Tregs reduced expression of proinflammatory cytokines and enhanced that of antiinflammatory cytokine both in vivo and in vitroLarge-dose Treg treatment significantly reduced the mRNA levels of proinflammatory MCP-1and IL-6in suprarenal aortic segments of ApoE-/-mice. Furthermore, the protein expression of MCP-1, IL-6and ICAM-1in aortic tissue assessed by immunohistochemistry or western blot was significantly lower in the large-dose Tregs groups than that in control, small-dose Tregs and PC groups. In HASMCs, we found the mRNA and protein expression of MCP-1, IL-6and ICAM-1significantly reduced after co-culture with Tregs than control or CD4+CD25-T cells.In vivo experiment, the protein level of the antiinflammatory IL-10was significantly increased in abdominal aortas of ApoE-/-mice with large-dose Treg treatment than control, small-dose Tregs or PC groups. Moreover, ELISA revealed the levels of IL-10in the3groups of Treg-treated HASMCs significantly higher than in control or CD25-groups.4.5Tregs lowered MMP-2and MMP-9expression in vivo and in vitroIt indicated a more reduction in MMP2and MMP9mRNA expression of aortas assessed by RT-PCR in angiotensin II-treated ApoE-/-mice with large-dose Treg delivery than those in the control, small-dose Tregs and PC groups. Meanwhile, protein expression of MMP-2 and MMP-9by immunohistochemistry or western blot analysis revealed a similar pattern. In HASMCs, the mRNA and protein levels of MMP-2and MMP-9were significantly decreased with Treg treatment in comparison with conntrol and CD25-group.4.6Tregs reduced ROS generationWe measured ROS generation in aortic tissues of ApoE-/-mice by dihydroethidium, which was weak signals with large-dose Treg treatment than those in control, small-dose Tregs and PC groups. The result showed that Tregs provided antioxidant protection in aneurysm formation.4.7Tregs inhibit apoptosis in angiotonin Ⅱ-induced aneurysmWe found TUNEL positivity greatly decreased in aortic media in large-dose Tregs group than those in control, small-dose Tregs and PC groups, which suggests that Treg treatment inhibited AngⅡ-induced apoptosis of aortic issues.In vitro experiment, FasL expression was significantly inhibited in Tregs treated-HASMCs than those in control and CD25-groups.4.8Tregs inhibited COX-2expressionIn this study, we found COX-2protein expression markedly decreased in the large-dose Tregs groups than those in the control, small-dose Tregs and PC groups. In HASMCs, Tregs at the3concentrations significantly decreased COX-2protein level assayed by western blot..4.9Tregs suppressed the activation of NF-κB pathwayAfter coculture with Tregs, we determined NF-κB p65protein expression in angiotonin II-stimulated HASMCs, As compared with control and CD25-group,3concentration of Tregs treatment dramatically inhibited the levels of NF-κB p65. Thus, Tregs suppressed the activation of the NF-κB and ERK1/2pathway.5. Conclusion(1) Tregs protected against angiotensin II induced AAA in ApoE-/-mice. Tregs treatment signifiantly the incidence and severity of AAA in animal models.(2) The potential mechanism may involve reduced macrophage accumulation and proinflammatory cytokine expression, enhanced antiinflammatory cytokine concentration, decreased MMP-2and MMP-9levels, preservation of medical SMCs and aortic wall elastin, as well as suppression of apoptosis and oxidative stress. Tregs inhibited the activation of NF-κB pathway, which may be the mechanism of its protective effect.