Masafumi Takeda

Oral Anti-CD3 Antibody Treatment Induces Regulatory T Cells and Inhibits the Development of Atherosclerosis in Mice

Background— Accumulating evidence suggests that several subsets of regulatory T cells that actively mediate immunologic tolerance play crucial roles in atherogenesis. Recently, orally administered anti-CD3 monoclonal antibody has been shown as an inducer of novel regulatory T cells expressing latency-associated peptide (LAP) on their surface, which potently prevents systemic autoimmunity. In the present study, we hypothesized that oral anti-CD3 antibody treatment may inhibit atherosclerosis in mice. Methods and Results— Six-week-old apolipoprotein E–deficient mice on a standard diet were orally given anti-CD3 antibody or control immunoglobulin G on 5 consecutive days, and atherosclerosis was assessed at age 16 weeks. Oral administration of anti-CD3 antibody significantly reduced atherosclerotic lesion formation and accumulations of macrophages and CD4+ T cells in the plaques compared with controls. We observed a significant increase in LAP+ cells and CD25+Foxp3+ cells in the CD4+ T-cell population in anti-CD3–treated mice, in association with increased production of the antiinflammatory cytokine transforming growth factor-β and suppressed T-helper type 1 and type 2 immune responses. Neutralization of transforming growth factor-β in vivo abrogated the preventive effect of oral anti-CD3 antibody. Conclusions— Our findings indicate the atheroprotective role of oral anti-CD3 antibody treatment in mice via induction of a regulatory T-cell response. These findings suggest that oral immune modulation may represent an attractive therapeutic approach to atherosclerosis.

Oral Administration of an Active Form of Vitamin D3 (Calcitriol) Decreases Atherosclerosis in Mice by Inducing Regulatory T Cells and Immature Dendritic Cells With Tolerogenic Functions

Objective—To determine whether the administration of an active form of vitamin D3 (calcitriol) could prevent atherosclerosis through anti-inflammatory actions. Methods and Results—Recent clinical studies have shown that lack of vitamin D3 is a risk factor for cardiovascular events. Oral calcitriol administration decreased atherosclerotic lesions, macrophage accumulation, and CD4+ T-cell infiltration at the aortic sinus, when compared with the corresponding observations in control mice. We observed a significant increase in Foxp3+ regulatory T cells and a decrease in CD80+CD86+ dendritic cells (DCs) in the mesenteric lymph nodes, spleen, and atherosclerotic lesions in oral calcitriol-treated mice in association with increased interleukin 10 and decreased interleukin 12 mRNA expression. CD11c+ DCs from the calcitriol group showed reduced proliferative activity of T lymphocytes, suggesting the suppression of DC maturation. Neutralization of CD25 in vivo revealed that calcitriol inhibited atherosclerosis mainly in a regulatory T cell-dependent manner but also partly because of a decrease in DC maturation. Conclusion—Oral calcitriol treatment could prevent the development of atherosclerosis by changing the function or differentiation of DCs and regulatory T cells. These findings suggest that intestinal and systemic immune modulation by calcitriol may be a potentially valuable therapeutic approach against atherosclerosis.

Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration

To realize cardiac regeneration using human induced pluripotent stem cells (hiPSCs), strategies for cell preparation, tissue engineering and transplantation must be explored. Here we report a new protocol for the simultaneous induction of cardiomyocytes (CMs) and vascular cells [endothelial cells (ECs)/vascular mural cells (MCs)], and generate entirely hiPSC-engineered cardiovascular cell sheets, which showed advantageous therapeutic effects in infarcted hearts. The protocol adds to a previous differentiation protocol of CMs by using stage-specific supplementation of vascular endothelial cell growth factor for the additional induction of vascular cells. Using this cell sheet technology, we successfully generated physically integrated cardiac tissue sheets (hiPSC-CTSs). HiPSC-CTS transplantation to rat infarcted hearts significantly improved cardiac function. In addition to neovascularization, we confirmed that engrafted human cells mainly consisted of CMs in >40% of transplanted rats four weeks after transplantation. Thus, our HiPSC-CTSs show promise for cardiac regenerative therapy.

Orally Administered Eicosapentaenoic Acid Induces Rapid Regression of Atherosclerosis Via Modulating the Phenotype of Dendritic Cells in LDL Receptor-Deficient Mice

Objective— Eicosapentaenoic acid (EPA) has been shown to have beneficial effects on cardiovascular diseases, although the precise mechanism is unknown. We investigated the effect of EPA on the regression of atherosclerosis. Methods and Results— LDL-receptor–deficient mice were fed a high-cholesterol diet for 8 weeks to build up aortic sinus atherosclerotic lesions and then were fed a normal diet with or without 5% EPA for 4 weeks. Atherosclerotic lesions were histologically assessed, and immunologic assays were performed. EPA treatment significantly regressed atherosclerosis (−22.7%, P<0.05) and decreased the content of macrophages, CD4+ T cells, and dendritic cells (DCs) in atherosclerotic lesions, though only changing the chow never induced the regression. Flow cytometric analysis revealed that EPA increased immature DCs (CD11c+ CD80− CD86−), increased the indoleamine 2,3-dioxygenase (IDO) in DCs, and decreased the number of CD4+ T cells. In the presence of the IDO inhibitor, the beneficial effects of EPA on regression were inhibited, suggesting that the effect of EPA was mainly mediated through IDO. Conclusion— In addition to lowering plasma cholesterol, EPA regressed atherosclerosis probably due to modulation of DC phenotype and reduction in T cell numbers. The present findings might partly explain the beneficial effects of EPA in clinics and support clinical evidence.

Augmentation of Vascular Remodeling by Uncoupled Endothelial Nitric Oxide Synthase in a Mouse Model of Diabetes Mellitus

Objective—Diabetes mellitus is associated with increased oxidative stress, which induces oxidation of tetrahydrobiopterin (BH4) in vessel wall. Without enough BH4, eNOS is uncoupled to L-arginine and produces superoxide rather than NO. We examined the role of uncoupled eNOS in vascular remodeling in diabetes. Methods and Results—Diabetes mellitus was produced by streptozotocin in C57BL/6J mice. Under stable hyperglycemia, the common carotid artery was ligated, and neointimal formation was examined 4 weeks later. In diabetic mice, the neointimal area was dramatically augmented. This augmentation was associated with increased aortic superoxide formation, reduced aortic BH4/dihydrobiopterin (BH2) ratio, and decreased plasma nitrite and nitrate (NOx) levels compared with nondiabetic mice. Chronic BH4 treatment (10 mg/kg/d) reduced the neointimal area in association with suppressed superoxide production and inflammatory changes in vessels. BH4/BH2 ratio in vessel wall was preserved, and plasma NOx levels increased. Furthermore, in the presence of diabetes, overexpression of bovine eNOS resulted in augmentation of neointimal area, accompanied by increased superoxide production in the endothelium. Conclusions—In diabetes, increased oxidative stress by uncoupled NOSs, particularly eNOS, causes augmentation of vascular remodeling. These findings indicate restoration of eNOS coupling has an atheroprotective benefit in diabetes.

Regression of atherosclerosis with anti-CD3 antibody via augmenting a regulatory T-cell response in mice

AIMS Although recent animal studies have investigated the cellular and molecular mechanisms underlying the process of atherosclerosis regression, it remains unknown whether adaptive immune responses including T cells are involved in this process. We investigated the role of T cells in atherosclerosis regression. METHODS AND RESULTS LDL receptor-deficient mice were fed a high-cholesterol diet for 8 weeks to form atherosclerotic lesions and were then changed to a standard diet, and atherosclerosis was assessed 4 weeks later. Just before changing the diet, the mice received an iv injection of anti-CD3 antibody (CD3-Ab) or control immunoglobulin G for 5 consecutive days. CD3-Ab treatment regressed atherosclerosis and decreased the accumulation of macrophages and CD4(+) T cells in the plaques. CD3-Ab treatment also dramatically reduced CD4(+) T cells and increased the proportion of regulatory T cells (Tregs). Depletion of Tregs by anti-CD25 antibody injection abolished the regression of atherosclerosis seen in CD3-Ab-treated mice, indicating the essential role for Tregs in this process. CONCLUSION CD3-Ab treatment induced rapid regression of established atherosclerosis via reducing CD4(+) T cells and increasing the proportion of Tregs. These findings suggest that therapeutic intervention for T-cell-mediated immune responses may represent a novel strategy to induce atherosclerosis regression in combination with lipid-lowering therapy.

CD3 Antibody and IL-2 Complex Combination Therapy Inhibits Atherosclerosis by Augmenting a Regulatory Immune Response

Background Accumulating evidence suggests that the balance between pathogenic effector T cells (Teffs) and regulatory T cells (Tregs) may be important for controlling atherosclerotic disease. We hypothesized that a combination therapy with anti‐CD3 antibody (CD3‐Ab) and IL‐2/anti‐IL‐2 monoclonal antibody complex (IL‐2 complex) aimed at increasing the ratio of Tregs to Teffs would effectively inhibit atherosclerosis in mice. Methods and Results We treated apolipoprotein E‐deficient mice fed a high‐cholesterol diet with vehicle, CD3‐Ab, IL‐2 complex, or their combination. Mice receiving the combination therapy had markedly reduced atherosclerotic lesions than mice treated with CD3‐Ab or IL‐2 complex alone. In addition, a striking increase in the Treg/Teff ratio of lymphoid organs and atherosclerotic lesions, along with plaque stabilization characterized by decreased macrophage content and increased collagen content was observed. The combination treatment also markedly reduced splenic Ly6Chigh inflammatory monocytes and might induce a favorable macrophage phenotype change in atherosclerotic lesions. Conclusions Our results indicate that in addition to suppressing Teff responses, enhancing Treg‐mediated immune responses is more efficacious in preventing atherosclerosis, suggesting a novel therapeutic approach for atherosclerosis.

UVB Exposure Prevents Atherosclerosis by Regulating Immunoinflammatory Responses

Objective— UVB irradiation is an established treatment for immunoinflammatory cutaneous disorders and has been shown to suppress cutaneous and systemic inflammatory diseases through modulation of the adaptive immune response. However, it remains unknown whether UVB irradiation prevents an immunoinflammatory disease of arteries such as atherosclerosis. Approach and Results— Here, we show that UVB exposure inhibits the development and progression of atherosclerosis in atherosclerosis-prone mice by expanding and enhancing the functional capacity of CD4+ forkhead box P3+ regulatory T cells and regulating proatherogenic T-cell responses. Experimental studies in Langerhans cell–depleted mice revealed that epidermal Langerhans cells play a critical role in UVB-dependent induction of CD4+ forkhead box P3+ regulatory T cells, suppression of proatherogenic T-cell responses, and prevention of atherosclerotic plaque development. Conclusions— Our findings suggest the skin immune system as a novel therapeutic target for atherosclerosis and provide a novel strategy for the treatment and prevention of atherosclerosis.

Regulatory T Cells and Tolerogenic Dendritic Cells as Critical Immune Modulators in Atherogenesis

Innate and adaptive immunity has been shown to be critically involved in the pathogenesis of atherosclerosis. In particular, immune suppression mediated by regulatory T cells (Tregs) or tolerogenic dendritic cells (DCs) serves as a vital mechanism for regulating pathogenic chronic inflammation in atherogensis, suggesting that promotion of endogenous regulatory immune responses could be a possible therapeutic approach to suppress atherosclerotic disease. In this review, we discuss the possible role of Tregs and tolerogenic DCs in the prevention of atherosclerosis and the promising strategies to prevent or cure atherosclerotic disease by modulating regulatory immune responses mediated by these suppressor cells.

Sarcomere gene variants act as a genetic trigger underlying the development of left ventricular noncompaction

BackgroundLeft ventricular noncompaction (LVNC) is a primary cardiomyopathy with heterogeneous genetic origins. The aim of this study was to elucidate the role of sarcomere gene variants in the pathogenesis and prognosis of LVNC.Methods and resultsWe screened 82 Japanese patients (0–35 years old), with a diagnosis of LVNC, for mutations in seven genes encoding sarcomere proteins, by direct DNA sequencing. We identified variants in a significant proportion of cases (27%), which were associated with poor prognosis (p = 0.012), particularly variants in TPM1, TNNC1, and ACTC1 (p = 0.012). To elucidate the pathological role, we developed and studied human-induced pluripotent stem cells (hiPSCs) from a patient carrying a TPM1 p.Arg178His mutation, who underwent heart transplantation. These cells displayed pathological changes, with mislocalization of tropomyosin 1, causing disruption of the sarcomere structure in cardiomyocytes, and impaired calcium handling. Microarray analysis indicated that the TPM1 mutation resulted in the down-regulation of the expression of numerous genes involved in heart development, and positive regulation of cellular process, especially the calcium signaling pathway.ConclusionsSarcomere genes are implicated as genetic triggers in the development of LVNC, regulating the expression of numerous genes involved in heart development, or modifying the severity of disease.