Takuo Emoto

Foxp3+ Regulatory T Cells Play a Protective Role in Angiotensin II–Induced Aortic Aneurysm Formation in Mice

Although regulatory T cells (Tregs) have been shown to play a protective role in abdominal aortic aneurysm (AAA) formation, it remains unclear whether expansion of endogenous Foxp3+ Tregs prevents AAA. In the current study, we determined the effects of endogenous Foxp3+ Treg expansion or depletion in an experimental model of AAA. We continuously infused 12-week-old apolipoprotein E–deficient mice fed a high-cholesterol diet with angiotensin II (n=60) or normal saline (n=12) by implanting osmotic mini-pumps and evaluated AAA formation at 16 weeks. The angiotensin II–infused mice received interleukin-2/anti–interleukin-2 monoclonal antibody complex (interleukin-2 complex; n=31) or PBS (n=29). Eighty-one percent of angiotensin II–infused mice developed AAA, with 42% mortality possibly because of aneurysm rupture. Interleukin-2 complex treatment systemically increased the number of Foxp3+ Tregs and significantly decreased the incidence (52%) and mortality (17%) of AAA. Immunohistochemical analysis showed reduced accumulation of macrophages and increased numbers of Foxp3+ Tregs in aneurysmal tissues, suggesting that expansion of Tregs may suppress local inflammation in the vessel wall and provide protection against AAA formation. Furthermore, genetic depletion of Foxp3+ Tregs led to a significant increase in the mortality of AAA, suggesting the protective role of Foxp3+ Tregs against AAA. Our findings suggest that Foxp3+ Tregs may play a protective role in AAA formation and that promotion of an endogenous regulatory immune response may be a potentially valuable therapeutic approach for preventing AAA.

Intestinal Immunity and Gut Microbiota as Therapeutic Targets for Preventing Atherosclerotic Cardiovascular Diseases

Atherosclerosis is considered a chronic inflammatory disease and an intervention targeting the inflammatory process could be a new therapeutic strategy for preventing atherosclerotic cardiovascular diseases (CVD). We hypothesized that the intestine, which is considered the biggest immune organ in the human body, could be a therapeutic target for preventing CVD. We demonstrated that oral administration of anti-CD3 antibody or an active form of vitamin D3 reduced atherosclerosis in mice via induction of regulatory T cells and tolerogenic dendritic cells in the gut-associated lymphoid tissues. Similar to regulatory immune responses achieved by oral tolerance, our method had systemic effects that ultimately contributed towards atherosclerosis reduction. Recently, we have been interested in the gut microbiota, which have been reported as highly associated with intestinal immunity and systemic metabolic disorders, including obesity and diabetes. Notably, the guts of obese individuals are predominantly colonized by Firmicutes over Bacteroidetes. The association between atherosclerosis and microbiota has been attracting increased attention, and gut microbiota have been shown to participate in the metabolism of a proatherogenic compound called trimethylamine-N-oxide (TMAO) and aggravate CVD. Our investigation of the relationship between susceptibility to CVD and the gut microbiota revealed a characteristic flora type. Here, we discuss the evidence for the relationship between the gut microbiota and cardiometabolic diseases, and consider the gut microbiota as new potential therapeutic targets for treating CVD. (Circ J 2015; 79: 1882-1890).

Circulating intermediate CD14++CD16+monocytes are increased in patients with atrial fibrillation and reflect the functional remodelling of the left atrium

Aims A recent large clinical study demonstrated the association between intermediate CD14++CD16+monocytes and cardiovascular events. However, whether that monocyte subset contributes to the pathogenesis of atrial fibrillation (AF) has not been clarified. We compared the circulating monocyte subsets in AF patients and healthy people, and investigated the possible role of intermediate CD14++CD16+monocytes in the pathophysiology of AF. Methods and results This case–control study included 44 consecutive AF patients without systemic diseases referred for catheter ablation at our hospital, and 40 healthy controls. Patients with systemic diseases, including structural heart disease, hepatic or renal dysfunction, collagen disease, malignancy, and inflammation were excluded. Monocyte subset analyses were performed (three distinct human monocyte subsets: classical CD14++CD16−, intermediate CD14++CD16+, and non-classical CD14+CD16++monocytes). We compared the monocyte subsets and evaluated the correlation with other clinical findings. A total of 60 participants (30 AF patients and 30 controls as an age-matched group) were included after excluding 14 AF patients due to inflammation. Atrial fibrillation patients had a higher proportion of circulating intermediate CD14++CD16+monocytes than the controls (17.0 ± 9.6 vs. 7.5 ± 4.1%, P < 0.001). A multivariable logistic regression analysis demonstrated that only the proportion of intermediate CD14++CD16+monocytes (odds ratio: 1.316; 95% confidence interval: 1.095–1.582, P = 0.003) was independently associated with the presence of AF. Intermediate CD14++CD16+monocytes were negatively correlated with the left atrial appendage flow during sinus rhythm (r= −0.679, P = 0.003) and positively with the brain natriuretic peptide (r = 0.439, P = 0.015). Conclusion Intermediate CD14++CD16+monocytes might be closely related to the pathogenesis of AF and reflect functional remodelling of the left atrium.

Comparisons of detailed arterial healing response at seven months following implantation of an everolimus- or sirolimus-eluting stent in patients with ST-segment elevation myocardial infarction

BACKGROUND The difference of arterial healing response following everolimus-eluting stent (EES) or sirolimus-eluting stent (SES) implantation in patients with ST-segment elevated myocardial infarction (STEMI) has not been compared in detail. METHODS Thirty-five patients with STEMI were randomly implanted with an EES or SES (23 EES, 12 SES). At seven months, neointimal thickness (NIT) and strut malapposition were evaluated by optical coherence tomography (OCT) and the grade and heterogeneity of neointimal coverage (NIC) and development of intra-stent thrombi were evaluated by angioscopy. RESULTS No significant differences were noted in clinical events experienced by the two groups, although one patient with an EES died following a papillary muscle rupture and one patient with a SES experienced sub-acute stent thrombosis. On OCT, although the EES implants showed a greater NIT than the SES implants (94.8 ± 88.8 μm vs 65.6 ± 63.3 μm, P<0.0001), both the EES and SES showed an excellent suppression of neointimal proliferation in the culprit lesion of STEMI. The frequency of uncovered and malapposed struts of EES was significantly lower than that of SES (2.7% vs. 15.7%, P<0.0001, 0.7% vs. 2.3%, P<0.0001, respectively). The ratio of stents fully covered with neointima of EES group was significantly higher than that of SES group (P=0.04). Angioscopic analysis also showed greater dominant NIC grade with homogenous NIC in EES than in SES (P=0.03, P=0.0002, respectively). The incidence of massive intra-stent thrombus of EES was lower than that of SES (P=0.05). CONCLUSION For patients with STEMI, EES may promote better arterial healing response than SES.

Overexpression of Cytotoxic T-Lymphocyte–Associated Antigen-4 Prevents Atherosclerosis in Mice

Objective— Although T-cell–mediated chronic inflammation contributes to atherosclerosis development, the role of a negative regulatory molecule cytotoxic T-lymphocyte–associated antigen-4 (CTLA-4) in atherosclerosis is poorly understood. We investigated the effects of CTLA-4 overexpression on atherosclerosis in apolipoprotein E–deficient (Apoe −/−) mice. Approach and Results— We generated CTLA-4 transgenic (CTLA-4-Tg)/Apoe −/− mice that display constitutive cell surface and intracellular expression of CTLA-4 in T cells and assessed atherosclerosis at age 16 weeks. CTLA-4 overexpression significantly reduced atherosclerotic lesion formation and intraplaque accumulation of macrophage and CD4+ T cells in the aortic root compared with controls. CTLA-4-Tg/Apoe −/− mice showed decreased numbers of effector CD4+ T cells and decreased expression of costimulatory molecules CD80 and CD86, ligands for CTLA-4, and a costimulatory molecule CD28, on CD11c+ dendritic cells compared with controls. Consistent with in vivo findings, in vitro experiments revealed that CD4+ T cells from CTLA-4-Tg/Apoe −/− mice showed decreased proliferative capacity and proinflammatory cytokine production, downregulated CD80 expression on CD11c+ dendritic cells, and suppressed the proliferation of other T cells by limiting the costimulatory pathway. Moreover, CD11c+ dendritic cells from CTLA-4-Tg/Apoe −/− mice showed reduced proliferative activity of T cells in vitro, suggesting the suppression of dendritic cell maturation in vivo. Conclusions— CTLA-4 regulates atherosclerosis by suppressing proatherogenic immune responses and could be an attractive therapeutic target for atherosclerosis.

Possible association between non-invasive parameter of flow-mediated dilatation in brachial artery and whole coronary plaque vulnerability in patients with coronary artery disease.

BACKGROUND Despite being a relatively widely-used non-invasive parameter of endothelial dysfunction, little is known regarding the relationship between flow-mediated dilatation (FMD) and coronary plaque vulnerability in patients with coronary artery disease (CAD). METHODS 111 CAD patients (age; 68.9 ± 9.3) who underwent both coronary intervention and FMD were enrolled. Spectral analyses of intravascular ultrasound radiofrequency data for both culprit and non-culprit lesions were performed using Virtual Histology software. Plaque burden was described based on fibrotic, fibro-fatty, dense calcium, and necrotic core (NC) components, and thin-cap fibroatheroma (TCFA) was defined as focal NC rich (> 10%) plaques touching the lumen with a percent-plaque volume exceeding 40%. RESULTS Averaged %FMD was 2.86 ± 2.03% (median 2.27%, 25th 1.40%, 75th 4.20%). NC volumes were negatively correlated with log%FMD for both culprit and non-culprit lesions (P = 0.001, r = 0.31 and P = 0.03, r = 0.21, respectively). We divided the patients into three tertiles according to %FMD; 38 were lower (≤ 1.75%), 41 were middle (> 1.75%, but ≤ 3.5%), and 32 were upper tertile (> 3.5%). The prevalence rate of TCFA increased with decreasing %FMD tertile and the incidence of major adverse cardiac events was significantly higher in lower %FMD tertile. Multivariate logistic regression analyses showed that the most powerful predictive factor for TCFA was log%FMD (P < 0.0001), and ROC curve analysis identified %FMD of < 2.81% (AUC = 0.82, sensitivity: 91.2%, specificity: 66.7%) as the optimal cut-off point for predicting the presence of TCFA. CONCLUSIONS Impaired endothelial function in brachial arteries may be associated with whole coronary plaque vulnerability and poor clinical outcome in patients with CAD.

Commensal bacteria at the crossroad between cholesterol homeostasis and chronic inflammation in atherosclerosis

The gut microbiota were shown to play critical roles in the development of atherosclerosis, but the detailed mechanism is limited. The purpose of this study is to clarify the influence of gut microbiota on atherogenesis via lipid metabolism and systemic inflammation. Germ-free or conventionally raised (Conv) ApoE-deficient (ApoE−/−) mice were fed chow diet and euthanized at 20 weeks of age. We found that the lack of gut microbiota in ApoE−/− mice caused a significant increase in the plasma and hepatic cholesterol levels compared with Conv ApoE−/− mice. The absence of gut microbiota changed the bile acid composition in the ileum, which was associated with activation of the enterohepatic fibroblast growth factor 15, fibroblast growth factor receptor 4 axis, and reduction of cholesterol 7α-hydroxylase and hepatic bile acid synthesis, resulting in the accumulation of liver cholesterol content. However, we found that the lack of microbiota caused a significant reduction in atherosclerotic lesion formation compared with Conv ApoE−/− mice, which might be associated with the attenuation of lipopolysaccharide-mediated inflammatory responses. Our findings indicated that the gut microbiota affected both hypercholesterolemia and atherogenesis in mice.

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.

Impact of CD14 ++ CD16 + monocytes on coronary plaque vulnerability assessed by optical coherence tomography in coronary artery disease patients with well-regulated lipid levels

BACKGROUND AND AIMS This study examined the impact of CD14++CD16+ monocytes on coronary plaque vulnerability, as assessed by optical coherence tomography (OCT), and investigated their association with daily glucose fluctuation. Although increased CD14++CD16+ monocyte levels have been reported to increase cardiovascular events, their impact on coronary plaque vulnerability in coronary artery disease (CAD) patients with or without diabetes mellitus (DM) remains unclear. METHODS This prospective observational study included 50 consecutive patients with CAD, receiving lipid-lowering therapy and undergoing coronary angiography and OCT. Patients were divided into 3 tertiles according to the CD14++CD16+ monocyte percentages assessed by flow cytometry. Standard OCT parameters were assessed for 97 angiographically intermediate lesions (diameter stenosis: 30-70%). Daily glucose fluctuation was analyzed by measuring the mean amplitude of glycemic excursion (MAGE). RESULTS CD14++CD16+ monocytes negatively correlated with fibrous cap thickness (r = -0.508, p < 0.01). The presence of thin-cap fibroatheroma (TCFA) was increased stepwise according to the tertile of CD14++CD16+ monocytes (0 [tertile 1] vs. 5 [tertile 2] vs. 10 [tertile 3], p < 0.01). CD14++CD16+ monocytes were a significant determinant of TCFA (OR 1.279, p = 0.001). In non-DM patients, a significant relationship was found between CD14++CD16+ monocytes and MAGE (r = 0.477, p = 0.018). CONCLUSIONS CD14++CD16+ monocytes were associated with coronary plaque vulnerability in CAD patients with well-regulated lipid levels both in DM and non-DM patients. Cross-talk between glucose fluctuation and CD14++CD16+ monocytes may enhance plaque vulnerability, particularly in non-DM patients. CD14++CD16+ monocytes could be a possible therapeutic target for coronary plaque stabilization.

Oral administration of the lactic acid bacterium Pediococcus acidilactici attenuates atherosclerosis in mice by inducing tolerogenic dendritic cells

The intestinal microbiota appears to play an important role in the development of atherosclerosis. We investigated the effect of the probiotic lactic acid bacterium Pediococcus acidilactici R037 on atherosclerosis using apolipoprotein E-deficient (ApoE−/−) mice. Six-week-old ApoE−/− mice were orally administered R037 six times a week. Mice treated with R037 for 12 weeks exhibited markedly attenuated atherosclerotic lesions in the aortic root (2.3 ± 0.15 × 105 µm2 vs. 3.3 ± 0.29 × 105 µm2, respectively; P < 0.01; n = 15–17 each group). The expression of Ki-67 in CD4+ T cells, the population of interferon γ-producing CD4+ T cells in the spleen, and pro-inflammatory cytokine production from splenic lymphocytes were significantly decreased in R037-treated mice. Interestingly, splenic dendritic cells (DCs) isolated from R037-treated mice suppressed CD4+ T-cell proliferation and pro-inflammatory cytokine production ex vivo, suggesting that R037 treatment induced tolerogenic DCs. Programmed cell death ligand 1 expression in DCs was significantly enhanced in R037-treated mice, which might explain the immunosuppressive effect of DCs at least in part. These results indicate that R037 attenuates atherosclerosis by inducing tolerogenic DCs, which suppress Th1-driven inflammation and the proliferative activity of CD4+ T cells. Our findings may provide a novel therapeutic approach for the prevention of atherosclerosis based on dietary supplementation with probiotics.