Tomoyuki Kita

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.

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.

Induction of mRNA for low-density lipoprotein receptors in heterozygous Watanabe heritable hyperlipidemic rabbits treated with CS-514 (pravastatin) and cholestyramine

We administered CS-514, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, alone and in combination with cholestyramine to heterozygous Watanabe heritable hyperlipidemic rabbits. This rabbit model for heterozygous familial hypercholesterolemia has hepatic low-density lipoprotein receptors that are assumed to be half as many as in normal rabbits. CS-514 alone lowered plasma low-density lipoprotein cholesterol levels by 50%, and in combination with cholestyramine, it lowered levels by 80%. The membrane-binding assay showed these drugs caused 1.5- and 1.8-fold increases in the number of hepatic low-density lipoprotein receptors, respectively. We also measured the amount of mRNA for low-density lipoprotein receptor by S1 nuclease protection assay in the same livers as above. These drugs induced mutant mRNA for the low-density lipoprotein receptor, which has an in-flame deletion of 12 nucleotides, as well as normal receptor mRNA. CS-514 alone produced a 1.8-fold increase in the amount of mRNA for the normal receptor and a 2.3-fold increase for the mutant mRNA, whereas CS-514 in combination with cholestyramine produced 1.9- and 3.1-fold increases, respectively. We conclude that CS-514 induces mRNA for the low-density lipoprotein receptor, subsequently increasing the receptor protein in the liver, and then reduces the levels of plasma cholesterol, and that the induction is augmented when the drug is administered in combination with cholestyramine.

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.

Torsades de Pointes with QT prolongation related to donepezil use.

An 83-year-old female, who had a history of anterior myocardial infarction, was treated for Alzheimers disease with donepezil. She suffered from repeated diarrhea and vomiting, and experienced syncope. She was admitted to our hospital and was diagnosed with acute colitis and syncope. On admission, her heart rate was 54 beats/min with regular rhythm. Laboratory data showed a low plasma potassium level. Electrocardiogram (ECG) showed poor R progression, ST elevation, negative T in precordial leads, and marked QT prolongation. Transthoracic echocardiogram showed the enlargement of the left atrium and aneurysmal area at the apex. Torsades de Pointes (TdP) with syncope and convulsion were confirmed on ECG monitoring twice after admission. We treated her with potassium chloride and started magnesium sulfate and lidocaine, and then added isoprenaline injection. After these treatments, her heart rate increased and we did not detect TdP again. With the aging population in Japan, prescriptions for donepezil are increasing. We have to be vigilant for syncope in patients taking donepezil, which is possibly related to QT prolongation and TdP.

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.

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.

Induction of Endothelial Platelet-Derived Growth Factor-B-Chain and Intercellular Adhesion Molecule-1 by Lysophosphatidylcholinea

Lysophosphatidylcholine (lyso-PC) is a major phospholipid component of atherogenic lipoproteins. Lyso-PC has been shown to differentially upregulate the adhesion molecules, such as VCAM-1 and ICAM-1, as well as smooth muscle growth factors, such as PDGF-A, B chains and HB-EGF gene expression in various cultured endothelial cells. In this paper, we demonstrate increased expression of cell- and matrix-associated forms of PDGF-B protein elicited by lyso-PC and further characterized potential signal transduction mechanisms responsible for lyso-PC-induced human umbilical vein endothelial cell. Cycloheximide inhibited PDGF-B but not ICAM-1 mRNA induction by lyso-PC, suggesting the dependence on de novo protein synthesis for PDGF-B, but not ICAM-1. A protein kinase C (PKC) inhibitor did not block lyso-PC-induced increases in PDGF-B or ICAM-1 mRNA. The elevated level of cAMP blocked both PDGF-B and ICAM-1 upregulation by lyso-PC. However cAMP-elevating agents did not suppress ICAM-1 upregulation by PMA. Taken together, PDGF-B and ICAM-1 gene induction by lyso-PC may involve different signaling mechanisms; however, both appear to be independent of PMA-regulatable PKC activation but are suppressed by increased levels of intracellular cAMP.

Probucol pretreatment enhances the chemotaxis of mouse peritoneal macrophages.

To investigate the effects of probucol on macrophage chemotaxis, we preincubated mouse peritoneal macrophages with probucol for 20 hours in vitro and using a modified Boyden chamber system compared their chemotactic responses with those of control macrophages that were preincubated with vehicle. Probucol pretreatment enhanced the macrophage chemotactic responses to zymosan-activated serum, acetylated low density lipoprotein (LDL), and native LDL. Probucol pretreatment also enhanced the basal migration observed when there was no stimulant in the lower chamber of a modified Boyden chamber. The chemoattracting potency of native LDL was weaker than that of zymosan-activated serum in control macrophages; however, both substances became equally potent when the macrophages were preincubated with probucol. The degree of the enhancement to native LDL after probucol preincubation reached fourfold to eightfold. The fashion of the enhanced migration of macrophages to native LDL after preincubation with probucol was predominantly chemotactic rather than chemokinetic. Time-course experiments revealed that it took more than 12 hours of probucol preincubation to show clearly enhanced macrophage chemotaxis to native LDL. Macrophages preincubated with probucol together with cycloheximide showed markedly reduced chemotaxis compared with macrophages preincubated only with probucol. Probucol pretreatment also enhanced macrophage chemotactic responses to high density lipoprotein, oxidized LDL, and lipoprotein-deficient serum.(ABSTRACT TRUNCATED AT 250 WORDS)

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.