Carvedilol suppresses ryanodine receptor-dependent Ca2+ bursts in human neurons bearing PSEN1 variants found in early onset Alzheimer's disease.

Seizures are increasingly being recognized as the hallmark of Alzheimer's disease (AD). Neuronal hyperactivity can be a consequence of neuronal damage caused by abnormal amyloid ß (Aß) depositions. However, it can also be a cell-autonomous phenomenon causing AD by Aß-independent mechanisms. Various studies using animal models have shown that Ca2+ is released from the endoplasmic reticulum (ER) via type 1 inositol triphosphate receptors (InsP3R1s) and ryanodine receptors (RyRs). To investigate which is the main pathophysiological mechanism in human neurons, we measured Ca2+ signaling in neural cells derived from three early-onset AD patients harboring Presenilin-1 variants (PSEN1 p.A246E, p.L286V, and p.M146L). Of these, it has been reported that PSEN1 p.A246E and p.L286V did not produce a significant amount of abnormal Aß. We found all PSEN1-mutant neurons, but not wild-type, caused abnormal Ca2+-bursts in a manner dependent on the calcium channel, Ryanodine Receptor 2 (RyR2). Indeed, carvedilol, an RyR2 inhibitor, and VK-II-86, an analog of carvedilol without the ß-blocking effects, sufficiently eliminated the abnormal Ca2+ bursts. In contrast, Dantrolene, an inhibitor of RyR1 and RyR3, and Xestospongin c, an IP3R inhibitor, did not attenuate the Ca2+-bursts. The Western blotting showed that RyR2 expression was not affected by PSEN1 p.A246E, suggesting that the variant may activate the RyR2. The RNA-Seq data revealed that ER-stress responsive genes were increased, and mitochondrial Ca2+-transporter genes were decreased in PSEN1A246E cells compared to the WT neurons. Thus, we propose that aberrant Ca2+ signaling is a key link between human pathogenic PSEN1 variants and cell-intrinsic hyperactivity prior to deposition of abnormal Aß, offering prospects for the development of targeted prevention strategies for at-risk individuals.

NLRP3 Inflammasome Activation Through Heart-Brain Interaction Initiates Cardiac Inflammation and Hypertrophy During Pressure Overload.

BACKGROUND: Mechanical stress on the heart, such as high blood pressure, initiates inflammation and causes hypertrophic heart disease. However, the regulatory mechanism of inflammation and its role in the stressed heart remain unclear. IL-1ß (interleukin-1ß) is a proinflammatory cytokine that causes cardiac hypertrophy and heart failure. Here, we show that neural signals activate the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3) inflammasome for IL-1ß production to induce adaptive hypertrophy in the stressed heart. METHODS: C57BL/6 mice, knockout mouse strains for NLRP3 and P2RX7 (P2X purinoceptor 7), and adrenergic neuron-specific knockout mice for SLC17A9, a secretory vesicle protein responsible for the storage and release of ATP, were used for analysis. Pressure overload was induced by transverse aortic constriction. Various animal models were used, including pharmacological treatment with apyrase, lipopolysaccharide, 2'(3')-O-(4-benzoylbenzoyl)-ATP, MCC950, anti-IL-1ß antibodies, clonidine, pseudoephedrine, isoproterenol, and bisoprolol, left stellate ganglionectomy, and ablation of cardiac afferent nerves with capsaicin. Cardiac function and morphology, gene expression, myocardial IL-1ß and caspase-1 activity, and extracellular ATP level were assessed. In vitro experiments were performed using primary cardiomyocytes and fibroblasts from rat neonates and human microvascular endothelial cell line. Cell surface area and proliferation were assessed. RESULTS: Genetic disruption of NLRP3 resulted in significant loss of IL-1ß production, cardiac hypertrophy, and contractile function during pressure overload. A bone marrow transplantation experiment revealed an essential role of NLRP3 in cardiac nonimmune cells in myocardial IL-1ß production and cardiac phenotype. Pharmacological depletion of extracellular ATP or genetic disruption of the P2X7 receptor suppressed myocardial NLRP3 inflammasome activity during pressure overload, indicating an important role of ATP/P2X7 axis in cardiac inflammation and hypertrophy. Extracellular ATP induced hypertrophic changes of cardiac cells in an NLRP3- and IL-1ß-dependent manner in vitro. Manipulation of the sympathetic nervous system suggested sympathetic efferent nerves as the main source of extracellular ATP. Depletion of ATP release from sympathetic efferent nerves, ablation of cardiac afferent nerves, or a lipophilic ß-blocker reduced cardiac extracellular ATP level, and inhibited NLRP3 inflammasome activation, IL-1ß production, and adaptive cardiac hypertrophy during pressure overload. CONCLUSIONS: Cardiac inflammation and hypertrophy are regulated by heart-brain interaction. Controlling neural signals might be important for the treatment of hypertensive heart disease.

STING, a cytosolic DNA sensor, plays a critical role in atherogenesis: a link between innate immunity and chronic inflammation caused by lifestyle-related diseases.

AIMS: Lifestyle-related diseases promote atherosclerosis, a chronic inflammatory disease; however, the molecular mechanism remains largely unknown. Endogenous DNA fragments released under over-nutrient condition provoke sterile inflammation through the recognition by DNA sensors. Here, we investigated the role of stimulator of interferon genes (STING), a cytosolic DNA sensor, in atherogenesis. METHODS AND RESULTS: Apolipoprotein E-deficient (Apoe-/-) mice fed a western-type diet (WTD), a hypercholesterolaemic mouse model, showed higher STING expression and markers for DNA damage such as γH2AX, p53, and single-stranded DNA (ssDNA) accumulation in macrophages in the aorta compared with wild-type (WT) mice. The level of cGAMP, a STING agonist, in the aorta was higher in Apoe-/- mice. Genetic deletion of Sting in Apoe-/- mice reduced atherosclerotic lesions in the aortic arch, lipid, and macrophage accumulation in plaques, and inflammatory molecule expression in the aorta compared with the control. Pharmacological blockade of STING using a specific inhibitor, C-176, ameliorated atherogenesis in Apoe-/- mice. In contrast, bone marrow-specific STING expression in Apoe-/- mice stimulated atherogenesis. Expression or deletion of STING did not affect metabolic parameters and blood pressure. In vitro studies revealed that STING activation by cGAMP or mitochondrial DNA accelerated inflammatory molecule expression (e.g. TNF-α or IFN-ß) in mouse and human macrophages. Activation of nuclear factor-κB and TANK binding kinase 1 was involved in STING-associated vascular inflammation and macrophage activation. Furthermore, human atherosclerotic lesions in the carotid arteries expressed STING and cGAMP. CONCLUSION: Stimulator of interferon genes stimulates pro-inflammatory activation of macrophages, leading to the development of atherosclerosis. Stimulator of interferon genes signalling may serve as a potential therapeutic target for atherosclerosis.

Roles of Epicardial Adipose Tissue in the Pathogenesis of Coronary Atherosclerosis　- An Update on Recent Findings.

Adipose tissue serves not only as an energy store or a mechanical cushion, but also as an endocrine organ. Recent evidence revealed that perivascular adipose tissue is involved in vascular homeostasis and pathophysiology of adjacent arteries by producing various adipokines. Epicardial adipose tissue (EAT) is located between the surface of the heart and the visceral layer of the pericardium and surrounds the coronary arteries. Many clinical studies suggest that an increase in EAT volume is associated with coronary artery disease. It has been reported that exercise and some antidiabetic drugs can reduce EAT volume. In this review, we outline recent findings on the roles of EAT in the pathogenesis of coronary atherosclerosis.

Biodegradable Extremely-Small-Diameter Vascular Graft Made of Silk Fibroin can be Implanted in Mice.

AIM: Synthetic vascular grafts are widely used in surgical revascularization, mainly for medium- to large-sized vessels. However, synthetic grafts smaller than 6 mm in diameter are associated with a high incidence of thrombosis. In this study, we evaluated silk fibroin, a major protein of silk, with high biocompatibility and biodegradability, as a useful material for extremely-small-diameter vascular grafts. METHODS: A small-sized (0.9 mm inner diameter) graft was braided from a silk fibroin thread. The right carotid arteries of 8- to 14-week-old male C57BL/6 mice were cut at the midpoint, and fibroin grafts (5- to 7-mm in length) were transplanted using a cuff technique with polyimide cuffs. The grafts were harvested at different time points and analyzed histologically. RESULTS: CD31＋ endothelial cells had already started to proliferate at 2 weeks after implantation. At 4 weeks, neointima had formed with α-smooth muscle actin+ cells, and the luminal surface was covered with CD31＋endothelial cells. Mac3＋ macrophages were accumulated in the grafts. Graft patency was confirmed at up to 6 months after implantation. CONCLUSION: This mouse model of arterial graft implantation enables us to analyze the remodeling process and biocompatibility of extremely-small-diameter vascular grafts. Biodegradable silk fibroin might be applicable for further researches using genetically modified mice.

Vildagliptin, a DPP-4 Inhibitor, Attenuates Endothelial Dysfunction and Atherogenesis in Nondiabetic Apolipoprotein E-Deficient Mice.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are novel antidiabetic agents with possible vascular protection effects. Endothelial dysfunction is an initiation step in atherogenesis. The purpose of this study was to investigate whether vildagliptin (Vilda) attenuates the development of endothelial dysfunction and atherosclerotic lesions in nondiabetic apolipoprotein E-deficient (ApoE-/-) mice. Eight-week-old nondiabetic ApoE-/- mice fed a Western-type diet received Vilda (50 mg/kg/day) for 20 weeks or 8 weeks. After 20 weeks of treatment, Vilda administration reduced atherogenesis in the aortic arch as determined by en face Sudan IV staining compared with the vehicle group (P < 0.05). Vilda also reduced lipid accumulation (P < 0.05) and vascular cell adhesion molecule-1 (VCAM-1) expression (P < 0.05) and tended to decrease macrophage infiltration (P = 0.05) into atherosclerotic plaques compared with vehicle. After 8 weeks of treatment, endothelium-dependent vascular reactivity was examined. Vilda administration significantly attenuated the impairment of endothelial function in nondiabetic ApoE-/- mice compared with the vehicle group (P < 0.05). Vilda treatment did not alter metabolic parameters, including blood glucose level, in both study protocols. To investigate the mechanism, aortic segments obtained from wild-type mice were incubated with exendin-4 (Ex-4), a glucagon-like peptide-1 (GLP-1) analog, in the presence or absence of lipopolysaccharide (LPS). Ex-4 attenuated the impairment of endothelium-dependent vasodilation induced by LPS (P < 0.01). Furthermore, Ex-4 promoted phosphorylation of eNOS at Ser1177 which was decreased by LPS in human umbilical endothelial cells (P < 0.05). Vilda inhibited the development of endothelial dysfunction and prevented atherogenesis in nondiabetic ApoE-/- mice. Our results suggested that GLP-1-dependent amelioration of endothelial dysfunction is associated with the atheroprotective effects of Vilda.

Pleural thickening on screening chest X-rays: a single institutional study.

Although pleural thickening is a common finding on routine chest X-rays, its radiological and clinical features remain poorly characterized. Our investigation of 28,727 chest X-rays obtained from annual health examinations confirmed that pleural thickening was the most common abnormal radiological finding. In most cases (92.2%), pleural thickening involved the apex of the lung, particularly on the right side; thus, it was defined as a pulmonary apical cap. Pleural thickening was more common in males than in females and in current smokers or ex-smokers than in never smokers. The prevalence increased with age, ranging from 1.8% in teenagers to 9.8% in adults aged 60 years and older. Moreover, pleural thickening was clearly associated with greater height and lower body weight and body mass index, suggesting that a tall, thin body shape may predispose to pleural thickening. These observations allowed us to speculate about the causative mechanisms of pleural thickening that are attributable to disproportionate perfusion, ventilation, or mechanical forces in the lungs.

Toll-Like Receptor 9 Plays a Pivotal Role in Angiotensin II-Induced Atherosclerosis.

Background Toll-like receptor ( TLR ) 9 recognizes bacterial DNA , activating innate immunity, whereas it also provokes inflammation in response to fragmented DNA released from mammalian cells. We investigated whether TLR 9 contributes to the development of vascular inflammation and atherogenesis using apolipoprotein E-deficient ( Apoe -/-) mice. Methods and Results Tlr9-deficient Apoe -/- ( Tlr9 -/- Apoe -/-) mice and Apoe -/- mice on a Western-type diet received subcutaneous angiotensin II infusion (1000 ng/kg per minute) for 28 days. Angiotensin II increased the plasma level of double-stranded DNA, an endogenous ligand of TLR 9, in these mice. Genetic deletion or pharmacologic blockade of TLR 9 in angiotensin II-infused Apoe -/- mice attenuated atherogenesis in the aortic arch ( P<0.05), reduced the accumulation of lipid and macrophages in atherosclerotic plaques, and decreased RNA expression of inflammatory molecules in the aorta with no alteration of metabolic parameters. On the other hand, restoration of TLR 9 in bone marrow in Tlr9 -/- Apoe -/- mice promoted atherogenesis in the aortic arch ( P<0.05). A TLR 9 agonist markedly promoted proinflammatory activation of Apoe -/- macrophages, partially through p38 mitogen-activated protein kinase signaling. In addition, genomic DNA extracted from macrophages promoted inflammatory molecule expression more effectively in Apoe -/- macrophages than in Tlr9 -/- Apoe -/- macrophages. Furthermore, in humans, circulating double-stranded DNA in the coronary artery positively correlated with inflammatory features of coronary plaques determined by optical coherence tomography in patients with acute myocardial infarction ( P<0.05). Conclusions TLR 9 plays a pivotal role in the development of vascular inflammation and atherogenesis through proinflammatory activation of macrophages. TLR 9 may serve as a potential therapeutic target for atherosclerosis.

Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia.

Background: Peripheral artery disease causes significant functional disability and results in impaired quality of life. Ischemic tissue injury releases various endogenous ligands for Toll-like receptors (TLRs), suggesting the involvement of TLRs in blood flow recovery. However, the role of TLR9, which was originally known as a sensor for bacterial DNA, remains unknown. This study investigated the role of TLR9 in blood flow recovery in the ischemic limb using a mouse hind-limb ischemia model. Methods and Results: Unilateral femoral artery ligation was performed in TLR9-deficient (Tlr9 -/-) mice and wild-type mice. In wild-type mice, femoral artery ligation significantly increased mRNA expression of TLR9 in the ischemic limb (P < 0.001) and plasma levels of cell-free DNA (cfDNA) as determined by single-stranded DNA (ssDNA) (P < 0.05) and double-stranded DNA (dsDNA) (P < 0.01), which are endogenous ligands for TLR9, compared with the sham-operated group. Laser Doppler perfusion imaging demonstrated significantly improved ratio of blood flow in the ischemic to non-ischemic limb in Tlr9 -/- mice compared with wild-type mice at 2 weeks after ligation (P < 0.05). Tlr9 -/- mice showed increased capillary density and reduced macrophage infiltration in ischemic limb. Genetic deletion of TLR9 reduced the expression of TNF-α, and attenuated NF-κB activation in ischemic muscle compared with wild-type mice (P < 0.05, respectively) at 3 days after the surgery. ODN1826, a synthetic agonistic oligonucleotide for TLR9, or plasma obtained from mice with ischemic muscle promoted the expression of TNF-α in wild-type macrophages (P < 0.05), but not in Tlr9 -/- macrophages. ODN1826 also activated NF-κB signaling as determined by the degradation of IκBα in wild-type macrophages (P < 0.05), but not in Tlr9 -/- macrophages. In vitro experiments using human umbilical vein endothelial cells demonstrated that TNF-α, or conditioned medium obtained from wild-type macrophages treated with ODN1826 accelerated cell death as determined by MTS assay (P < 0.05 and P < 0.01, respectively). Conclusion: Our results suggest that ischemic muscle releases cfDNA, which activates TLR9 and enhances inflammation, leading to impairment of blood flow recovery in the ischemic limb. cfDNA-TLR9 signaling may serve as a potential therapeutic target in ischemic limb disease.

Ticagrelor, a P2Y12 antagonist, attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein-E-deficient mice.

BACKGROUND AND AIMS: Ticagrelor reduces cardiovascular events in patients with acute coronary syndrome (ACS). Recent studies demonstrated the expression of P2Y12 on vascular cells including endothelial cells, as well as platelets, and suggested its contribution to atherogenesis. We investigated whether ticagrelor attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein E-deficient (apoe-/-) mice. METHODS: Eight-week-old male apoe-/- mice were fed a western-type diet (WTD) supplemented with 0.1% ticagrelor (approximately 120 mg/kg/day). Non-treated animals on WTD served as control. Atherosclerotic lesions were examined by en-face Sudan IV staining, histological analyses, quantitative RT-PCR analysis, and western blotting. Endothelial function was analyzed by acetylcholine-dependent vasodilation using aortic rings. Human umbilical vein endothelial cells (HUVEC) were used for in vitro experiments. RESULTS: Ticagrelor treatment for 20 weeks attenuated atherosclerotic lesion progression in the aortic arch compared with control (p < 0.05). Ticagrelor administration for 8 weeks attenuated endothelial dysfunction (p < 0.01). Ticagrelor reduced the expression of inflammatory molecules such as vascular cell adhesion molecule-1, macrophage accumulation, and lipid deposition. Ticagrelor decreased the phosphorylation of JNK in the aorta compared with control (p < 0.05). Ticagrelor and a JNK inhibitor ameliorated impairment of endothelium-dependent vasodilation by adenosine diphosphate (ADP) in wild-type mouse aortic segments. Furthermore, ticagrelor inhibited the expression of inflammatory molecules which were promoted by ADP in HUVEC (p < 0.001). Ticagrelor also inhibited ADP-induced JNK activation in HUVEC (p < 0.05). CONCLUSIONS: Ticagrelor attenuated vascular dysfunction and atherogenesis through the inhibition of inflammatory activation of endothelial cells. These effects might be a potential mechanism by which ticagrelor decreases cardiovascular events in patients with ACS.

Roles of Perivascular Adipose Tissue in the Pathogenesis of Atherosclerosis.

Traditionally, it is believed that white adipose tissues serve as energy storage, heat insulation, and mechanical cushion, whereas non-shivering thermogenesis occurs in brown adipose tissue. Recent evidence revealed that adipose tissue secretes many types of cytokines, called as adipocytokines, which modulate glucose metabolism, lipid profile, appetite, fibrinolysis, blood pressure, and inflammation. Most of the arteries are surrounded by perivascular adipose tissue (PVAT). PVAT has been thought to be simply a structurally supportive tissue for vasculature. However, recent studies showed that PVAT influences vasodilation and vasocontraction, suggesting that PVAT regulates vascular tone and diameter. Adipocytokines secreted from PVAT appear to have direct access to the adjacent arterial wall by diffusion or via vasa vasorum. In fact, PVAT around atherosclerotic lesions and mechanically-injured arteries displayed inflammatory cytokine profiles, suggesting that PVAT functions to promote vascular lesion formation. Many clinical studies revealed that increased accumulation of epicardial adipose tissue (EAT), which surrounds coronary arteries, is associated with coronary artery disease. In this review article, we will summarize recent findings about potential roles of PVAT in the pathogenesis of atherosclerosis, particularly focusing on a series of basic and clinical studies from our laboratory.

A variant of death-receptor 3 associated with rheumatoid arthritis interferes with apoptosis-induction of T cell.

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology. To unravel the molecular mechanisms in RA, we performed targeted DNA sequencing analysis of patients with RA. This analysis identified a variant of the death receptor 3 (DR3) gene, a member of the family of apoptosis-inducing Fas genes, which contains four single-nucleotide polymorphisms (SNPs) and a 14-nucleotide deletion within exon 5 and intron 5. We found that the deletion causes the binding of splicing regulatory proteins to DR3 pre-mRNA intron 5, resulting in a portion of intron 5 becoming part of the coding sequence, thereby generating a premature stop codon. We also found that this truncated DR3 protein product lacks the death domain and forms a heterotrimer complex with wildtype DR3 that dominant-negatively inhibits ligand-induced apoptosis in lymphocytes. Myelocytes from transgenic mice expressing the human DR3 variant produced soluble truncated DR3, forming a complex with TNF-like ligand 1A (TL1A), which inhibited apoptosis induction. In summary, our results reveal that a DR3 splice variant that interferes with ligand-induced T cell responses and apoptosis may contribute to RA pathogenesis.

Inhibition of activated factor X by rivaroxaban attenuates neointima formation after wire-mediated vascular injury.

Accumulating evidence suggests that activated factor X (FXa), a key coagulation factor, plays an important role in the development of vascular inflammation through activation of many cell types. Here, we investigated whether pharmacological blockade of FXa attenuates neointima formation after wire-mediated vascular injury. Transluminal femoral artery injury was induced in C57BL/6 mice by inserting a straight wire. Rivaroxaban (5mg/kg/day), a direct FXa inhibitor, was administered from one week before surgery until killed. At four weeks after surgery, rivaroxaban significantly attenuated neointima formation in the injured arteries compared with control (P<0.01). Plasma lipid levels and blood pressure were similar between the rivaroxaban-treated group and non-treated group. Quantitative RT-PCR analyses demonstrated that rivaroxaban reduced the expression of inflammatory molecules (e.g., IL-1ß and TNF-α) in injured arteries at seven days after surgery (P<0.05, respectively). In vitro experiments using mouse peritoneal macrophages demonstrated that FXa increased the expression of inflammatory molecules (e.g., IL-1ß and TNF-α), which was blocked in the presence of rivaroxaban (P<0.05). Also, in vitro experiments using rat vascular smooth muscle cells (VSMC) demonstrated that FXa promoted both proliferation and migration of this cell type (P<0.05), which were blocked in the presence of rivaroxaban. Inhibition of FXa by rivaroxaban attenuates neointima formation after wire-mediated vascular injury through inhibition of inflammatory activation of macrophages and VSMC.

Teneligliptin, a dipeptidyl peptidase-4 inhibitor, attenuated pro-inflammatory phenotype of perivascular adipose tissue and inhibited atherogenesis in normoglycemic apolipoprotein-E-deficient mice.

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) inhibitors have various cellular effects that are associated with vascular protection. Here, we examined whether teneligliptin alters the pro-inflammatory phenotype of perivascular adipose tissue (PVAT) and inhibits atherogenesis. METHODS AND RESULTS: Teneligliptin (60mg/kg/day) was administered orally to apolipoprotein-E-deficient (ApoE-/-) mice for 20weeks. Teneligliptin significantly inhibited the development of atherosclerosis in the aortic arch compared with vehicle (P<0.05), without alteration of blood glucose level or blood pressure. Histological analyses demonstrated that teneligliptin decreased lipid deposition and MCP-1 expression (P<0.05, respectively), and tended to decrease macrophage accumulation in atherosclerotic plaques. The results of quantitative RT-PCR analysis demonstrated that teneligliptin reduced the expression of inflammatory molecules such as TNF-α and MCP-1 in the abdominal aorta. Furthermore, teneligliptin reduced the expression of a macrophage marker and Nox-4, a major NADPH oxidase subunit in adipocytes, in PVAT around the aortic arch. Administration of teneligliptin for 8weeks ameliorated endothelium-dependent vasodilation and reduced oxidative stress as determined by urinary 8-OHdG excretion (P<0.05) compared with vehicle. In vitro experiments demonstrated that exendin-4 (Ex-4), a GLP-1 analog, decreased the expression of inflammatory molecules in RAW264.7 cells. Also, Ex-4 decreased Nox4 expression in 3T3-L1 adipocytes. CONCLUSION: Teneligliptin inhibited atherogenesis with attenuation of the inflammatory phenotype in PVAT. A GLP-1 analog suppressed pro-inflammatory activation of macrophages and adipocytes. Suppression of the pro-inflammatory phenotype of PVAT might contribute, at least partially, to the cardioprotective effects of teneligliptin.

Combination of n-3 polyunsaturated fatty acids reduces atherogenesis in apolipoprotein E-deficient mice by inhibiting macrophage activation.

BACKGROUND AND AIMS: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are major components of n-3 polyunsaturated fatty acids (n-3 PUFAs) which inhibit atherogenesis, although few studies have examined the effects of the combination of EPA and DHA on atherogenesis. The aim of this study was to investigate whether DHA has additional anti-atherosclerotic effects when combined with EPA. METHODS: Male 8-week-old apolipoprotein E-deficient (Apoe-/-) mice were fed a western-type diet supplemented with different amounts of EPA and DHA; EPA (2.5%, w/w), low-dose EPA + DHA (2.5%, w/w), or high-dose EPA + DHA (5%, w/w) for 20 weeks. The control group was fed a western-type diet containing no n-3 PUFA. Histological and gene expression analysis were performed in atherosclerotic lesions in the aorta. To address the mechanisms, RAW264.7 cells were used. RESULTS: All n-3 PUFA treatments significantly attenuated the development and destabilization of atherosclerotic plaques compared with the control. The anti-atherosclerotic effects were enhanced in the high-dose EPA + DHA group (p < 0.001), whereas the pure EPA group and low-dose EPA + DHA group showed similar results. EPA and DHA additively attenuated the expression of inflammatory molecules in RAW264.7 cells stimulated with LPS. DHA or EPA + DHA suppressed LPS-induced toll-like receptor 4 (TLR4) expression in lipid rafts on RAW264.7 cells (p < 0.05). Lipid raft disruption by methyl-ß-cyclodextrin suppressed mRNA expression of inflammatory molecules in LPS-stimulated macrophages. CONCLUSION: n-3 PUFAs suppressed atherogenesis. DHA combined with EPA had additional anti-inflammatory effects and inhibited atherogenesis in Apoe-/- mice. The reduction of TLR4 expression in lipid rafts in macrophages by DHA might be involved in this mechanism, at least partially.

Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance.

Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9 (-/-) ) macrophages. Fat-fed Tlr9 (-/-) mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9 (-/-) mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography-determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance.

Dipeptidyl peptidase-4 inhibitor, linagliptin, ameliorates endothelial dysfunction and atherogenesis in normoglycemic apolipoprotein-E deficient mice.

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) inhibitors have vasoprotective effects. This study investigated whether a recently approved DPP-4 inhibitor, linagliptin (Lina), suppresses atherogenesis in non-diabetic apolipoprotein-E deficient (ApoE(-/-)) mice, and examined its effects on endothelial function. METHODS AND RESULTS: Lina (10mg/kg/day) was administered orally to ApoE(-/-) mice for 20 weeks. Lina reduced atherogenesis without alteration of metabolic parameters including blood glucose level compared with control (P<0.05). Results of immunohistochemical analyses and quantitative RT-PCR demonstrated that Lina significantly decreased inflammatory molecule expression and macrophage infiltration in the atherosclerotic aorta. Lina administration to ApoE(-/-) mice for 9 weeks ameliorated endothelium-dependent vasodilation compared with that in untreated mice. Plasma active glucagon-like peptide-1 (GLP-1) level was significantly higher in the treated group (P<0.05). Exendin-4 (Ex-4), a GLP-1 analog, ameliorated endothelium-dependent vasodilation impaired by palmitic acid (PA) in wild-type mouse aortic segments. Ex-4 promoted phosphorylation of eNOS(Ser1177) and Akt, both of which were abrogated by PA, in human umbilical vein endothelial cells. In addition, Lina administration to ApoE(-/-) mice decreased oxidative stress, as determined by urinary 8-OHdG secretion and NADPH oxidase subunit expression in the abdominal aorta. CONCLUSION: Lina inhibited atherogenesis in non-diabetic ApoE(-/-) mice. Amelioration of endothelial dysfunction associated with a reduction of oxidative stress by GLP-1 contributes to the atheroprotective effects of Lina.

Rivaroxaban, a novel oral anticoagulant, attenuates atherosclerotic plaque progression and destabilization in ApoE-deficient mice.

OBJECTIVE: Activated factor X (FXa) plays a key role in the coagulation cascade, whereas accumulating evidence suggests that it also contributes to the pathophysiology of chronic inflammation on the vasculature. In this study, we assessed the hypothesis that rivaroxaban (Riv), a direct FXa inhibitor, inhibits atherogenesis by reducing macrophage activation. METHODS AND RESULTS: Expression levels of PAR-1 and PAR-2, receptors for FXa, increased in the aorta of apolipoprotein E-deficient (ApoE(-/-)) mice compared with wild-type mice (P < 0.01, P < 0.05, respectively). Administration of Riv (5 mg/kg/day) for 20 weeks to 8-week-old ApoE(-/-) mice reduced atherosclerotic lesion progression in the aortic arch as determined by en-face Sudan IV staining compared with the non-treated group (P < 0.05) without alteration of plasma lipid levels and blood pressure. Histological analyses demonstrated that Riv significantly decreased lipid deposition, collagen loss, macrophage accumulation and matrix metallopeptidase-9 (MMP-9) expression in atherosclerotic plaques in the aortic root. Quantitative RT-PCR analyses using abdominal aorta revealed that Riv significantly reduced mRNA expression of inflammatory molecules, such as MMP-9, tumor necrosis factor-α (TNF-α). In vitro experiments using mouse peritoneal macrophages or murine macrophage cell line RAW264.7 demonstrated that FXa increased mRNA expression of inflammatory molecules (e.g., interleukin (IL)-1ß and TNF-α), which was blocked in the presence of Riv. CONCLUSIONS: Riv attenuates atherosclerotic plaque progression and destabilization in ApoE(-/-) mice, at least in part by inhibiting pro-inflammatory activation of macrophages. These results indicate that Riv may be particularly beneficial for the management of atherosclerotic diseases, in addition to its antithrombotic activity.

HMGB1 plays a critical role in vascular inflammation and lesion formation via toll-like receptor 9.

OBJECTIVE: Endogenous ligands such as high-mobility group box 1 (HMGB1) and nucleic acids are released by dying cells and bind to Toll-like receptors (TLRs). As TLR9 is involved in both microbial and sterile inflammation by detecting both bacterial and endogenous DNA, we investigated its role in inflammation and lesion formation in a mouse model of vascular injury. METHODS AND RESULTS: C57BL/6 (WT) and TLR9 KO mice were subjected to wire-mediated vascular injury. Anti-HMGB1 antibody and purified HMGB1 protein were chronically delivered around the injured arteries by gelatin hydrogel, and neointima formation at 4 weeks after injury was evaluated. In addition, the same vascular injury was performed in bone-marrow chimeric mice (WT bone marrow into TLR KO mice; TLR9 KO bone marrow into WT mice). We also evaluated the production of inflammatory cytokines by mouse macrophages in response to HMGB1 and CpG-ODN. In wild-type mice after vascular injury, anti-HMGB1 antibody significantly reduced neointima formation and HMGB1 protein accelerated neointima hyperplasia. HMGB1 failed to accelerate lesion formation in TLR9 KO mice. The bone marrow transplantation study revealed that TLR9 in bone marrow-derived cells played a fundamental role in neointima formation. In vitro, HMGB1 and CpG-ODN synergistically induced the production of inflammatory cytokines by macrophages. CONCLUSIONS: HMGB1 serves as an endogenous mediator of inflammation and lesion formation via the TLR9 pathway in response to vascular injury. Blockade of HMGB1 and/or TLR9 may represent a novel approach to treating atherosclerosis.