Natural Killer T Cells Are Involved in Atherosclerotic Plaque Instability in Apolipoprotein-E Knockout Mice.

The infiltration and activation of macrophages as well as lymphocytes within atherosclerotic lesion contribute to the pathogenesis of plaque rupture. We have demonstrated that invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes that recognize glycolipid antigens, play a crucial role in atherogenesis. However, it remained unclear whether iNKT cells are also involved in plaque instability. Apolipoprotein E (apoE) knockout mice were fed a standard diet (SD) or a high-fat diet (HFD) for 8 weeks. Moreover, the SD- and the HFD-fed mice were divided into two groups according to the intraperitoneal injection of α-galactosylceramide (αGC) that specifically activates iNKT cells or phosphate-buffered saline alone (PBS). ApoE/Jα18 double knockout mice, which lack iNKT cells, were also fed an SD or HFD. Plaque instability was assessed at the brachiocephalic artery by the histological analysis. In the HFD group, αGC significantly enhanced iNKT cell infiltration and exacerbated atherosclerotic plaque instability, whereas the depletion of iNKT cells attenuated plaque instability compared to PBS-treated mice. Real-time PCR analyses in the aortic tissues showed that αGC administration significantly increased expressional levels of inflammatory genes such as IFN-γ and MMP-2, while the depletion of iNKT cells attenuated these expression levels compared to those in the PBS-treated mice. Our findings suggested that iNKT cells are involved in the exacerbation of plaque instability via the activation of inflammatory cells and upregulation of MMP-2 in the vascular tissues.

The disruption of invariant natural killer T cells exacerbates cardiac hypertrophy and failure caused by pressure overload in mice.

NEW FINDINGS: What is the central question of this study? We questioned whether the disruption of invariant natural killer T (iNKT) cells exacerbates left ventricular (LV) remodelling and heart failure after transverse aortic constriction in mice. What are the main findings and their importance? Pressure overload induced by transverse aortic constriction increased the infiltration of iNKT cells in mouse hearts. The disruption of iNKT cells exacerbated LV remodelling and hastened the transition from hypertrophy to heart failure, in association with the activation of mitogen-activated protein kinase signalling. Activation of iNKT cells modulated the immunological balance in this process and played a protective role against LV remodelling and failure. ABSTRACT: Chronic inflammation is involved in the development of cardiac remodelling and heart failure (HF). Invariant natural killer T (iNKT) cells, a subset of T lymphocytes, have been shown to produce various cytokines and orchestrate tissue inflammation. The pathophysiological role of iNKT cells in HF caused by pressure overload has not been studied. In the present study, we investigated whether the disruption of iNKT cells affected this process in mice. Transverse aortic constriction (TAC) and a sham operation were performed in male C57BL/6J wild-type (WT) and iNKT cell-deficient Jα18 knockout (KO) mice. The infiltration of iNKT cells was increased after TAC. The disruption of iNKT cells exacerbated left ventricular (LV) remodelling and hastened the transition to HF after TAC. Histological examinations also revealed that the disruption of iNKT cells induced greater myocyte hypertrophy and a greater increase in interstitial fibrosis after TAC. The expressions of interleukin-10 and tumour necrosis factor-α mRNA and their ratio in the LV after TAC were decreased in the KO compared with WT mice, which might indicate that the disruption of iNKT cells leads to an imbalance between T-helper type 1 and type 2 cytokines. The phosphorylation of extracellular signal-regulated kinase was significantly increased in the KO mice. The disruption of iNKT cells exacerbated the development of cardiac remodelling and HF after TAC. The activation of iNKT cells might play a protective role against HF caused by pressure overload. Targeting the activation of iNKT cells might thus be a promising candidate as a new therapeutic strategy for HF.

[The clinical condition and treatment of diseases associated with sudden death].

The Hokkaido Medical Society is a group of doctors and medical researchers in Hokkaido. Its purpose is to contribute to medicine and to the improvement of medical treatment. This symposium was carried out in order to inform citizens about the condition known as sudden death. We hypothesize that the incidence of sudden death tends to increase in line with the incidence of metabolic syndrome. Approximately four hundred patients were transported to our hospital by ambulance in a state of cardiopulmonary arrest (CPA) last year. The number of CPA patients who are treated in our hospital has increased in comparison to the previous decade. The theme of this year is "The clinical condition and treatment of diseases associated with sudden death" in view of the above mentioned situation. In 2015, it was reported that sudden death occurred in an American pilot and that the co-pilot was forced to make an emergency landing. Interestingly, sudden death can ever sometimes occur in pilots who undergo regular physical examinations. Numerous diseases and conditions are associated with sudden death, including: acute myocardial infarction, irregular pulse, cardiac insufficiency, cerebrovascular disease, aortic dissection and choking. We are of the opinion that the frequency of sudden death is very high in the fields of emergency medicine, cardiovascular medicine, cardiovascular surgery and neurosurgery. In this symposium, we presented and explained the condition that is known as sudden death and the current state of treatment of sudden death in emergency medicine, cardiovascular medicine, cardiovascular surgery and neurosurgery departments of the Hokkaido University Graduate School of Medicine in October, 2015. We hope that the symposium will help the citizen audience to understand the condition and treatment of sudden death, and also to help prevent sudden death.

AST-120 ameliorates lowered exercise capacity and mitochondrial biogenesis in the skeletal muscle from mice with chronic kidney disease via reducing oxidative stress.

BACKGROUND: Exercise capacity and quality of life are markedly impaired in chronic kidney disease (CKD). Increased plasma uremic toxins such as indoxyl sulfate (IS), which induce oxidative stress, may be involved in this process. An oral adsorbent, AST-120, can reduce circulating IS, however, its effects on skeletal muscle and exercise capacity have not been investigated in CKD. METHODS: Subtotal-nephrectomy or sham operation was performed in 8-week-old C57BL/6J mice. They were divided into two groups with or without 8% (w/w) of AST-120 in standard diet for 20 weeks. Sham, Sham + AST-120, CKD and CKD + AST-120 (n = 12, each group) were studied. We also conducted a C2C12 cell culture study to determine the direct effects of IS on oxidative stress. RESULTS: Plasma IS levels were significantly increased in CKD compared with Sham (1.05 ± 0.11 versus 0.21 ± 0.03 mg/dL, P <0.05), which was significantly ameliorated in CKD + AST-120 (0.41 ± 0.06 mg/dL). The running distance to exhaustion determined by treadmill tests was significantly reduced in CKD compared with Sham (267 ± 17 versus 427 ± 36 m, P <0.05), and this reduction was also significantly ameliorated in CKD + AST-120 (407 ± 38 m) without altering skeletal muscle weight. Citrate synthase activity and mitochondrial biogenesis gene were downregulated, and superoxide production was significantly increased in the skeletal muscle from CKD, and these changes were normalized in CKD + AST-120. Incubation of C2C12 cells with IS significantly increased NAD(P)H oxidase activity. CONCLUSIONS: The administration of AST-120 improved exercise capacity and mitochondrial biogenesis of skeletal muscle via reducing oxidative stress. AST-120 may be a novel therapeutic agent against exercise intolerance in CKD.

Activation of invariant natural killer T cells by α-galactosylceramide ameliorates myocardial ischemia/reperfusion injury in mice.

Invariant natural killer T (iNKT) cells orchestrate tissue inflammation via regulating various cytokine productions. However the role of iNKT cells has not been determined in myocardial ischemia/reperfusion (I/R) injury. The purpose of this study was to examine whether the activation of iNKT cells by α-galactosylceramide (α-GC), which specifically activates iNKT cells, could affect myocardial I/R injury. I/R or sham operation was performed in male C57BL/6J mice. I/R mice received the injection of either αGC (I/R+αGC, n=48) or vehicle (I/R+vehicle, n=49) 30 min before reperfusion. After 24h, infarct size/area at risk was smaller in I/R+αGC than in I/R+vehicle (37.8 ± 2.7% vs. 47.1 ± 2.5%, P<0.05), with no significant changes in area at risk. The numbers of infiltrating myeloperoxidase- and CD3-positive cells were lower in I/R+αGC. Apoptosis evaluated by TUNEL staining and caspase-3 protein was also attenuated in I/R+αGC. Myocardial gene expression of tumor necrosis factor-α and interleukin (IL)-1ß in I/R+αGC was lower to 46% and 80% of that in I/R+vehicle, respectively, whereas IL-10, IL-4, and interferon (IFN)-γ were higher in I/R+αGC than I/R+vehicle by 2.0, 4.1, and 9.6 folds, respectively. The administration of anti-IL-10 receptor antibody into I/R+αGC abolished the protective effects of αGC on I/R injury (infarct size/area at risk: 53.1 ± 5.2% vs. 37.4 ± 3.5%, P<0.05). In contrast, anti-IL-4 and anti-IFN-γ antibodies did not exert such effects. In conclusion, activated iNKT cells by αGC play a protective role against myocardial I/R injury through the enhanced expression of IL-10. Therapies designed to activate iNKT cells might be beneficial to protect the heart from I/R injury.

Activation of natural killer T cells ameliorates postinfarct cardiac remodeling and failure in mice.

RATIONALE: Chronic inflammation in the myocardium is involved in the development of left ventricular (LV) remodeling and failure after myocardial infarction (MI). Invariant natural killer T (iNKT) cells have been shown to produce inflammatory cytokines and orchestrate tissue inflammation. However, no previous studies have determined the pathophysiological role of iNKT cells in post-MI LV remodeling. OBJECTIVE: The purpose of this study was to examine whether the activation of iNKT cells might affect the development of LV remodeling and failure. METHODS AND RESULTS: After creation of MI, mice received the injection of either α-galactosylceramide (αGC; n=27), the activator of iNKT cells, or phosphate-buffered saline (n=31) 1 and 4 days after surgery, and were followed during 28 days. Survival rate was significantly higher in MI+αGC than MI+PBS (59% versus 32%, P<0.05). LV cavity dilatation and dysfunction were significantly attenuated in MI+αGC, despite comparable infarct size, accompanied by a decrease in myocyte hypertrophy, interstitial fibrosis, and apoptosis. The infiltration of iNKT cells were increased during early phase in noninfarcted LV from MI and αGC further enhanced them. It also enhanced LV interleukin (IL)-10 gene expression at 7 days, which persisted until 28 days. AntienIL-10 receptor antibody abrogated these protective effects of αGC on MI remodeling. The administration of αGC into iNKT cell-deficient Jα18(-/-) mice had no such effects, suggesting that αGC was a specific activator of iNKT cells. CONCLUSIONS: iNKT cells play a protective role against post-MI LV remodeling and failure through the enhanced expression of cardioprotective cytokines such as IL-10.

Identification and further differentiation of subendocardial and transmural myocardial infarction by fast strain-encoded (SENC) magnetic resonance imaging at 3.0 Tesla.

OBJECTIVES: To investigate whether subendocardial and transmural myocardial infarction can be identified and differentiated using the peak circumferential and longitudinal strains measured by fast strain-encoded (SENC). METHODS: Nineteen patients with ischemic heart diseases underwent imaging with fast SENC and late gadolinium enhancement (LGE) MRI at 3 T. Fast SENC measurements were performed in three short-axis slices (basal, mid-ventricular and apical levels) and one long-axis view (four-chamber) to assess peak longitudinal and circumferential systolic strains. RESULTS: All patients showed myocardial infarction with an average of 7 positive LGE segments. A total of 304 segments for longitudinal strains (LS) and 114 segments for circumferential strains (CS) could be analysed. Positive LGE segments showed lower peak CS and LS compared with the no LGE segments (P < 0.0001 for both). Segments with subendocardial infarction showed reduced CS and LS compared with the no LGE segments (P < 0.0001 for both). There was a significant difference in CS between subendocardial and transmural infarct segments (P = 0.03), but no significant difference in LS between them (P = 0.64). CONCLUSIONS: Fast SENC can identify old myocardial infarction and differentiate subendocardial from transmural infarction.

Natural killer T cells are involved in adipose tissues inflammation and glucose intolerance in diet-induced obese mice.

BACKGROUND: Macrophage and lymphocyte infiltration in adipose tissue may contribute to the pathogenesis of obesity-mediated metabolic disorders. Natural killer T (NKT) cells, which integrate proinflammatory cytokines, have been demonstrated in the atherosclerotic lesions and in visceral adipose tissue. OBJECTIVE: To determine whether NKT cells are involved in glucose intolerance and adipose tissue inflammation in diet-induced obese mice. METHODS AND RESULTS: Male beta(2)-microglobulin knockout (KO) mice lacking NKT cells and C57BL/6J (wild-type) mice were fed with a high-fat diet (HFD) for 13 weeks [corrected]. Body weight and visceral obesity were comparable between wild-type and KO mice. However, macrophage infiltration was reduced in adipose tissue and glucose intolerance was significantly ameliorated in KO mice. To further confirm that NKT cells are involved in these abnormalities, alpha-galactosylceramide, 0.1 microg/g body weight, which specifically activates NKT cells, was administered after 13 weeks of HFD feeding. alpha-Galactosylceramide significantly exacerbated glucose intolerance and macrophage infiltration as well as cytokine gene expression in adipose tissue. CONCLUSIONS: NKT cells play a crucial role in the development of adipose tissue inflammation and glucose intolerance in diet-induced obesity.

Impact of statins on modulation by insulin of expression of plasminogen activator inhibitor type-1.

OBJECTIVE: Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic inhibitor of fibrinolysis and a known determinant of cardiovascular risk. That its expression is stimulated by insulin in HepG2 human hepatoma cells has been shown earlier. Others have found that increased expression of PAI-1 is a risk factor for acute myocardial infarction. Pleiotropic (noncholesterol lowering) effects of statins seem to reduce cardiovascular risk. This study was designed to determine whether insulin stimulation of PAI-1 expression is attenuated by statins, and if so to explore mechanisms that are responsible for the attenuation. METHODS: PAI-1 protein in the conditioned media was assayed by western blotting, and PAI-1 mRNA expression was measured by real-time reverse transcriptase polymerase chain reactions. RESULTS: Insulin (0.001-10 micromol/l) increased accumulation of PAI-1 protein in the conditioned media and PAI-1 mRNA expression in HepG2 cells. A transient transfection assay of the human PAI-1 promoter-luciferase construct demonstrated that insulin increased PAI-1 promoter activity. Increased PAI-1 mRNA was attenuated significantly by U0126 and PD98059, specific inhibitors of mitogen-activated protein kinase. In contrast, GF109203X, an inhibitor of the protein kinase C pathway, and LY294002, an inhibitor of phosphatidylinositol 3-kinase, exerted no effects. Simvastatin (10 micromol/l), known to translocate membrane-bound sterol regulatory element-binding protein to nuclei, attenuated PAI-1 mRNA expression induced by insulin. It did not affect baseline PAI-1 mRNA expression. Intraperitoneal injection of insulin (0.1 IU/kg) increased concentrations of PAI-1 antigen in plasma in mice within 3 h, correlating with hepatic PAI-1 mRNA expression. CONCLUSION: Insulin-mediated augmented expression of PAI-1 may be amenable to suppression attributable to pleiotropic effects of statins, potentially diminishing cardiovascular risk in patients with insulin resistance.

Allograft inflammatory factor-1 augments macrophage phagocytotic activity and accelerates the progression of atherosclerosis in ApoE-/- mice.

Allograft inflammatory factor (AIF)-1, originally cloned from a rat heart allograft under chronic rejection, is induced in various inflammatory conditions including atherosclerosis. Using mouse AIF-1 transfected macrophages and AIF-1 transgenic (AIF-1 Tg) mice, we analyzed the influence of AIF-1 overexpression on macrophage phagocytosis and the development of atherosclerosis. The AIF-1 transfectants showed significantly increased phagocytosis of latex beads and E. coli BioParticles as well as incorporation of acetylated low-density lipoprotein (LDL) compared to those of vector controls. Concordant results were obtained with elicited peritoneal exudate cells from AIF-1 Tg mice. When AIF-1 Tg mice were crossbred with apolipoprotein E knockout mice (ApoE-/-), these AIF-1 Tg ApoE-/- mice developed significantly increased atherosclerotic lesions compared to ApoE-/- mice. These results suggest that enhanced AIF-1 expression leads to augmented incorporation of degenerated LDL by macrophages and promotes development of atherosclerotic vasculopathy.

Platelet aggregability in patients with hypertension treated with angiotensin II type 1 receptor blockers.

AIM: Cardiovascular events associated with hypertension often involve thrombosis. Increased platelet activity is one of the risk factors of cardiovascular diseases. Antithrombotic properties of antihypertensive agents are not fully characterized. Angiotensin II type 1 receptor blockers (ARBs) are widely used for the treatment of hypertension. Some ARBs can provoke antiaggregatory effects on platelets in vitro. Whether ARBs can inhibit platelet aggregation was tested in hypertensive patients in vivo. METHODS: Platelet aggregation was assessed by the highly sensitive particle counting method using laser-light scattering. RESULTS: Large platelet aggregation induced by adenosine diphosphate (ADP, 3 microM) was 2.6+/-0.4 (x10(7)) (SE) in hypertensive patients treated with losartan (72+/-3 years old, n=10) while it was 3.9+/-0.6 in hypertensive patients treated with candesartan (70+/-5 years old, n=6; p=0.056). Large platelet aggregation induced by thromboxane A2 receptor agonist, U46619 (10 microM), was 2.8+/-0.5 (x10(7)) in hypertensive patients treated with losartan while it was 5.1+/-0.9 in hypertensive patients treated with candesartan (p=0.033). Clinical characteristics including the control of blood pressure did not differ between the two groups (losartan 136+/-5/73+/-3 mmHg vs. candesartan 135+/-4/76+/-5). CONCLUSION: Thus, losartan may have the possibility to inhibit platelet activation in patients with hypertension independent of blood pressure reduction. Antiaggregatory properties may be independent of angiotensin II type 1 receptor or of antihypertensive actions. The favorable effects of losartan on reduction of adverse cardiovascular events among hypertensive patients may be at least partly mediated by inhibition of platelet activation.

Natural killer T cells accelerate atherogenesis in mice.

We have investigated the potential role of CD1d-restricted natural killer T (NKT) cells in the development of atherosclerosis in mice. When fed an atherogenic diet (AD), NKT cell-deficient CD1d(-/-) mice had significantly smaller atherosclerotic lesions than AD-fed C57BL/6 (wild-type [WT]) mice. A significant reduction in atherosclerotic lesions was also demonstrated in AD-fed, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice reconstituted with CD1d(-/-) bone marrow cells compared with the lesions observed in Ldlr(-/-)mice reconstituted with WT marrow cells. In addition, repeated injections of alpha-GalCer or the related glycolipid OCH to apolipoprotein E knockout (apoE(-/-)) mice during the early phase of atherosclerosis significantly enlarged the lesion areas compared with mice injected with vehicle control. However, administering alpha-GalCer to apoE(-/-) mice with established lesions did not significantly increase the lesion area but considerably decreased the collagen content. Atherosclerosis development in either AD-fed WT or apoE(-/-) mice was associated with the presence of Valpha14Jalpha18 transcripts in the atherosclerotic arterial walls, indicating that NKT cells were recruited to these lesions. Thioglycolate-elicited macrophages pulsed with oxidized low-density lipoproteins expressed enhanced CD1d levels and induced NKT cells to produce interferon-gamma, a potentially proatherogenic T-helper 1 (TH1) cytokine. Collectively, we conclude that NKT cells are proatherogenic in mice.

Induction of plasminogen activator inhibitor-1 in endothelial cells by basic fibroblast growth factor and its modulation by fibric acid.

Plasminogen activator inhibitor-1 (PAI-1) inhibits fibrinolysis and proteolysis. Basic fibroblast growth factor (bFGF) stimulates angiogenesis, which requires regional proteolysis. Because modulation of vasculopathy requires tight control of proteolysis, effects of bFGF on PAI-1 expression in endothelial cells (ECs) were characterized. bFGF increased PAI-1 mRNA and accumulation of PAI-1 protein in conditioned media in human umbilical vein ECs. The bFGF-mediated increase in PAI-1 mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. The rate of decrease in PAI-1 mRNA after actinomycin D treatment was not affected by bFGF. Transient transfection assays of the human PAI-1 promoter-luciferase construct demonstrated that bFGF-induced PAI-1 transcription was dependent on the elements within the -313 to -260 bp relative to the transcription start site. This region contains an E26 transformation specific 1 (Ets-1)-like site. Electrophoretic mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of the Ets-1-like transcription factor to the PAI-1 promoter. Nucleotide substitution to disrupt the Ets-1-like site reduced bFGF-stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator-activated receptor-alpha, inhibited basal and bFGF-stimulated PAI-1 expression. By inducing PAI-1 expression from ECs, bFGF may control proteolysis and fibrinolysis in vessel walls.