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.

S-1-Propenylcysteine promotes IL-10-induced M2c macrophage polarization through prolonged activation of IL-10R/STAT3 signaling

Atherosclerosis is a chronic inflammatory disease that may lead to the development of serious cardiovascular diseases. Aged garlic extract (AGE) has been reported to ameliorate atherosclerosis, although its mode of action remains unclear. We found that AGE increased the mRNA or protein levels of arginase1 (Arg1), interleukin-10 (IL-10), CD206 and hypoxia-inducible factor 2α (HIF2α) and decreased that of CD68, HIF1α and inducible nitric oxide synthase in the aorta and spleen of apolipoprotein E knockout mice. We also found that S-1-propenylcysteine (S1PC), a characteristic sulfur compound in AGE, increased the level of IL-10-induced Arg1 mRNA and the extent of M2c-like macrophage polarization in vitro. In addition, S1PC increased the population of M2c-like macrophages, resulting in suppressed the population of M1-like macrophages and decreased lipopolysaccharide-induced production of pro-inflammatory cytokines. These effects were accompanied by prolonged phosphorylation of the IL-10 receptor α (IL-10Rα) and signal transducer and activator of transcription 3 (STAT3) that inhibited the interaction between IL-10Rα and Src homology-2-containing inositol 5’-phosphatase 1 (SHIP1). In addition, administration of S1PC elevated the M2c/M1 macrophage ratio in senescence-accelerated mice. These findings suggest that S1PC may help improve atherosclerosis due to its anti-inflammatory effect to promote IL-10-induced M2c macrophage polarization.

Abstract P435: Probiotic Supplements Could Prevent Diet-induced Mitochondrial Dysfunction In The Liver Hepatocytes Of Apolipoprotein-e Knockout Mice

A poor diet can modify the intestinal microbiota, known as gut dysbiosis, which causes hepatocyte mitochondrial dysfunction through the release of endotoxins in the portal vein. Mitochondrial dysfunction in the liver leads to non-alcoholic fatty liver disease (NAFLD). Epidemiological evidence shows that most deaths from NAFLD are related to cardiovascular diseases (CVD) demonstrated by impaired vasodilatation as well as increased arterial stiffness, calcification, inflammation, and intimal-medial thickness. Therefore, the objective of our study was to investigate the potential of probiotic supplements to prevent a diet-induced mitochondrial dysfunction in hepatocytes. Apolipoprotein-E knockout mice—genetically modified animals that develop dyslipidemia—were placed in 3 diet groups including a control, western style (high in fats and carbohydrates), as well as a low-carbohydrate high-protein diet. Each group was then further divided in 3 subgroups based on the dose of probiotics administered, including a control, low (0.5 B/dose) and high (5 B/dose) dose of probiotics, for a total of 9 groups (n= 8-12 animals/group). The probiotic supplements contained a 1:1 ratio of Lactobacillus Helveticus and Bifidobacterium Bifidum . The bodyweight and water intake were monitored weekly. After 6 weeks, the liver mitochondria were analyzed by high resolution respirometry using a sequential substrate addition protocol (malate, octanoylcarnitine, ADP, glutamate, pyruvate, cytochrome C, succinate and FCCP) to investigate mitochondrial maximal respiration, leak, membrane damage and uncoupling. Our preliminary results show that the groups that received a high dose of probiotics gained less weight, had a higher respiration following the addition of ADP (a biomarker of complex V efficiency) and a lower respiration following FCCP (a biomarker of mitochondrial uncoupling), as compared to control. Supplements of probiotics could promote weight loss and prevent a diet-induced mitochondrial dysfunction in liver hepatocytes, an important risk factor for NAFLD and therefore, CVD. This project could translate into future research directions in preventative medicine, for example by investigating the potential of probiotics in the prevention of mitochondrial dysfunction in other organs, including the heart and the vascular system.

Abstract 38: Radiation Exposure And Coronary Atherosclerosis: Differential Effect Of The Radiation Site

Background: Thoracic radiation is commonly used in breast cancer, Hodgkin’s lymphoma, head and neck and lung cancer patients. Accelerated coronary artery atherosclerosis is a common complication of thoracic radiation as a result of unintended cardiac radiation. It is unclear however whether specific areas of the heart are more susceptible to the effects of radiation (RT). In this study we hypothesize that accelerated development of lesions post RT is dependent upon differential sensitivity of specific areas of the heart to the effects of RT. Methods: Male Apolipoprotein E knockout mice on a high fat diet received 16Gy cardiac RT targeted to the whole or partial (apical or basal) region of the heart at 9 or 16 weeks of age (n=5 per group). Atherosclerotic lesions in H&E stained slides and inflammatory infiltrates in the hearts by IHC were assessed 8 weeks following RT and compared to control unirradiated mice. Results: Our studies show that: (1) Subendocardial atherosclerotic lesions at the base of heart in mice irradiated at 9 weeks of age after basal irradiation (7.8±2.49) were comparable to whole heart irradiation (12.2±3.29). (2) A greater number of atherosclerotic lesions were present in the basal coronary arteries (29.33±5.48 vs 9±2.70) and basal subendocardial vasculature (6.66±2.07 vs 0.2±0.2) after irradiation of the cardiac base as compared to unirradiated controls in mice irradiated at 16 weeks of age. (3) Apical or whole heart irradiation had no impact on the development of lesions in the basal region of the hearts of older mice. (4) IL-6 was significantly increased in the serum of mice 6 hours post basal cardiac irradiation (105.10±17.56 pg/ml) when compared to unirradiated controls (29.85±11.63 pg/ml), demonstrating an acute inflammatory response. (5) Infiltration of inflammatory cells (CD45 and CD3) and enhanced expression of endothelial adhesion molecules (CD31) were differentially and locally regulated based upon the site of irradiation. Conclusion: Our results indicate that the base of the heart is more prone to development of RT induced atherosclerotic lesions likely due to acute and delayed inflammatory responses. Avoiding this area from direct radiation exposure may improve the quality of life for cancer patients receiving thoracic RT.

Angiotensin Receptor Blocker and Neprilysin Inhibitor Suppresses Cardiac Dysfunction by Accelerating Myocardial Angiogenesis in Apolipoprotein E-Knockout Mice Fed a High-Fat Diet

Hypothesis. Myocardial angiogenesis is important for maintaining cardiac contractile function in patients with cardiac hypertrophy. Evidence shows that angiotensin receptor blocker and neprilysin inhibitors (ARNIs) improve heart failure. The present study investigated the myocardial angiogenic effect of valsartan plus sacubitril in early-stage cardiac dysfunction. Materials and Methods. Male apolipoprotein E-knockout mice fed a high-fat diet were divided into control (CTL), valsartan (30 mg/kg) (VAL), sacubitril (30 mg/kg) (SAC), and valsartan plus sacubitril (30 mg/kg each) (VAL/SAC) groups after 4 weeks of prefeeding and were subsequently treated for 12 weeks. Results. The VAL/SAC group exhibited significantly higher serum brain natriuretic peptide levels; more subtle changes in left ventricular systolic diameter, fractional shortening, and ejection fraction, and significantly higher expression levels of natriuretic peptide precursor B and markers of angiogenesis, including clusters of differentiation 34, vascular endothelial growth factor A, and monocyte chemotactic protein 1, than the CTL group. Conclusions. Valsartan plus sacubitril preserved left ventricular systolic function in apolipoprotein E-knockout mice fed a high-fat diet. This result suggests that myocardial angiogenic factors induced by ARNI might provide cardioprotective effects.