Inhibition of Atherosclerotic Plaque Development by Oral Administration of α-Glucosyl Hesperidin and Water-Dispersible Hesperetin in Apolipoprotein E Knockout Mice.

OBJECTIVE: Hesperidin, an abundant flavonoid in citrus fruit, and its aglycone, hesperetin, have been reported to possess various physiological activities, including antioxidant, anti-inflammatory, hypolipidemic, and antihypertensive activities. In this study, we investigated whether α-glucosyl hesperidin and water-dispersible hesperetin have protective effects on atherosclerotic progression in apolipoprotein E knockout (Apo-E KO) mice. METHODS: Ten-week-old male Apo-E KO mice were randomly assigned a regular high-fat diet, a high-fat diet with 0.5% α-glucosyl hesperidin, or a high-fat diet with 0.1% water-dispersible hesperetin for 12 weeks. Measurement of plasma total cholesterol levels, histological staining of aortic root, and immunohistochemistry for macrophages were performed to evaluate atherosclerotic plaque formation. Vascular reactivity of mouse aortic rings was also measured. RESULTS: Both α-glucosyl hesperidin and water-dispersible hesperetin reduced plasma total cholesterol level. They also reduced plaque formation area, adipose deposition, and macrophage infiltration into atherosclerotic lesion. Vascular-endothelium-dependent relaxation in response to acetylcholine was improved in both experimental diet groups compared to the high-fat diet group. CONCLUSIONS: Our study suggests that both α-glucosyl hesperidin and water-dispersible hesperetin exert protective effects on atherosclerotic progression in Apo-E KO mice because they exhibit hypolipidemic activity, reduce inflammation through macrophages, and prevent endothelial dysfunction.

Enhanced inflammation in epicardial fat in patients with coronary artery disease.

It has been hypothesized that epicardial fat, a local visceral fat depot with close proximity to coronary arteries, may serve as a source of inflammatory cytokines and cells in coronary atherosclerotic lesions. Here, we characterized infiltration of inflammatory cells and expression of adipocytokines in epicardial adipose tissue in patients with and without coronary artery disease (CAD). Pare samples were obtained from epicardial and subcutaneous adipose tissue during elective cardiac surgery (CAD, n = 8; non-CAD, n = 9). Inflammatory cell infiltration was investigated by immunohistochemical staining using antibodies against CD3, CD4, CD8 and CD68. Expression of adipocytokines was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. Infiltration of macrophages and CD8-positive T cells in the epicardial adipose tissue in the CAD group was greater than that in the non-CAD group. In contrast, there was no significant difference between the two groups in the number of inflammatory cells in subcutaneous adipose tissue. No statistical difference could be found between the CAD group and the non-CAD group in the expression levels of adiponectin and inflammatory cytokines in epicardial adipose tissue. Our findings suggest that inflammatory cell infiltration is enhanced in epicardial adipose tissue, but not in subcutaneous fat, in patients with coronary artery disease. Chronic inflammation in epicardial fat may influence the pathogenesis of coronary atherosclerosis.

Expression of cystatin C prevents oxidative stress-induced death in PC12 cells.

Cystatin C, an inhibitor of cysteine proteinases, is suggested to be involved in oxidative stress-induced apoptosis of cultured CNS neurons and various neuronal diseases in vivo; however, little is known about its mechanism of action. To address the role cystatin C plays in oxidative stress-induced neuronal cell death, we established PC12 cell lines that stably expressed rat cystatin C. These cystatin C-expressing PC12 cells showed remarkable resistance to high (50%) oxygen atmosphere. This resistance correlate with expression levels of cystatin C, demonstrating that cystatin C has a protective effect on high oxygen-induced cell death. In contrast, in a normal (20%) oxygen atmosphere neither control nor cystatin C-expressing PC12 cells showed a significant change in the number of living cells, indicating that cystatin C does not play an important role in the regulation of cellular proliferation. Furthermore, the cystatin C-expressing cell line also resisted other oxidative stresses, including glutamate- and 13-L-hydroperoxylinoleic acid (LOOH)-induced cell death. These results demonstrate that cystatin C has protective effects against various oxidative stresses that induce cell death.

Estrogen enhances depolarization-induced glutamate release through activation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase in cultured hippocampal neurons.

Changes in synaptic efficacy are considered necessary for learning and memory. Recently, it has been suggested that estrogen controls synaptic function in the central nervous system. However, it is unclear how estrogen regulates synaptic function in central nervous system neurons. We found that estrogen potentiated presynaptic function in cultured hippocampal neurons. Chronic treatment with estradiol (1 or 10 nm) for 24 h significantly increased a high potassium-induced glutamate release. The estrogen-potentiated glutamate release required the activation of both phosphatidylinositol 3-kinase and MAPK. The high potassium-evoked release with or without estradiol pretreatment was blocked by tetanus neurotoxin, which is an inhibitor of exocytosis. In addition, the reduction in intensity of FM1-43 fluorescence, which labeled presynaptic vesicles, was enhanced by estradiol, suggesting that estradiol potentiated the exocytotic mechanism. Furthermore, protein levels of synaptophysin, syntaxin, and synaptotagmin (synaptic proteins, respectively) were up-regulated by estradiol. We confirmed that the up-regulation of synaptophysin was blocked by the MAPK pathway inhibitor, U0126. These results suggested that estrogen enhanced presynaptic function through the up-regulated exocytotic system. In this study, we propose that estrogen reinforced excitatory synaptic transmission via potentiated-glutamate release from presynaptic sites.

Exendin-4, a glucagon-like peptide-1 receptor agonist, attenuates neointimal hyperplasia after vascular injury.

Exendin-4 is a glucagon-like peptide-1 receptor agonist that has been used as a drug for treatment of type 2 diabetes. To investigate the effect of exendin-4 on the cardiovascular system, we investigated the impact of exendin-4 on neointimal hyperplasia of the femoral artery after vascular injury. We performed wire-mediated endovascular injury in C57BL/6 mice, followed by administration of exendin-4 24 nmol/kg/day via infusion pump. Four weeks after the injury, exendin-4 treatment significantly attenuated neointimal hyperplasia of the injured artery, although it did not affect glucose metabolism and lipid profile in wild-type mice. Immunofluorescence study revealed abundant expression of GLP-1 receptor on α-smooth muscle actin-positive cells in the injured vessel. Cell proliferation assay using rat aortic smooth muscle cells showed that exendin-4 reduced PDGF-BB induced smooth muscle cell proliferation through the cAMP/PKA pathway. Exendin-4 also inhibited TNFα production by peritoneal macrophages in response to inflammatory stimulus. Our findings indicate that a GLP-1 receptor agonist attenuated neointimal formation after vascular injury. GLP-1 receptor agonists or drugs that raise endogenous GLP-1 level might be effective in the treatment of vascular diseases.

Coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue.

OBJECTIVES: The purpose of this report was to assess the link between macrophage polarization in epicardial adipose tissue and atherosclerosis in patients with coronary artery disease (CAD). BACKGROUND: Macrophage accumulation enhances chronic inflammation in adipose tissue, but macrophage phenotypic change in human epicardial adipose tissue and its role in atherogenesis are unknown. METHODS: Samples were obtained from epicardial and subcutaneous adipose tissue during elective cardiac surgery (CAD, n = 38; non-CAD, n = 40). Infiltration of M1/M2 macrophages was investigated by immunohistochemical staining with antibodies against CD11c and CD206, respectively. Expression of pro- and anti-inflammatory adipocytokines in adipose tissue was evaluated by real-time quantitative polymerase chain reaction. RESULTS: Infiltration of macrophages and expression of pro- and anti-inflammatory cytokines were enhanced in epicardial fat of patients with CAD compared with that in non-CAD patients (p < 0.05). The ratio of M1/M2 macrophages was positively correlated with the severity of CAD (r = 0.312, p = 0.039). Furthermore, the expression of pro-inflammatory cytokines was positively correlated, and the expression of anti-inflammatory cytokines was negatively correlated with the ratio of M1/M2 macrophages in epicardial adipose tissue of CAD patients. By contrast, there was no significant difference in macrophage infiltration and cytokine expression in subcutaneous adipose tissue between the CAD and non-CAD groups. CONCLUSIONS: The ratio of M1/M2 macrophages in epicardial adipose tissue of CAD patients is changed compared with that in non-CAD patients. Human coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue.

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species.

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.