Aim: Cilostazol is clinically used as an inhibitor of platelet aggregation. Although several reports have demonstrated its anti-inflammatory effect, its effect on monocytes and their adhesive interaction to vascular endothelium remains unclear. We thus examined the potential role of cilostazol towards monocyte endothelial interaction under physiological flow conditions. Methods: THP-1 cells, a monocytic cell line, were pretreated with cilostazol (5 µM) for 48 hours. The cells were then perfused over TNF-alpha (5 µg/mL for 4 hours)-stimulated monolayers of human umbilical vein endothelial cells (HUVECs) at shear stress of 1.0 dyen/cm2. Results: TNF-α-activated HUVECs supported significantly more monocyte adhesion to HUVECs (7.32±1.25/HPF) compared to inactivated HUVECs (0.74±0.15/HPF), and the amount of adhesion to TNF-α-activated HUVECs was markedly reduced (3.63±0.55/HPF) when THP-1 cells were incubated in the presence of cilostazol at 5 µM. Interestingly, surface expressions of integrins were not dramatically changed after cilostazol treatment. Intracellular concentration of cAMP was significantly increased after cilostazol treatment, and treatment with Forskolin and Dibutyryl-cAMP, potent inducers of cAMP, dramatically increased THP-1 adhesion to HUVECs. Conclusion: These data suggest that cilostazol has a potential anti-inflammatory effect on monocyte-endothelial interactions via the upregulation of intracellular cAMP.
Aim: An extract of Ginkgo Biloba L. was shown to have preventive effects on cardiovascular disorders, but the molecular mechanisms of its actions remain to be elucidated. Since matrix metalloproteinases (MMPs) are implicated in the rupture of atherosclerotic plaques and the subsequent occurrence of acute coronary syndrome, we examined the effects of a leaf extract (Ginkgolon-24) on the production of MMP-1 in human coronary smooth muscle cells stimulated with oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal, which are factors proposed to play a pivotal role in atherogenesis. Methods: The production of MMP-1 and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 were estimated by immunoblotting. The tyrosine-phosphorylated form of platelet-derived growth factor receptor β (PDGFR-β) was analyzed by immunoprecipitation of the receptor followed by immunoblotting. Results: oxLDL and 4-hydroxynonenal accelerated the production of MMP-1 with the preceding phosphorylation of ERK1/2 and PDGFR-beta;. Pretreatment with Ginkgolon-24 inhibited the production of MMP-1 and phosphorylation of ERK1/2 induced by oxLDL and 4-hydroxynonenal, but did not affect the production and phosphorylation induced by phorbol ester. Furthermore, Ginkgolon-24 prevented tyrosine phosphorylation of the receptor induced by oxLDL and 4-hydroxynonenal. Conclusion: These results suggest that Ginkgo Biloba extract suppresses the oxLDL- and 4-hydroxynonenal-induced production of MMP-1, probably through the inhibition of PDGFR-β activation in human coronary smooth muscle cells.
Aim: The aim of this study was to investigate transcriptional activation in vascular SMC cultured under hypoxic conditions and high low-density lipoprotein (LDL) levels. Methods: We cultured vascular SMC under hypoxic conditions and high LDL levels, and RNA expression profiles for more than 5800 were analyzed by DNA microarray. We performed promoter sequence analysis of genes induced by the combination of hypoxia and high LDL level conditions. Results: In human coronary arterial SMC, the combination of hypoxia and high LDL level conditions induced the expression of 40 genes. Genes induced during the first 24 hours were known to be involved in inflammation, while late genes induced during 48 to 72 hours were composed primarily of genes involved in lipid and/or glucose metabolism. Promoter sequence analysis of these genes revealed that 39 of the 40 genes possessed multiple hypoxic response elements (HRE). The most induced gene in the combination of hypoxia and high LDL level conditions was the leptin gene. Functional analysis of the 3kb leptin promoter revealed that HRE at-166 mediated transcriptional activation by hypoxia, but 3kb reporter constructs can not reproduce the additive affect of LDL under hypoxia. Conclusion: These results support the hypothesis that an HRE-mediated mechanism may be involved in transcriptional activation during lipid deposition in vascular SMC induced by hypoxia and LDL loading, but additional mechanisms may be involved in the synergistic action induced by LDL.
Aim: This study was investigated to characterize the activation mechanism of a mitogen-activated protein (MAP) kinase superfamily in diabetes in aortae and cultured vascular smooth muscle cells (VSMCs) from rats. Methods: Male Sprague-Dawley rats were used for this procedure, and diabetes was induced by streptozotocin injection at 50 mg/kg. After 6 weeks, the thoracic aortae from normal and diabetic rats were removed for detection of the MAP kinase superfamily by immunoblot analysis. Results: In aortae, the protein levels of extracellular signal-regulated protein kinase (ERK)-1, c-jun NH2-terminal protein kinase (JNK)-1 and -2, and p38 increased significantly more in diabetic rats than in normal rats. In contrast, phosphorylated protein levels of ERK-1 and -2, JNK-1, and p38 were significantly more elevated in diabetic rats than in normal rats. In VSMCs from normal rats, a high concentration of glucose cultured for three days significantly increased the phosphorylated protein levels of ERKs and p38, but not JNKs, without any change of these protein levels. Serum interleukin (IL)-1β was significantly higher in diabetic rats than in normal rats. Several types of proinflammatory cytokine dose-dependently phosphorylated the levels of ERKs, JNK-1, and p38, but not JNK-2, in VSMCs from normal rats. In cells from diabetic rats, phosphorylated protein levels of ERKs and p38 were significantly elevated by IL-1β. In addition, interferon-γ phosphorylated the levels of ERKs in diabetic cells more than in normal cells. Conclusion: Our results suggest that, under diabetic conditions, the MAP kinase superfamily was activated by different pathways in the vasculature; i.e., ERKs and p38 might be mainly phosphorylated by a complex of high concentrations of glucose and of several types of proinflammatory cytokines, but the phosphorylation of JNK-1 might depend on the concentration of proinflammatory cytokines such as IL-1β, and/or additional unknown factors, except glucose.
Aim: To investigate whether the perinatal and postnatal exposure of mice to bisphenol A (BPA) caused the development of obesity and/or hyperlipidemia. Methods: Pregnant mice were exposed to BPA in drinking water at concentrations of either 1 µg/mL (LD group) or 10 µg/mL (HD group) from gestation day 10 and throughout the lactating period. After weaning, the pups were allowed free access to drinking water containing the appropriate concentrations of BPA. The body weight, adipose tissue weight, and serum lipid levels were measured in the offspring at postnatal day 31. Results: In females, the mean body weight increased by 13% in the LD group (p<0.05) and 11% in the HD group (p<0.05) compared with the control group. The mean adipose tissue weight increased by 132% in the LD group (p<0.01). The mean total cholesterol level increased by 33% in the LD group (p<0.01) and 17% in the HD group (p<0.05). In males, the mean body weight and mean adipose tissue weight increased by 22% (p<0.01) and 59% (p<0.01), respectively, in the HD group compared with the control group. The mean triacylglycerol level increased by 34% in the LD group (p<0.05). Conclusions: The continuous exposure of mice to BPA during the perinatal and postnatal periods caused the development of obesity and hyperlipidemia.
Aim: We investigated the effects of a calorie-restricted low-carbohydrate diet on glucose and lipid metabolism, and body fat distribution, especially on the secretion of leptin and lipoprotein lipase from adipose tissue in Otsuka Long Evans Tokushima Fatty (OLETF) rats. Methods: Forty-three week-old male OLETF rats were randomized into three groups (n=6 per group): the HC group (HC) was fed a diet with 60% carbohydrate; the LC group (LC) with 30% carbohydrate; and the P-HC group (P-HC) with 60% carbohydrate and pioglitazone (0.1%). The total calorie intake was restricted to 70% of the average intake from each diet (60 kcal/day). The diets were continued for 8 weeks. Results: Similar decreases in body weight and serum glucose were observed in the three groups. Serum insulin concentration was significantly decreased in LC and P-HC compared to HC. Serum total cholesterol and triglycerides decreased significantly (p<0.05) in LC and P-HC compared to HC. The decrease of visceral fat area measured by computed tomography was greatest in LC among the three groups. At the end of the diet, leptin secretion from visceral adipose tissue and lipoprotein lipase (LPL) activity in subcutaneous adipose tissue were significantly higher in LC and P-HC compared to HC (p<0.05). Conclusion: These results indicate that under calorie-restricted conditions, low carbohydrates are much more effective than high carbohydrates in improving insulin sensitivity.
Aim: Adiponectin reportedly reduces insulin resistance. Exercise has also been shown to lessen insulin resistance, although it is not well known whether exercise increases levels of adiponectin and/or its receptors nor whether it effects are dependent on exercise intensity and/or period. We previously reported that blood adiponectin levels increased by 150% in animals that exercised at a rate of 30 m/min for 60 minutes, 2 days per week, and adiponectin receptor 1 (AdipoR1) mRNA levels in muscle increased up to 4 times in response to exercise at a rate of 25 m/min for 30 min, 5 days per week for 12 weeks. Methods: In light of this information, we examined the effects of short-term exercise on adiponectin, and adiponectin receptor levels in rats, using ELISA and real-time PCR. Results: Our data showed that adiponectin mRNA levels in adipose tissue increased by 280% in rats exercised at a rate of 30 m/min for 60 minutes for 2 weeks and correlated with the exercise time periods. No effects of short-term exercise on adiponectin receptor 1 mRNA in muscle were observed. Conclusion: Thus, long-term exercise may be required to regulate adiponectin receptor 1 mRNA expression in muscle and adiponectin mRNA expression in adipose tissue.