A Randomized Controlled Trial Comparing the Effects of Sitagliptin and Glimepiride on Endothelial Function and Metabolic Parameters: Sapporo Athero-Incretin Study 1 (SAIS1).

OBJECTIVES: The DPP-4 inhibitors are incretin-related drugs that improve hyperglycemia in a glucose-dependent manner and have been reported to exert favorable effects on atherosclerosis. However, it has not been fully elucidated whether DPP-4 inhibitors are able to improve endothelial function in patients with type 2 diabetes. Therefore, we investigated the efficacy of sitagliptin, a DPP-4 inhibitor, on endothelial function and glycemic metabolism compared with that of the sulfonylurea glimepiride. MATERIALS AND METHODS: In this multicenter, prospective, randomized parallel-group comparison study, 103 outpatients with type 2 diabetes (aged 59.9 ± 9.9 years with HbA1c levels of 7.5 ± 0.4%) with dietary cure only and/or current metformin treatment were enrolled and randomly assigned to receive sitagliptin or glimepiride therapy once daily for 26 weeks. Flow-mediated dilation (FMD), a comprehensive panel of hemodynamic parameters (Task Force® Monitor), and serum metabolic markers were assessed before and after the treatment. RESULTS: During the study period, no statistically significant change in %FMD was seen in both groups (sitagliptin, 5.6 to 5.6%; glimepiride, 5.6 to 6.0%). Secretory units of islets in transplantation, TNF-α, adiponectin and biological antioxidant potential significantly improved in the sitagliptin group, and superoxide dismutase also tended to improve in the sitagliptin group, while improvements in HbA1c levels were similar between groups. Cardiac index, blood pressure and most other metabolic parameters were not different. CONCLUSIONS: Regardless of glycemic improvement, early sitagliptin therapy did not affect endothelial function but may provide favorable effects on beta-cell function and on inflammatory and oxidative stress in patients with type 2 diabetes without advanced atherosclerosis. TRIAL REGISTRATION: UMIN Clinical Trials Registry System UMIN 000004955.

TNF-α production in NKT cell hybridoma is regulated by sphingosine-1-phosphate: implications for inflammation in atherosclerosis.

OBJECTIVES: Natural killer T (NKT) cells are unique T lymphocytes that recognize glycolipid antigen and produce various cytokines. NKT cells accelerate atherosclerosis in mice. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid and regulates T-lymphocyte trafficking. We aimed to determine the effects of S1P on the production of proinflammatory cytokine, tumor necrosis factor (TNF)-α, in NKT cell hybridomas and mouse NKT cells. MATERIALS AND METHODS: NKT cell hybridomas and sorted mouse NKT cells were stimulated with S1P and α-galactosylceramide (α-GalCer), the major ligand to produce cytokines in NKT cells. TNF-α mRNA expression and protein production were determined by real-time PCR and ELISA, respectively. Cell migration was assayed using chemotaxicell. Plasma S1P was measured using HPLC. RESULTS: Hybridomas expressed S1P receptors, S1P1, S1P2, and S1P4. S1P and α-GalCer increased TNF-α mRNA expression and protein production. S1P enhanced TNF-α induction by α-GalCer. S1P receptor antagonists decreased the TNF-α mRNA expression induced by S1P. FTY720, an immunosuppressive S1P receptor modulator, also decreased the TNF-α mRNA expression. The migration of NKT cell hybridomas was increased by S1P. FTY720 reduced the migration induced by S1P. S1P also increased the TNF-α mRNA expression in mouse NKT cells. Plasma TNF-α levels in patients with high plasma S1P (≥500 nmol/l) were higher than those in patients with low S1P (<500 nmol/l). CONCLUSION: S1P binds to S1P receptors in NKT cells and enhances TNF-α production. TNF-α overproduction may induce atherogenic inflammatory responses. S1P may serve as a novel therapeutic target for amelioration of vascular inflammatory diseases.

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.

Olmesartan, but not amlodipine, improves endothelium-dependent coronary dilation in hypertensive patients.

OBJECTIVES: We aimed to compare the effects of the angiotensin II receptor blocker (ARB) olmesartan versus the calcium channel blocker (CCB) amlodipine on coronary endothelial dysfunction in patients with hypertension. BACKGROUND: Angiotensin II receptor blockers are thought to have greater beneficial effects than CCBs on coronary vasomotion by directly blocking action of angiotensin II. METHODS: Twenty-six patients with untreated essential hypertension were prospectively assigned to treatment with either olmesartan (27.7 +/- 12.4 mg/day, n = 13) or amlodipine (5.6 +/- 1.5 mg/day, n = 13) for 12 weeks. Changes of corrected myocardial blood flow (DeltaMBF) and coronary vascular resistance (DeltaCVR) from rest to cold pressor were measured by using 15O-water and positron emission tomography before and after treatment. Blood biomarkers including lipids, glucose, insulin, high-sensitivity C-reactive protein, interleukin-6, tumor necrosis factor-alpha, and superoxide dismutase (SOD) were also measured. RESULTS: Olmesartan and amlodipine reduced blood pressure (BP) to the same extent (-28.7 +/- 16.2 mm Hg vs. -26.7 +/- 10.8 mm Hg). In the olmesartan group, DeltaMBF tended to be greater (-0.15 +/- 0.19 ml/g/min vs. 0.03 +/- 0.17 ml/g/min, p = 0.09 by 2-way analysis of variance), and DeltaCVR was significantly decreased (7.9 +/- 23.5 mm Hg/[ml/g/min] vs. -16.6 +/- 18.0 mm Hg/[ml/g/min], p < 0.05) after treatment, whereas these parameters did not change in the amlodipine group (DeltaMBF: -0.15 +/- 0.12 ml/g/min vs. -0.12 +/- 0.20 ml/g/min; DeltaCVR: 6.5 +/- 18.2 mm Hg/[ml/g/min] vs. 4.8 +/- 23.4 mm Hg/[ml/g/min]). Serum SOD activity tended to increase (4.74 +/- 4.77 U/ml vs. 5.57 +/- 4.74 U/ml, p = 0.07 by 2-way analysis of variance) only in the olmesartan group. CONCLUSIONS: Olmesartan, but not amlodipine, improved endothelium-dependent coronary dilation in hypertensive patients independent of BP reduction. These beneficial effects on coronary vasomotion might be via an antioxidant property of ARBs.

Plasma interleukin-6 and tumor necrosis factor-alpha can predict coronary endothelial dysfunction in hypertensive patients.

Coronary endothelial function is impaired in hypertension; however, the severity of this impairment varies among patients. We aimed to identify the predictors of coronary endothelial dysfunction among clinical variables related to hypertension and atherosclerosis. Twenty-seven untreated, uncomplicated essential hypertensive patients and 10 age-matched healthy controls were studied prospectively. Myocardial blood flow (MBF) was measured by using (15)O-water positron emission tomography (PET) at rest and during a cold pressor test (CPT). Coronary vascular resistance (CVR) during CPT was used as a marker of coronary endothelial function. Serum low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides, malondialdehyde-LDL, homeostasis model assessment, high-sensitivity C-reactive protein (hs-CRP), and plasma interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha were also measured. CVR during CPT was significantly higher in hypertensive patients than in healthy controls (114+/-26 vs. 94+/-12 mmHg/[mL/g/min]; p<0.05). By univariate analysis, CVR during CPT was correlated with LDL cholesterol (r=0.38, p<0.05), IL-6 (r=0.46, p<0.02), and TNF-alpha (r=0.39, p<0.05) in hypertensive patients. By multivariate analysis, IL-6 and TNF-alpha were significant independent predictors of CVR during CPT. Elevated plasma IL-6 and TNF-alpha levels were independent predictors of coronary endothelial dysfunction in hypertensive patients. These results suggest that plasma IL-6 and TNF-alpha might be useful for identifying the high risk subgroup of hypertensive patients with coronary endothelial dysfunction and provide an important clue to link systemic inflammation to the development of coronary atherosclerosis.

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.

Elevated plasma plasminogen activator inhibitor type-1 is an independent predictor of coronary microvascular dysfunction in hypertension.

BACKGROUND: Elevated plasma plasminogen activator inhibitor-1 (PAI-1) is related to cardiovascular events, but its role in subclinical coronary microvascular dysfunction remains unknown. Thus, in the present study it was investigated whether elevated plasma PAI-1 activity is associated with coronary microvascular dysfunction in hypertensive patients. METHODS AND RESULTS: Thirty patients with untreated essential hypertension and 10 age-matched healthy controls were studied prospectively. Myocardial blood flow (MBF) was measured by using (15)O-water positron emission tomography. Clinical variables associated with atherosclerosis (low-density lipoprotein-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, homeostasis model assessment (HOMA-IR), and PAI-1 activity) were assessed to determine their involvement in coronary microvascular dysfunction. Adenosine triphosphate (ATP)-induced hyperemic MBF and coronary flow reserve (CFR) were significantly lower in hypertensive patients than in healthy controls (ATP-induced MBF: 2.77+/-0.82 vs 3.49+/-0.71 ml x g(-1) x min(-1); p<0.02 and CFR: 2.95 +/-1.06 vs 4.25+/-0.69; p<0.001). By univariate analysis, CFR was positively correlated with HDL-cholesterol (r=0.46, p<0.02), and inversely with HOMA-IR (r=-0.39, p<0.05) and PAI-1 activity (r=-0.61, p<0.001). By multivariate analysis, elevated PAI-1 activity remained a significant independent determinant of diminished CFR. CONCLUSIONS: Elevated plasma PAI-1 activity was independently associated with coronary microvascular dysfunction, which suggests that plasma PAI-1 activity is an important clue linking hypofibrinolysis to the development of atherosclerosis.

Hepatocyte growth factor regulates E box-dependent plasminogen activator inhibitor type 1 gene expression in HepG2 liver cells.

OBJECTIVE: We sought to determine the etiologic mechanism of pleiotropic growth factor, hepatocyte growth factor (HGF), as a regulator of hepatic synthesis of plasminogen activator inhibitor (PAI)-1, the physiological inhibitor of fibrinolysis and a potential inducer of atherothrombosis. METHODS AND RESULTS: HGF increased PAI-1 mRNA expression and PAI-1 protein accumulation in the conditioned media of human liver-derived HepG2 cells, and increased hepatic PAI-1 mRNA expression in vivo in mice. HGF-inducible PAI-1 mRNA was attenuated by U0126, a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, and genistein, an inhibitor of tyrosine kinase. HGF increased the human PAI-1 promoter (-829 to +36 bp) activity, and deletion and mutation analysis uncovered a functional E box (5'-CACATG-3') at positions -158 to -153 bp. Electrophoretic mobility shift assays demonstrated that this E box binds upstream stimulatory factors (USFs). HGF phosphorylated USFs through MAPK and tyrosine kinase pathways. Co-transfection of USF1 expression vector increased PAI-1 promoter activity. Sterol regulatory element-binding protein-1 attenuated HGF-inducible PAI-1 promoter activity. CONCLUSIONS: Because USFs are involved in the regulation of carbohydrates and lipid metabolism, HGF-mediated PAI-1 production may provide a novel link between atherothrombosis and metabolic derangements. Targeting HGF signaling pathway may modulate the thrombotic risk in high-risk patients.

Interleukin-6 and mevastatin regulate plasminogen activator inhibitor-1 through CCAAT/enhancer-binding protein-delta.

OBJECTIVE: We sought to determine the etiologic mechanism of proinflammatory cytokine, interleukin-6 (IL-6), and statin as regulators of synthesis of plasminogen activator inhibitor-1 (PAI-1), the physiological fibrinolysis inhibitor and an acute-phase reactant. METHODS AND RESULTS: Transient transfection and luciferase assay in HepG2 human hepatoma-derived cells demonstrated that IL-6 increased PAI-1 promoter activity and mevastatin decreased IL-6-inducible response. Systematic deletion assay of the promoter demonstrated that the region (-239 to -210 bp) containing a putative CCAAT/enhancer-binding protein (C/EBP) binding site was necessary. Point mutation in this site abolished the IL-6-inducible response. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that C/EBPalpha, C/EBPbeta, and C/EBPdelta were involved in protein-DNA complex formation in intact cells. Deoxyribonuclease (DNase) I footprinting analysis revealed that 5' flanking region (-232 to -210 bp) is acute-phase response protein-binding site. C/EBPdelta binding activity was increased by IL-6 and attenuated by mevastatin. Mevastatin attenuated IL-6-mediated increase of C/EBPdelta protein in the nuclear extracts. IL-6 also increased PAI-1 and C/EBPdelta mRNA in mouse primary hepatocytes. CONCLUSIONS: IL-6 increases hepatic PAI-1 expression mediated by the -232- to -210-bp region of the promoter containing a C/EBPdelta binding site. Vascular protection by statins may be partly mediated through regulation of CEBPdelta and consequent modulation of PAI-1 expression.

Increased expression of plasminogen activator inhibitor-1 by mediators of the acute phase response: a potential progenitor of vasculopathy in hypertensives.

Hypertension is an important risk factor for coronary atherosclerosis, which is accelerated by inflammation and diminished fibrinolysis. We have previously shown that levels of plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of fibrinolysis, are increased with atherogenic metabolic derangement. Because the liver is one of the major sources of circulating PAI-1, we here examined the effects of two proinflammatory cytokines, interleukin (IL)-1beta, and IL-6, on PAI-1 production in a human hepatoma cell line, HepG2. IL-1beta (1 ng/ml) and IL-6 (1 ng/ml) increased the accumulation of PAI-1 in the conditioned media over 24 h (IL-1beta: 2.1 +/- 0.2 (mean +/- SD) fold over the control; IL-6:1.4 +/- 0.2 fold; Western blot, p < 0.05). The increase in PAI-1 protein accumulation correlated with the increased expression of PAI-1 mRNA (Northern blot). An HMG-CoA reductase inhibitor (mevastatin, 10 micromol/l) attenuated the PAI-1 production induced by IL-1beta and IL-6. The plasma PAI-1 activity level was higher in hypertensives than in normotensives (10.0 +/- 9.8 AU/ml vs. 6.2 +/- 4.5 AU/ml, p < 0.05). The plasma PAI-1 antigen level was also higher in hypertensives than in normotensives (30.9 +/- 22.4 ng/ml vs. 24.4 +/- 13.3 ng/ml, p < 0.05). Thus, 1) IL-1beta and IL-6 can increase PAI-1 production in hepatic cells and 2) mevastatin may exert anti-thrombotic effects by decreasing the PAI-1 protein production induced by these proinflammatory cytokines. These results provide further insights into how inflammation is involved in the atherothrombotic complications observed in hypertensives, which may be ameliorated by HMG-CoA reductase inhibitors.

Association of cardiovascular risk factors and endothelial dysfunction in japanese hypertensive patients: implications for early atherosclerosis.

Although hypertension, hyperlipidemia, diabetes and smoking are known risk factors of atherosclerosis in Caucasians, their relative contributions to early atherosclerosis among Japanese are unknown. Decrease in flow-mediated dilation (FMD) of the brachial artery is a useful marker of endothelial dysfunction and early atherosclerosis. To evaluate the relative contribution of hypertension to early atherogenesis, we determined FMD, as well as plasma levels of tissue-type plasminogen activator (t-PA; a sensitive index of endothelial damage) and tumor necrosis factor (TNF)-a and interleukin (IL)-6 (established markers of inflammation) in normotensive and hypertensive patients under treatment. FMD was significantly reduced as the number of risk factors increased, suggesting that accumulations of risk factors were related to endothelial dysfunction. FMD was reduced in hypertensives (9.9 +/- 5.8 (SD) %) compared to normotensives (14.6 +/- 7.6, p<0.01) despite good blood pressure control (139 +/- 20/80 +/- 14 mmHg in hypertensives). Nitroglycerine-induced endothelium-independent vasodilation was not altered in hypertensives (16.0 +/- 6.3%) as compared to normotensives (16.7 +/- 5.8). Plasma t-PA, TNF-alpha, and IL-6 levels were increased in hypertensives despite good blood pressure control. Thus, hypertension alone is a high risk for early atherosclerosis. Persistent endothelial damage and moderate inflammation may increase the risk of early atherosclerosis synergistically under the presence of hypertension in Japanese.

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.

Relationships between brachial artery flow mediated dilation and carotid artery intima-media thickness in patients with suspected coronary artery disease.

The correlation of peripheral endothelial dysfunction and intima-media thickness (IMT) in patients with suspected coronary artery disease (CAD) has been unclear. Inflammation and thrombosis may play a role at early stages of atherosclerosis. Thus, early atherosclerosis was noninvasively examined morphologically by IMT of carotid arteries, and functionally by flow mediated dilation (FMD) of brachial arteries in patients who were suspected of CAD and had undergone coronary angiography. Plasma antigen levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, representative atherogenic cytokines, tissue factor (TF) and tissue factor pathway inhibitor (TFPI), markers of coagulation, and plasma activity level of plasminogen activator inhibitor type-1 (PAI-1), a marker of defective fibrinolysis, were measured. Patients with coronary atherosclerosis in one or more vessels with lesion > or = 50% had significantly reduced FMD compared with those with angiographically normal coronary arteries. Carotid artery IMT increased significantly only in patients with advanced coronary atherosclerosis in one or more vessels with lesion > or = 90%. Plasma antigen levels of IL-6 were significantly increased in patients with reduced FMD (< 5%) compared to those in patients with FMD between 10 and 15%. Plasma antigen levels of TF, total and free TFPI, and PAI-1 activity tended to increase with a reduction in FMD. Thus, (1) FMD was reduced at early stages of CAD while IMT was increased in advanced CAD, and (2) inflammation and thrombosis may play a role in the early stages of the atherosclerotic process.