Serum CETP status is independently associated with reduction rates in LDL-C in pitavastatin-treated diabetic patients and possible involvement of LXR in its association.

BACKGROUND: Statins decrease cholesteryl ester transfer protein (CETP) levels, which have been positively associated with hepatic lipid content as well as serum low density lipoproteins-cholesterol (LDL-C) levels. However, the relationship between the CETP status and statin-induced reductions in LDL-C levels has not yet been elucidated in detail. We herein examined the influence of the CETP status on the lipid-reducing effects of pitavastatin in hypercholesterolemic patients with type 2 diabetes mellitus as well as the molecular mechanism underlying pitavastatin-induced modifications in CETP levels. METHODS: Fifty-three patients were treated with 2 mg of pitavastatin for 3 months. Serum levels of LDL-C, small dense (sd) LDL-C, and CETP were measured before and after the pitavastatin treatment. The effects of pitavastatin, T0901317, a specific agonist for liver X receptor (LXR) that reflects hepatic cholesterol contents, and LXR silencing on CETP mRNA expression in HepG2 cells were also examined by a real-time PCR assay. RESULTS: The pitavastatin treatment decreased LDL-C, sdLDL-C, and CETP levels by 39, 42, and 23%, respectively. Despite the absence of a significant association between CETP and LDL-C levels at baseline, baseline CETP levels and its percentage change were an independent positive determinant for the changes observed in LDL-C and sdLDL-C levels. The LXR activation with T0901317 (0.5 µM), an in vitro condition analogous to hepatic cholesterol accumulation, increased CETP mRNA levels in HepG2 cells by approximately 220%, while LXR silencing markedly diminished the increased expression of CETP. Pitavastatin (5 µM) decreased basal CETP mRNA levels by 21%, and this was completely reversed by T0901317. CONCLUSION: Baseline CETP levels may predict the lipid-reducing effects of pitavastatin. Pitavastatin-induced CETP reductions may be partially attributed to decreased LXR activity, predictable by the ensuing decline in hepatic cholesterol synthesis. TRIAL REGISTRATION: UMIN Clinical Trials Registry ID UMIN000019020.

Verotoxin-1 stimulation of macrophage-like THP-1 cells up-regulates tissue factor expression through activation of c-Yes tyrosine kinase: Possible signal transduction in tissue factor up-regulation.

Verotoxin (VT)-producing Escherichia coli (E. coli) O157:H7 infections are frequently complicated by thrombotic angiopathy, hemolytic uremic syndrome (HUS) and neurological symptoms. The present data demonstrate that VT-1 (Shiga toxin) stimulation of macrophage-like THP-1 cells up-regulates the activity, antigen and mRNA levels of tissue factor (TF), a key cofactor of the coagulation-inflammation-thrombosis circuit. This up-regulation is accompanied by phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase), IkappaB kinase beta (IKKbeta) and extracellular signal-regulated kinase 2 (ERK2). Changes in TF mRNA levels were in parallel with the activation of NF-kappaB/Rel and Egr-1 activation, but not with AP-1. Inhibition of PI3-kinase attenuated VT-1-induced phosphorylation of IKKbeta and ERK2, and the up-regulation of TF mRNA levels. VT-1 stimulation rapidly activated c-Yes tyrosine kinase, a member of the Src family. Treatment of the cells with c-Yes antisense oligos attenuated the VT-1-induced phosphorylation of PI3-kinase, IKKbeta and ERK2, activations of NF-kappaB/Rel and Egr-1, and up-regulation of TF mRNA levels. These results suggest that VT-1-induced macrophage stimulation activates c-Yes, which then up-regulates TF expression through activation of the IKKbeta/proteasome/NF-kappaB/Rel and MEK/ERK2/Egr-1 pathways via activation of PI3-kinase. Induction of macrophage TF expression by VT-1 may play an important role in the acceleration of the coagulation-inflammation-thrombosis circuit during infections by VT-producing E. coli.

Pitavastatin-induced thrombomodulin expression by endothelial cells acts via inhibition of small G proteins of the Rho family.

OBJECTIVE: 3-hydroxyl-3-methyl coenzyme A reductase inhibitors (statins) can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. The main feature of the antithrombotic properties of endothelial cells is an increase in the expression of thrombomodulin (TM) without induction of tissue factor (TF) expression. We investigated the effect of statins on the expression of TM and TF by endothelial cells. METHODS AND RESULTS: The incubation of endothelial cells with pitavastatin led to a concentration- and time-dependent increase in cellular TM antigen and mRNA levels. In contrast, the expression of TF mRNA was not induced under the same conditions. A nuclear run-on study revealed that pitavastatin accelerates TM transcription rate. The stimulation of TM expression by pitavastatin was prevented by either mevalonate or geranylgeranylpyrophosphate. Specific inhibition of geranylgeranyltransferase-I and Rac/Cdc42 by GGTI-286 and Clostridium sordellii lethal toxin, respectively, enhanced TM expression, whereas inactivation of Rho by Clostridium botulinum C3 exoenzyme was ineffective. CONCLUSIONS: Statins regulate TM expression via inhibition of small G proteins of the Rho family; Rac/Cdc42. A statin-mediated increase in TM expression by endothelial cells may contribute to the beneficial effects of statins on endothelial function.

Effect of cilostazol on impaired vasodilatory response of the brachial artery to ischemia in smokers.

The vascular endothelial function of smokers is known to be impaired. This study investigated whether cilostazol could improve the vasodilatory response of the brachial artery to ischemia, an indicator of endothelial function, in ten male smokers. Endothelium-dependent vasodilatation and endothelium-independent vasodilatation of the brachial artery were measured in 11 male non-smokers and 20 male smokers with matching age and weight. The results showed that the vasodilatory response to reactive hyperemia was significantly smaller in the smokers (4.8 +/- 1.6%) when compared to that in the non-smokers (7.6 +/- 2.5%) (p = 0.0013). However, no significant difference in the vasodilatory response to isosorbide dinitrate was observed between the two groups. In addition, there were no significant differences in serum lipid, Lp (a), or blood homocysteine between the smokers and non-smokers. When 150 mg/day of cilostazol was administered for two weeks, the vasodilatory response to reactive hyperemia significantly improved (4.2 +/- 1.2% to 7.8 +/- 3.5%, p = 0.0032). The increased vasodilatory response to reactive hyperemia by cilostazol was reduced after cessation of the drug (4.5 +/- 1.5%). These findings suggest that cilostazol improves vascular endothelial dysfunction in smokers.

Thrombomodulin expression by THP-1 but not by vascular endothelial cells is upregulated by pioglitazone.

Thrombomodulin-protein C pathway is a major anti-thrombotic mechanism present in endothelial cells (EC), and an important modulator of inflammation. Peroxisomal proliferator activated receptor-gamma (PPARgamma) expressed in monocytes/macrophages may have a role in cell differentiation. Since the expression of thrombomodulin (TM) by monocytes is upregulated during differentiation into macrophages, we investigated the effect of pioglitazone, a thiazolidinedione (TZD) that is a synthetic ligand of PPARgamma, on the expression of TM by a human monocyte/macrophage cell line; human acute monocytic leukemia (THP-1) cells. Pioglitazone dose-dependently upregulated TM antigen expression by THP-1 cells accompanied by an upregulation of TM cofactor activity for thrombin-dependent protein C activation. Thrombomodulin mRNA expression in THP-1 cells was also upregulated by pioglitazone, whereas tissue factor (TF) mRNA expression was not induced at all. Treatment cells with a natural PPARgamma ligand, 15-deoxy-delta12,14-prostaglandin J(2) (PGJ2), also enhanced TM protein expression. PGF(2alpha) an agent known to inactivate PPARgamma, diminished the stimulatory effect of pioglitazone and PGJ2 on TM protein expression. In contrast, pioglitazone had no effect on TM antigen expression by human umbilical vein ECs. These results suggest that PPARgamma activation in macrophages may counteract potentially prothrombotic and putative inflammatory properties in activated macrophages.

Function of hormone-sensitive lipase in diacylglycerol-protein kinase C pathway.

To explore the functional effects of hormone-sensitive lipase (HSL) in diacylglycerol (DAG) metabolism, Chinese hamster ovary cells were stably transfected with rat HSL cDNA (wt-HSL), inactive mutant S423A-HSL cDNA (S423A) and pcDNA3 vector alone (Ct). [(14)C]Glucose-incorporation into triglyceride (TG) was 75% lower in the presence or absence of insulin in cells expressing wt-HSL compared to Ct or S423A. [(14)C]Glucose-incorporation into DAG was 33% lower without insulin and 51% lower with insulin in cells expressing wt-HSL compared to Ct or S423A. Insulin stimulated glucose-incorporation into DAG 2.2-fold in S423A and Ct cells, whereas only a 50% increase was observed in cells expressing wt-HSL. Phospholipase C-mediated release of DAG from membrane phospholipids was reduced 70% in cells expressing wt-HSL compared to Ct or S423A. Western blot analysis showed that membrane-bound protein kinase C (PKC)-alpha and -epsilon were decreased 40-50% in cells expressing wt-HSL grown in high glucose with insulin. These data show that HSL potentially hydrolyzes cellular DAG generated either by de novo synthesis from glucose or release from membrane phospholipids by phospholipase C, resulting in a reduction in the translocation of DAG-sensitive PKCs.

Myocardial metabolic regulation through peroxisome proliferator-activated receptor alpha after myocardial infarction.

Although peroxisome proliferator-activated receptor alpha (PPARalpha) is closely associated with myocardial fatty acid metabolism, the pathophysiological role of PPARalpha in myocardial infarction (MI) is not yet known. The aim of the present study was to clarify the relationship between cardiac energy metabolism and PPARalpha expression in the remodelling of myocardium after MI. We assayed the expression of PPARalpha and several metabolic genes in cultured cardiac cells (myocytes and nonmyocytes) and in MI hearts. PPARalpha was strongly expressed in cardiac myocytes but not in nonmyocytes (mainly fibroblasts). In MI rats, PPARalpha and PPARalpha-regulated genes (lipoprotein lipase, heart-type fatty acid binding protein, long-chain acyl-CoA dehydrogenase and uncoupling protein-3) were decreased concomitantly, whereas uncoupling protein-2 was not decreased in severely ischemic regions. Immunohistochemical staining for PPARalpha was less decreased in borderline myocardium than in sham-operated hearts. Furthermore, in electron microscopic study, there were no lipid droplet accumulations in surviving myocardium after MI. Our results suggest that the reduced expression of PPARalpha is closely related to that of fatty acid metabolism genes in infarcted myocardium, and PPARalpha may play an important role in cardiac energy metabolism during remodelling after MI.

[Diabetic nephropathy and plasminogen activator inhibitor 1 in urine samples].

Plasminogen activator inhibitor-1 (PAI-1) may contribute to renal fibrosis because of its involvement in matrix (ECM) accumulation through inhibition of plasmin-dependent ECM degradation. The aim of this study is to determine urinary PAI-1 concentrations and its intrarenal localization in patients with various renal diseases and to identify inducers for PAI-1 expression in human cultured proximal renal tubular cells (HRCs). Urinary PAI-1 concentrations were significantly higher in patients with overt diabetic nephropathy (DN, n=36) than in proliferative glomerulonephritis (PGN, n=8), nephrotic syndrome (NS, n=10) and healthy controls (n=12). Urinary PAI-1 concentrations (ng/gCr) were directly correlated with urinary N-acetyl glucosaminidase (NAG) levels (r=0.58, p<0.05). As for intrarenal localization of PAI-1 antigen, strong stainings for PAI-1 were observed in proximal tubular cells of renal biopsy samples from patients with DN, while no stainings for PAI-1 were found in renal tissues of PGN or NS. Immunoblot analysis revealed the presence of PAI-1 protein in whole cell lyzates from HRCs grown to semiconfluency. Exposure of growth-arrested HRCs with hypoxia (1% O2) or TNF-alpha (10 ng/ml) for 24 hours increased the secretion rate of PAI-1 protein by about 2.0-fold, while 24-hour treatment with high glucose (450 mg/dl) did not increase PAI-1 secretion at all, compared with that of the control cells under normal glucose (100 mg/dl) and normoxia (18% O2). These findings suggest that PAI-1 expression is upregulated especially in the proximal renal tubular cells of DN, which may be explained partially by hypoxia and inflammatory cytokines but not high glucose.

Serum level of triglycerides is a potent risk factor comparable to LDL cholesterol for coronary heart disease in Japanese patients with type 2 diabetes: subanalysis of the Japan Diabetes Complications Study (JDCS).

CONTEXT: Risk factors for cardiovascular complications in Japanese patients with diabetes have not been fully elucidated. OBJECTIVE: Our objective was to determine incidence of and risk factors for coronary heart disease (CHD) and stroke in Japanese diabetic patients. DESIGN AND SETTINGS: We conducted a prospective study at 59 hospitals throughout Japan. PATIENTS: Patients included 940 men and 831 women with type 2 diabetes (mean age, 58.2 yr) without a history of cardiovascular complications who were followed for a median of 7.86 yr. INTERVENTION: This was an observational study. MAIN OUTCOME MEASURES: Incidence of CHD and stroke was evaluated. RESULTS: Incidences of CHD and stroke per 1000 person-years were 9.59 and 7.45, respectively, whereas those of myocardial and brain infarctions were 3.84 and 6.29, respectively. Multivariate Cox analysis revealed that the serum log-transformed triglyceride level was a potent and independent predictor of CHD [hazard ratio (HR) = 1.54; 95% confidence interval (CI) = 1.22-1.94 per 1 sd increase), comparable to low-density lipoprotein (LDL) cholesterol (HR = 1.49; 95% CI = 1.25-1.78 per 1 sd increase). Triglycerides and LDL cholesterol linearly and continuously increased CHD risk, and subjects in the top third for both had markedly high risks of CHD, and their effects were possibly additive. However, serum triglycerides worked independently of blood pressure levels. Systolic blood pressure was the only significant predictor for stroke except for age (HR = 1.31; 95% CI = 1.04-1.65, per 1 sd increase). CONCLUSIONS: In Japanese patients with type 2 diabetes, the serum triglyceride level was a leading predictor of CHD, comparable to LDL cholesterol. Because the serum triglyceride level is not a leading predictor of CHD in diabetic subjects in Western countries, ethnic group-specific strategies for prevention of diabetic macroangiopathy may be indicated.

Waist circumference as a cardiovascular and metabolic risk in Japanese patients with type 2 diabetes.

Excess waist circumference (WC) is a frequently used indicator of abdominal obesity and/or cardiovascular disease (CVD) risk. Nonetheless, search of the literature revealed no prospective studies on the association between WC and CVD events in diabetic patients. In this study, the clinical significance and implications of WC as a cardiovascular and metabolic risk indicator was prospectively investigated in Japanese patients with type 2 diabetes. For this purpose, baseline data on WC, hypertension, and dyslipidemia were collected and subsequent CVD (coronary heart disease and stroke) events during the following 8 years were studied in 1,424 Japanese type 2 diabetic patients, and the cross-sectional/longitudinal associations between WC and CVD risk factors/events were analyzed. Mean WC levels were significantly increased according to the number of coexisting risk factors. However, no significant difference in mean WC between subgroups with and without CVD events was noted, and excess WC alone was not predictive of subsequent CVD events either in male or female subjects even after adjustment for age, smoking, hypertension, and dyslipidemia. In female patients, excess WC (> or =80 cm) was predictive of CVD events only with the coexistence of hypertension. In Japanese diabetic patients, excess WC alone, although a good marker for clustering of CVD risk factors, did not raise the risk of CVD events unless accompanied by hypertension in female patients. Further investigations are necessary before WC as a risk factor can be utilized in clinical settings for the management of diabetes in this population.

Components of metabolic syndrome and their combinations as predictors of cardiovascular disease in Japanese patients with type 2 diabetes. Implications for improved definition. Analysis from Japan Diabetes Complications Study (JDCS).

AIM: The prognostic power of metabolic syndrome (MetS) in patients with diabetes has been studied with inconsistent results depending on the definition of MetS. To clarify the best combination of MetS components to predict future cardiovascular disease (CVD) events, we estimated CVD risk in Japanese patients with type 2 diabetes according to MetS components. METHODS: Patients were categorized according to the presence three MetS components in addition to hyperglycemia. hypertension, dyslipidemia and excess waist circumference (WC) (according to either Japanese or Asian cut-off values). Hazard ratios for CVD events were compared in patients with various categories of MetS components. RESULTS: At least two components of MetS were required for a significantly elevated risk for CVD; however, component combinations with significantly increased risk differed depending on gender or the WC cut-off value. Any two among 1) excess WC (men > or =90 cm, women > or =80 cm); 2) hypertension (systolic blood pressure > or =130 mmHg or diastolic blood pressure > or =85 mmHg or use of an antihypertensive agent); and 3) dyslipidemia (triglycerides > or =150 mg/dL or HDL-cholesterol <40 mg/dL or use of drug treatment) could be used to identify significantly higher risk (approximately twice) for CVD regardless of gender. CONCLUSIONS: The results suggest that the current MetS criteria should be modified when applied to patients with type 2 diabetes.

Oxidized phospholipids in oxidized low-density lipoprotein down-regulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RARbeta-RXRalpha heterodimers and Sp1 and Sp3 to their binding sequences in the TM promoter.

The present work investigated the mechanism for down-regulation of thrombomodulin (TM), an anticoagulant glycoprotein, on cultured umbilical vein endothelial cells (HUVECs) exposed to lipid extracts from oxidized low-density lipoprotein (ox-LDL). HUVECs exposed to phospholipid extracts, but not to free cholesterol, triglyceride, or cholesterol ester, isolated from ox-LDL reduced TM mRNA levels to nearly the same extent as native ox-LDL. Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC), but not native PAPC or a reduced form of ox-PAPC, markedly decreased TM mRNA levels. The apparent half-life (t 1/2 = 2.7 hours) of TM mRNA in control cells was not significantly different from that in cells exposed to ox-LDL or ox-PAPC. TM mRNA levels were regulated by transcriptional activation via a retinoid receptor beta (RARbeta). The binding activities of nuclear proteins from HUVECs treated with ox-LDL or ox-PAPC to the DR4 or stimulatory protein 1 (Sp1) sequence in the TM promoter were significantly reduced with decreased expression of RARbeta, retinoid X receptor alpha (RXRalpha), Sp1, and Sp3 in the nuclei. The promoter activity in HUVECs transfected with a reporter plasmid expressing the TM promoter with targeted deletions in the DR4 and Sp1 binding elements was decreased to about 20% of that with the wild-type construct. Treatment of the cells with ox-PAPC had no additional effect on the promoter activity. These results suggest that oxidized phospholipids in ox-LDL inhibit transcription of the TM gene in HUVECs by inhibiting the binding of RARbeta-RXRalpha heterodimer and Sp, including Sp1 and Sp3, to the DR4 element and Sp1 binding element, respectively, in the TM promoter with reduced expression of RARbeta, RXRalpha, and Sp1 and Sp3 in the nuclei.

Expression of thrombomodulin in human aortic smooth muscle cells with special reference to atherosclerotic lesion types and age differences.

Expression of thrombomodulin (TM) in atherosclerotic lesions of the human aorta (8 cases of diffuse intimal thickening, 4 fatty streaks, 11 atheromatous plaques, and 5 fibrous plaques) as well as in undiseased aortas of 5 infants obtained at autopsy was studied immunohistochemically using a novel polyclonal antibody against human TM. TM was expressed in intimal smooth muscle cells (SMC) besides endothelial cells and foamy macrophages in almost all patients (26/28). In addition, medial SMC in adult cases over 27 years of age expressed TM. In young adults with diffuse intimal thickening under 26 years of age, medial SMC showed no TM expression whereas intimal SMC did show it. Both intimal and medial SMC in infants showed no TM expression. An immunofluorescence method showed TM expression in cultured adult human SMC. These findings indicate that TM expression in SMC may depend on patient age as well as lesion type of atherosclerosis.

Cardiac gene expression profile and lipid accumulation in response to starvation.

Starvation induces many biochemical and histological changes in the heart; however, the molecular events underlying these changes have not been fully elucidated. To explore the molecular response of the heart to starvation, microarray analysis was performed together with biochemical and histological investigations. Serum free fatty acids increased twofold in both 16- and 48-h-fasted mice, and cardiac triglyceride content increased threefold and sixfold in 16- and 48-h-fasted mice, respectively. Electron microscopy showed numerous lipid droplets in hearts of 48-h-fasted mice, whereas fewer numbers of droplets were seen in hearts from 16-h-fasted mice. Expression of 11,000 cardiac genes was screened by microarrays. More than 50 and 150 known genes were detected by differential expression analysis after 16- and 48-h-fasts, respectively. Genes for fatty acid oxidation and gluconeogenesis were increased, and genes for glycolysis were decreased. Many other genes for metabolism, signaling/cell cycle, cytoskeleton, and tissue antigens were affected by fasting. These data provide a broad perspective of the molecular events occurring physiologically in the heart in response to starvation.