Vitamin D supplementation and bone turnover in advanced heart failure: the EVITA trial.

Low vitamin D status is common in patients with heart failure and may influence bone health. A daily vitamin D dose of 4000 IU (moderately high dose) for 3 years had however no effect on parameters of bone metabolism, even in patients with very low vitamin D status. INTRODUCTION: Low vitamin D status is common in patients with heart failure (HF) and has been related to disturbed bone turnover. The present study investigated the effect of a daily vitamin D3 dose of 4000 IU on bone turnover markers (BTMs) in patients with advanced HF and 25-hydroxyvitamin D (25OHD) concentrations < 75 nmol/L. METHODS: In this pre-specified secondary analysis of a randomized controlled trial, we assessed in 158 male HF patients (vitamin D group: n = 80; placebo group: n = 78) between-group differences in calciotropic hormones (25OHD, 1,25-dihydroxyvitamin D [1,25(OH)2D], intact parathyroid hormone [iPTH]), and BTMs (cross-linked C-telopeptide of type I collagen, bone-specific alkaline phosphatase, undercarboxylated osteocalcin). Comparisons were performed at the end of a 3-year vitamin D supplementation period with adjustments for baseline values. RESULTS: Compared with placebo, vitamin D increased 25OHD on average by 54.3 nmol/L. At study termination, 25OHD and 1,25(OH)2D were significantly higher (P < 0.001 and P = 0.007, respectively), whereas iPTH tended to be lower in the vitamin D group than in the placebo group (P = 0.083). BTMs were initially within their reference ranges and did not differ significantly between groups at study termination, neither in the entire study cohort nor when data analysis was restricted to the subgroup of patients with initial 25OHD concentrations < 30 nmol/L (n = 54) or to patients with initial hyperparathyroidism (n = 65) (all P values > 0.05). CONCLUSIONS: A daily vitamin D3 dose of 4000 IU did not influence BTMs. Data indicate that vitamin D supplementation will not lower bone turnover in male patients with heart failure.

Randomized supplementation of 4000 IU vitamin D3 daily vs placebo on the prevalence of anemia in advanced heart failure: the EVITA trial.

BACKGROUND: Low 25-hydroxyvitamin D (25OHD) levels (< 75 nmol/l) are inversely associated with anemia prevalence. Since anemia and low 25OHD levels are common in patients with heart failure (HF), we aimed to investigate whether vitamin D supplementation can reduce anemia prevalence in advanced HF. METHODS: EVITA (Effect of Vitamin D on Mortality in Heart Failure) is a randomized, placebo-controlled clinical trial in patients with initial 25OHD levels < 75 nmol/l. Participants received either 4000 IU vitamin D3 daily or a matching placebo for 36 months. A total of 172 patients (vitamin D group: n = 85; placebo group: n = 87) were investigated in this pre-specified secondary data analysis. Hemoglobin (Hb) and other hematological parameters were measured at baseline and study termination. Assessment of between-group differences in anemia prevalence and Hb concentrations was performed at study termination, while adjusting for baseline differences. RESULTS: In the vitamin D and placebo group, baseline proportions of patients with anemia (Hb < 12.0 g/dL in females and < 13.0 g/dL in males) were 17.2% and 10.6%, respectively (P = 0.19). At study termination, the proportion of patients with anemia in the vitamin D and placebo groups was 32.2% and 31.8%, respectively (P > 0.99). There was no between-group difference in change in the Hb concentrations (- 0.04 g/dL [95%CI:-0.53 to 0.45 g/dL]; P = 0.87). Results regarding anemia risk and Hb concentrations were similar in the subgroup of patients with chronic kidney disease (vitamin D group: n = 26; placebo group: n = 23). Moreover, results did not differ substantially when data analysis was restricted to patients with deficient baseline 25OHD levels. CONCLUSIONS: A daily vitamin D supplement of 4000 IU did not reduce anemia prevalence in patients with advanced HF. Data challenge the clinical relevance of vitamin D supplementation to increase Hb levels. TRIAL REGISTRATION: The study was registered at EudraCT (No. 2010-020793-42) and clinicaltrials.gov ( NCT01326650 ).

[New AHA and ACC guidelines on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk : Statement of the D•A•CH Society for Prevention of Cardiovascular Diseases, the Austrian Atherosclerosis Society and the Working Group on Lipids and Atherosclerosis (AGLA) of the Swiss Society for Cardiology]. / Neue AHA- und ACC-Leitlinie zur Risikoreduktion von Herz-Kreislauf-Erkrankungen durch Cholesterinsenkung : Stellungnahme der D•A•CH-Gesellschaft Prävention von Herz-Kreislauf-Erkrankungen e. V., der Österreichischen Atherosklerose Gesellschaft und der Arbeitsgruppe Lipide und Atherosklerose (AGLA) der Schweizer Gesellschaft für Kardiologie.

Guidelines for the reduction of cholesterol to prevent atherosclerotic vascular events were recently released by the American Heart Association and the American College of Cardiology. The authors claim to refer entirely to evidence from randomized controlled trials, thereby confining their guidelines to statins as the primary therapeutic option. The guidelines derived from these trials do not specify treatment goals, but refer to the percentage of cholesterol reduction by statin medication with low, moderate, and high intensity. However, these targets are just as little tested in randomized trials as are the cholesterol goals derived from clinical experience. The same applies to the guidelines of the four patient groups which are defined by vascular risk. No major statin trial has included patients on the basis of their global risk; thus the allocation criteria are also arbitrarily chosen. These would actually lead to a significant increase in the number of patients to be treated with high or maximum dosages of statins. Also, adhering to dosage regulations instead of cholesterol goals contradicts the principles of individualized patient care. The option of the new risk score to calculate lifetime risk up to the age of 80 years in addition to the 10-year risk can be appreciated. Unfortunately it is not considered in the therapeutic recommendations provided, despite evidence from population and genetic studies showing that even a moderate lifetime reduction of low-density lipoprotein (LDL) cholesterol or non-HDL cholesterol has a much stronger effect than an aggressive treatment at an advanced age. In respect to secondary prevention, the new American guidelines broadly match the European guidelines. Thus, the involved societies from Germany, Austria and Switzerland recommend continuing according to established standards, such as the EAS/ESC guidelines.

[Primary disorders of lipid metabolism: their place in current dyslipidemia guidelines and treatment innovations]. / Primäre Fettstoffwechselstörungen ­ Stellenwert in aktuellen Dyslipidämieleitlinien und therapeutische Innovationen.

According to current guidelines, the selection and intensity of lipid-effective therapies are based on the risk to be treated. The sole clinical categories of primary and secondary prevention of cardiovascular diseases result in over- and under-treatment, which may be a contributory cause of incomplete implementation of current guidelines in everyday practice. For the extent of benefit in cardiovascular outcome studies with lipid-lowering drugs, the importance of dyslipdemia for the pathogenesis of atherosclerosis-related diseases is crucial. Primary lipid metabolism disorders are characterized by life-long increased exposure to atherogenic lipoproteins. This article describes the relevance of new data for low density lipoprotein-effective therapy: inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), adenosine triphosphate (ATP) citrate lyase with bempedoic acid, and ANGPTL3 with special consideration of primary lipid metabolism disorders, which are insufficiently taken into account, or not taken into account at all, in current guidelines. This is due to their apparently low prevalence rate and thus the lack of large outcome studies. The authors also discuss the consequences of increased lipoprotein (a), which cannot be sufficiently reduced until the ongoing intervention studies examining antisense oligonucleotides and small interfering RNA (siRNA) against apolipoprotein (a) are completed. Another challenge in practice is the treatment of rare, massive hypertriglyceridemia, especially with the aim of preventing pancreatitis. For this purpose, the apolipoprotein C3 (ApoC3) antisense oligonucleotide volenasorsen is available, which binds to the mRNA for ApoC3 and lowers triglycerides by around three quarters.

Role of physician gender in drug therapy.

There is evidence that female patients receive less intensified drug therapy in many medical conditions than male patients. However, there are only limited data regarding the influence of physician gender on drug therapy. It has been shown, for example, that female physicians tend to adhere more closely to guideline-recommended pharmacotherapy compared to their male counterparts. In some medical conditions where drug therapy is only one among various components of a complex interplay of therapeutic regimes (e.g., diabetes, cardiovascular diseases, depression, pain management), female physicians seem to achieve better overall intermediate outcomes and some studies suggest that "better" drug therapy is provided by female compared to male physicians. The reasons for the overall better outcomes may be superior communication skills of female physicians, participatory decision making, and consequently improved drug adherence in addition to or in combination with more effective non-pharmacologic treatment results. It is impossible to distinguish between the individual contributions of drug- and nondrug-related influence on such improved outcomes and thus to determine whether they are due to unconfounded physician gender effects on drug therapy. There is until now in no area of medicine evidence to suggest that a patient will consistently receive higher quality of drug therapy by switching to a physician of a specific gender.

The whey fermentation product malleable protein matrix decreases triglyceride concentrations in subjects with hypercholesterolemia: a randomized placebo-controlled trial.

Malleable protein matrix (MPM) is a unique whey-derived ingredient obtained through a fermentation process using proprietary lactic acid bacteria strains from the Lactobacillus kefiranofaciens species. Because evidence from animal models suggests that MPM decreases serum lipid concentrations, the purpose of the present trial was to assess the hypothesis that MPM exerts lipid-lowering effects in humans. A total of 161 subjects (50% male; age 54.5 ± 9.8 yr, body mass index 26.3 ± 3.6 kg/m(2)) with hypercholesterolemia with baseline low-density lipoprotein cholesterol (LDL-C) levels of 181 ± 30 mg/dL and normal triglyceride (TG) levels (131 ± 55 mg/dL) were randomized to receive MPM (2 × 15 g/d) or matching placebo. A 6-wk run-in phase was followed by a double-blind 12-wk treatment phase after randomization. The data were analyzed on an intention-to-treat basis. The primary outcome measure was the percentage change of LDL-C. The secondary outcome measures were changes in TG and high-density lipoprotein cholesterol concentrations as well as changes in other cardiovascular risk factors. After 12 wk of treatment, the relative TG decrease from baseline reached 9.8%, whereas LDL-C was slightly decreased (by 1.5%) following MPM treatment compared with placebo in the intention-to-treat cohort. The treatment effect on TG reduction was much higher in the subset of subjects having TG levels at baseline of 150 mg/dL or above (n=42), reaching 20.0% compared with placebo. High-density lipoprotein cholesterol concentrations, blood pressure, and fasting blood glucose remained unchanged, whereas a positive treatment effect was seen on hemoglobin A(1c). The MPM product was tolerated well without severe adverse events. In conclusion, MPM has significant TG-lowering properties in subjects with combined hypercholesterolemia and higher TG levels. Its effects on LDL-C concentrations and glucose metabolism deserve further investigation.

Atherosclerotic disease location and disparities in the control and treatment of cardiovascular risk factors in patients with Type 2 diabetes.

AIMS: To assess whether differences exist in the control and intensity of medication treatment of cardiovascular risk factors in secondary prevention patients with Type 2 diabetes, depending on their atherosclerotic disease territory [coronary artery disease (CAD), cerebrovascular disease (CBVD) or peripheral arterial disease (PAD)]. METHODS: Cross-sectional analysis of 17 571 patients with Type 2 diabetes with prevalent atherosclerotic disease. Endpoints included uncontrolled cardiovascular disease (CVD) risk factors [systolic blood pressure (SBP) > or = 140 mmHg, low-density lipoprotein cholesterol > or = 3.4 mmol/l and glycated haemoglobin > or = 8.0%] and high intensity of medication treatment (defined as > or = 2 classes of anti-hypertensive agents, > or = 1 lipid-lowering agent or either insulin or > or = 2 oral glucose-lowering agents) in patients with uncontrolled CVD risk factors. Multiple-adjusted odds ratios were calculated for CAD, CBVD and PAD after adjusting for sex, age, body mass index, current smoking and diabetes duration. RESULTS: Proportions of patients with uncontrolled risk factors were significantly different among disease territories. Decreased odds of having lipids not controlled were observed in patients with CAD, while decreased odds of having systolic blood pressure not controlled were observed in patients with PAD. PAD was associated with the highest odds of hyperglycaemia not being controlled. High-intensity treatment was observed in lipid and blood glucose management but not in hypertension management, independent of disease location. CONCLUSIONS: In subjects with Type 2 diabetes and atherosclerotic disease, control of modifiable CVD risk factors but not intensity of medication treatment is modified by atherosclerotic disease territory. Intensity of medication treatment is different between risk factors.

Vitamin D supplementation of 4000 IU daily and cardiac function in patients with advanced heart failure: The EVITA trial.

BACKGROUND: Data regarding the effects of vitamin D on cardiac function are inconclusive. METHODS: In a post-hoc analysis of the EVITA (Effect of vitamin D on mortality in heart failure) trial, we investigated whether a daily vitamin D3 supplement of 4000 IU for three years affects echocardiography parameters like left ventricular end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), and LV ejection fraction (LVEF) in patients with advanced heart failure (HF) and 25­hydroxyvitamin D levels <75 nmol/L. Of 400 patients enrolled, 199 were assigned to vitamin D and 201 to placebo. We assessed time × treatment interaction effects using linear mixed models and analyzed in subgroups vitamin D effects at 12 and 36 months post-randomization using analysis of covariance with adjustments for baseline values. RESULTS: At baseline, values of LVEDD, LVESD, and LVEF were 67.5 ±â€¯10.5 mm, 58.9 ±â€¯12.0 mm, and 30.47 ±â€¯10.2%, respectively. There were no time × treatment interaction effects on LV echocardiographic parameters in the entire study cohort, neither at 12 months nor at 36 months post-randomization (P-values > 0.05). However, in the subgroup of patients aged ≥50 years, vitamin D treatment was associated with an increase in LVEF of 2.73% (95%CI: 0.14 to 5.31%) at 12 months post-randomization (n = 311). The increase was slightly attenuated to 2.60% (95%CI: -2.47 to 7.67%) at 36 months post-randomization (n = 242). CONCLUSION: Our data indicate that vitamin D supplementation does not significantly improve cardiac function in all patients with advanced HF. However, vitamin D probably improves LV function in HF patients aged ≥50 years.

Physician gender is associated with the quality of type 2 diabetes care.

OBJECTIVES: Patient gender influences the quality of medical care whilst the role of physician gender is not well established. To investigate the influence of physician gender on quality of care in patients with type 2 diabetes. DESIGN AND METHODS: Cross-sectional study in 51 053 outpatients (48.6% male), treated by 3096 office-based physicians (66.3% male; 74.0% general practitioners, 21.8% internists and 4.2% diabetologists). Outcome measures included processes of care, intermediate outcomes and medical management. Quality of care measures were based on current ADA guidelines. Hierarchical regression models were used to avoid case-mix bias and to correct for physician-level clustering. Adjusted odds ratios were calculated controlling for age, gender, disease duration and presence of atherosclerotic disease. RESULTS: The patients of female physicians were more often women, more obese, older and had more often atherosclerotic disease (34% in the total cohort). The patients of female physicians more often reached target values in glycaemic control (HbA1c < 6.5%; OR 1.14; 1.05-1.24, P = 0.002), blood lipoproteins (LDL-C < 100 mg dL(-1); OR 1.16; 1.06-1.27, P = 0.002), and blood pressure (systolic values < 130 mmHg; OR 1.11; 1.02-1.22, P = 0.018). They were more likely to receive antihypertensive drug therapy in general (OR 1.35; 1.24-1.46, P < 0.0001) and angiotensin converting enzyme (ACE) inhibitors in particular (OR 1.17; 1.09-1.25, P < 0.0001). The patients of female physicians less often performed glucose self-monitoring (OR 0.83; 0.76-0.91, P < 0.0001) and less often received oral hypoglycaemic agents (OR 0.88; 0.82-0.95, P = 0.001). CONCLUSIONS: Physician gender influences quality of care in patients with type 2 diabetes. Female physicians provide an overall better quality of care, especially in prognostically important risk management.

SREBP-1c gene polymorphism is associated with increased inhibition of cholesterol-absorption in response to ezetimibe treatment.

OBJECTIVE: Sterol regulatory binding proteins 1 and 2 (SREBPs) are transcription factors regulating lipid metabolism. A recent study has associated the CC genotype of the SREBP-1c polymorphism G952G with increased cholesterol synthesis. Further evidence suggests that SREBPs play a role in cholesterol absorption and that SREBP polymorphisms modulate the response to statin therapy. The present study examines whether the G952G polymorphism alters cholesterol synthesis and/or absorption and whether it modulates the response to widely used lipid-lowering drugs such as inhibitors of cholesterol synthesis (simvastatin) or absorption (ezetimibe). METHODS: Seventy-two healthy male subjects with LDL cholesterol <190 mg/dL participated in the study. Twenty four subjects were treated with ezetimibe (10 mg), simvastatin (40 mg) or their combination, respectively, for two weeks. Blood was drawn before and after the 2-week treatment period. RESULTS: Eleven CC homozygous carriers of the gene were found (15%). There were no differences in cholesterol synthesis or absorption between the CC homozygotes and the G allele-carriers, as measured by the ratios to cholesterol of serum lathosterol, desmosterol and cholestenol (synthesis markers) and cholestanol, sitosterol and campesterol (absorption markers). Ezetimibe had a significantly more potent effect in blocking cholesterol absorption in the CC homozygotes compared to the G-carriers ( P=0.002). CONCLUSIONS: The G/C (G952G) polymorphism of the SREBP-1 gene is not associated with cholesterol synthesis or absorption in a German male population. The CC homozygotes have a significantly increased response to the effects of ezetimibe on cholesterol absorption compared to the G allele-carriers, suggesting that SREBP-1 may be implicated in ezetimibe's mechanism of action.

Comparison of a new long-acting testosterone undecanoate formulation vs testosterone enanthate for intramuscular androgen therapy in male hypogonadism.

OBJECTIVE: To assess the efficacy and safety of a novel long-acting im testosterone undecanoate (TU) formulation in comparison with testosterone enanthate (TE). SUBJECTS AND METHODS: An open-label, randomized, prospective clinical trial in 40 hypogonadal men (baseline serum testosterone levels <5 nmol/l), randomly assigned to 250 mg TE/3 weeks (no.=20) or 1000 mg TU im every 6 to 9 weeks for 30 weeks (no.=20). Subsequently, 32/40 men continued the study for another 114 weeks, now receiving TU 1000 mg/12 weeks. RESULTS: TU and TE produced no statistically significant improvements in grip strength over the first 30 weeks, which only occurred after approximately 90 weeks when all subjects received TU. There were no changes in body mass index with TU and TE, neither in the follow-up period when all patients received TU. But ratios of waist to hip circumferences declined in the longer term. Total serum cholesterol, LDL cholesterol, and triglycerides declined over the first 30 weeks, while plasma HDL also declined. Plasma LDL decreased further under long-term TU therapy, while HDL then increased. Hemoglobin and hematocrit values significantly increased over the first 30 weeks in both treatment groups and then no further increase was observed. Levels did not exceed the upper limit of normal. In both treatment groups, serum prostate specific antigen levels rose slightly after 30 weeks, with no further increase over the first 12 months, remaining stable within the normal range. Plasma T before the following TU injection was above the lower limit of reference values. Four injections per year are adequate. CONCLUSIONS: Administration of TU every 12 weeks is at least as safe and efficacious for treatment of hypogonadal men as TE, with a substantially lower frequency of administration. Follow-up over 114 weeks, when all subjects received TU, showed an excellent profile of efficacy and safety.

Peroxisome proliferator-activated receptor alpha (PPARalpha) and athero-sclerosis.

The peroxisome proliferator-activated receptors (PPARs) alpha, beta/delta and gamma are ligand-activated transcription factors belonging to the nuclear receptor superfamily. PPARs heterodimerize with the retinoid X receptor (RXR) and modulate the function of many target genes. They were originally described as regulators of various metabolic, pathways, but have been recently found to also exert modulating actions in the vascular wall. PPARalpha is activated by endogenous ligands, such as polyunsaturated fatty acids and by synthetic agonists such as the fibrates. PPARalpha is expressed mainly in the liver, kidney and skeletal muscle and is involved in fatty acid oxidation. However, it is also expressed in vascular cells such as the endothelial cells, vascular smooth muscle cells and macrophages, where it exerts anti-inflammatory and antioxidant effects. Since atherosclerosis is both a chronic inflammatory and a lipid disorder and since PPARalpha is expressed in vascular cells and regulates the expression of genes involved in lipid metabolism and inflammation, PPARalpha activators may constitute useful agents for the prevention of atherosclerosis, beyond their effects on lipid metabolism. This review will focus on the functions of PPARalpha on lipid metabolism, on vascular inflammation and its relationship to atherosclerosis. Furthermore, the currently available preclinical and clinical data on PPARalpha activators as well as their future perspectives will be discussed.

Limitations of the reverse transcription-polymerase chain reaction method for the detection of carcinoembryonic antigen-positive tumor cells in peripheral blood.

To examine the limitations of reverse transcription (RT)-PCR for the detection of circulating tumor cells in blood, we established a RT-PCR for carcinoembryonic antigen (CEA). Whole blood (10(7) nucleated cells) was mixed with cells from the colon cancer cell line LS174T (concentrations ranging from 0 to 10(6) cells). RT-PCR was performed to detect CEA mRNA in blood under various conditions. Healthy blood donors (n = 24) were examined by the established method for detecting CEA mRNA in blood. We were able to show that there is a detection limit for RT-PCR of 10 tumor cells in total and of 1 tumor cell in 10(5) nucleated cells. To obtain these results, a high number of PCR cycles (first PCR, 30 cycles; nested PCR, 45 cycles) was required. Under these PCR conditions, we found a positive PCR signal in 33% of healthy blood donors (n = 8). To overcome this problem, we reduced the nested PCR to 35 cycles. At that point, none of the controls showed a positive signal for CEA, and there was a subsequent decrease of the detection limit to 1 tumor cell in 10(2)-10(3) nucleated cells, lower than the detection limit of an immunocytological examination (1 tumor cell in 10(4) nucleated cells). When the amplification was performed with the tumor cells only and with no nucleated blood cells present, under exactly the same conditions, there was still a detection limit of 1 tumor cell in 106 nucleated cells. Our data clearly show that there is a severe loss of expected sensitivity of RT-PCR if it is performed in blood or nucleated blood cells. We conclude that PCR for CEA mRNA expression is not more sensitive than immunocytology and is, furthermore, plagued by the problem of a high percentage of false positive results.

Treatment of Cardiovascular Risk Factors in Women.

Cardiovascular disease (CVD) is a leading cause of death for both women and men. Common traditional risk factors for CVD, such as hypercholesterolemia, hypertension and smoking have a high prevalence in women and in some cases a greater health impact compared with men. Nevertheless, risk factors are treated less often and less aggressively in women than in men, partly due to decreased awareness on the part of public health opinion makers, patients and physicians. About seventy five percent of all coronary heart disease deaths among women could be avoided if CVD risk factors like hypercholesterolemia, hypertension and smoking are adequately treated. This narrative review discusses the treatment of the 4 CVD risk factors, namely hypercholesterolemia, hypertension, smoking and diabetes. These risk factors were examined in the Framingham Heart study and years later they were found in the INTERHEART study to be the 4 most important risk factors for the development of CVD.

Lipid-induced changes in vascular smooth muscle cell membrane fluidity are associated with DNA synthesis.

In the present study, we examined whether changes in the membrane fluidity of vascular smooth muscle cells (VSMCs) alter their DNA synthesis. For this purpose, the membrane fluidity of the cells was modulated after treatment of VSMCs with 1,2-dioleoyl phosphatidylcholine (PC). Treatment of VSMCs with 1,2-dioleoyl PC-rich medium containing 10% heat-inactivated human serum and 3 mg/ ml 1,2-dioleoyl PC for 24 h resulted in an increase in VSMC membrane fluidity at all temperatures from 15 degrees to 40 degrees C as well as a 51% inhibition of DNA synthesis, compared with untreated cells. Remarkably, enrichment of VSMCs with 1,2-dioleoyl PC/cholesterol-rich medium containing 10% human serum, 3 mg/ml 1,2-dioleoyl PC and 2 mg/ml cholesterol restored both membrane fluidity and DNA synthesis to the levels of untreated cells. The present findings show an inverse association between increased membrane fluidity and cellular DNA synthesis.

Molecular mechanisms explaining the preventive effects of catechins on the development of proliferative diseases.

Various growth factors such as the platelet derived growth factor (PDGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) transduce their mitogenic signals through the activation of tyrosine kinase receptors (RTKs). Since enhanced RTK activity has been associated with the development of proliferative diseases such as atherosclerosis and cancer, there has been an increased interest recently in the development of small molecule RTK inhibitors for the prevention/treatment of the aforementioned diseases. Many cell culture and animal studies have shown that catechins, the main compounds of the green tea leaves, are potent natural inhibitors of several RTKs. In the present article we review the various molecular and cellular mechanisms through which catechins inhibit the growth factor-RTK-mediated signal transduction pathways and exert their antiproliferative/apoptotic effects.

Effects of authentic and VLDL hydrolysis-derived fatty acids on vascular smooth muscle cell growth.

There are contradictory findings regarding the effects of free fatty acids on vascular smooth muscle cell (VSMC) growth. In the present study we investigated the effects of fatty acids released from hydrolysis of human VLDL triglycerides by lipoprotein lipase and of the fatty acids most abundant in the hydrolysed VLDL, namely oleic, linoleic, palmitic and myristic acid, all non albumin-bound, on VSMC growth. The effect of fatty acids on VSMC growth was assessed by [(3)H]-thymidine incorporation, colourimetrically, by cell counting, by determination of the cytoplasmic histone-associated DNA fragments and the caspase 3 activity. The fatty acid concentrations were determined by gas chromatography-mass spectrometry. Stimulation of ERK1/2 and p38 was determined by the chemiluminescence Western blotting method. Incubation of VSMC with purified VLDL (100 microg ml(-1)) and lipoprotein lipase (35 u ml(-1)) led to almost complete cell death although the ERK1/2 and the p38 MAP kinases were stimulated. The EC(50) of oleic, linoleic, myristic and palmitic acid were 4.6+/-1.3, 2.4+/-0.2, 116+/-10 and 287+/-30 microM, respectively. The estimated EC(50) of myristic and palmitic acid when derived from hydrolysed VLDL were 10 and 8 times, respectively, lower than when used alone. Apoptosis was not involved in the fatty acid-induced VSMC growth suppression/death. We conclude that (a) non albumin-bound fatty acids cause VSMC necrosis in a dose-dependent manner with a parallel ERK1/2 and p38 stimulation, (b) unsaturated fatty acids are more toxic to VSMC than saturated, and (c) saturated fatty acids are more toxic to VSMC in the hydrolysed VLDL than when used individually.

Early intracellular signalling pathway of ethanol in vascular smooth muscle cells.

1. ERKs belong to MAP kinase family and are activated by several growth and stress factors. Although ethanol has been shown to modulate ERK1 and ERK2 (p44(mapk) and p42(mapk)) activity, it can also act as an antiproliferative agent in various mammalian cells. Since the nature of the antiproliferative effect of ethanol in VSMCs has not been defined, we examined its effects on growth and on early intracellular events normally induced by growth factors in VSMCs. 2. Measurement of cytosolic Ca(2+) and pH in cell monolayers was performed using fura-2/AM and BCECF/AM, respectively. The effect of ethanol on VSMCs growth was assessed by [(3)H]-thymidine incorporation, by cell counting and by determination of the caspase 3 activity. Stimulation of ERK1 and ERK2 was examined by the chemiluminescence Western blotting method. The expression of c-fos was quantitated by Northern blotting. Determination of inositolphosphates was performed after labelling of VSMCs with myo-[2-(3)H]-inositol and separation of inositolphosphates by HPLC. 3. Ethanol (0.3 - 1.0% v v(-1), 17 - 170 mM) induced a dose-dependent maximal stimulation of p44(mapk)/p42(mapk) at 30 min and expression of c-fos mRNA with a maximum at 120 min. Intracellular events upstream to MAP kinase, like an increase in [Ca(2+)](i), activation of the Na(+)/H(+) exchanger and formation of phosphoinositol metabolites were also markedly activated by ethanol. Treatment of VSMCs with ethanol for 3 - 5 min induced an increase in DNA synthesis whereas treatment of the cells for more than 30 min was toxic. Caspase 3 activity was not modulated by ethanol treatment of VSMCs. 4. We may postulate that the activation of these mitogenic signals including the elevation of DNA synthesis reflects a cell effort to protect itself against the toxic effects of ethanol.

Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle alpha-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis.

The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth. We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet-derived growth factor beta-receptor (PDGF-Rbeta). The effect of PDGF-BB on VSMCs growth was assessed by [(3)H]-thymidine incorporation. Tyrosine phosphorylation of PDGF-Rbeta, PLC-gamma1, ERK1 and ERK2, p125(FAK) and paxillin as well as Sm alpha-actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm alpha-actin filaments, paxillin and PDGF-Rbeta was performed by immunfluorescence microscopy. Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm alpha-actin filament array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125(FAK) and paxillin but decreased the content of a Sm alpha-actin protein. Maximal decrease of 70% was observed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRNA level. CTX treatment completely abolished PDGF-BB stimulated DNA-synthesis although PDGF-Rbeta level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rbeta, PI 3'-K, PLC-gamma1 and ERK1/2 indicating an action of cyclic AMP on PDGF-beta receptor. We conclude that although cyclic AMP attenuates the PDGF-Rbeta mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF-BB-induced DNA synthesis in VSMCs.

Troglitazone and rosiglitazone induce apoptosis of vascular smooth muscle cells through an extracellular signal-regulated kinase-independent pathway.

Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. The peroxisome proliferator-activated receptor gamma (PPARgamma) ligands thiazolidinediones have been reported to induce apoptosis in macrophages and in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis in VSMC, cultured rat VSMC were treated with increasing doses of the thiazolidinedione analogues troglitazone (TRO) and rosiglitazone (RSG). Both ligands induced cell death in a concentration-dependent manner (EC50 12.1+/-3.3 microM and 1.43+/-0.39 microM, respectively), causing almost complete cell death at the highest concentrations (100 microM and 10 microM for TRO and RSG, respectively), along with an expected parallel decrease in [3H]thymidine uptake into cell DNA (EC50 6.7+/-2.4 microM and 0.75+/-0.19 microM, respectively). The cell count was determined by the coulter counter principle. Furthermore two apoptotic markers were measured, the caspase 3 activity and the cytoplasmic histone-associated DNA fragments, both of which were significantly increased when the aforementioned high concentrations were used. This indicates that apoptosis is involved in the TRO- and RSG-induced VSMC growth suppression. The same concentrations of TRO and RSG caused an unexpected stimulation of the extracellular signal-regulated response kinases 1 and 2 (ERK1/2) and stimulated the p38 mitogenic-activated protein (MAP) kinase as determined by Western blotting. In order to establish whether the proapoptotic effects of TRO and RSG are mediated through ERK1/2 activation, we used the selective MAP kinase kinase (MEK) inhibitor PD98059 (20 microM), which suppressed the TRO- and RSG-induced ERK1/2 activation but did not abolish their proapoptotic effects. We conclude that the thiazolidinedione analogues TRO and RSG induce cell death due to apoptosis in VSMC through an ERK1/2-independent pathway.