Ton J. Rabelink

Metabolic and Additional Vascular Effects of Thiazolidinediones

Several cardiovascular risk factors (dyslipidaemia, hypertension, glucose intolerance, hypercoagulability, obesity, hyperinsulinaemia and low-grade inflammation) cluster in the insulin resistance syndrome. Treatment of these individual risk factors reduces cardiovascular complications. However, targeting the underlying pathophysiological mechanisms of the insulin resistance syndrome is a more rational treatment strategy to further improve cardiovascular outcome.Our understanding of the so-called cardiovascular dysmetabolic syndrome has been improved by the discovery of nuclear peroxisome proliferator-activated receptors (PPARs). PPARs are ligand-activated transcription factors belonging to the nuclear receptor superfamily. As transcription factors, PPARs regulate the expression of numerous genes and affect glycaemic control, lipid metabolism, vascular tone and inflammation. Activation of the subtype PPAR-γ improves insulin sensitivity. Expression of PPAR-γ is present in several cell types involved in the process of atherosclerosis. Thus, modulation of PPAR-γ activity is an interesting therapeutic approach to reduce cardiovascular events.Thiazolidinediones are PPAR-γ agonists and constitute a new class of pharmacological agents for the treatment of type 2 (non-insulin-dependent) diabetes mellitus. Two such compounds are currently available for clinical use: rosiglitazone and pioglitazone. Thiazolidinediones improve insulin sensitivity and glycaemic control in patients with type 2 diabetes. In addition, improvement in endothelial function, a decrease in inflammatory conditions, a decrease in plasma levels of free fatty acids and lower blood pressure have been observed, which may have important beneficial effects on the vasculature.Several questions remain to be answered about PPAR-γ agonists, particularly with respect to the role of PPAR-γ in vascular pathophysiology. More needs to be known about the adverse effects of thiazolidinediones, such as hepatotoxicity, increased low-density lipoprotein cholesterol levels and increased oedema. The paradox of adipocyte differentiation with weight gain concurring with the insulin-sensitising effect of thiazolidinediones is not completely understood. The decrease in blood pressure induced by thiazolidinedione treatment seems incompatible with an increase in the plasma volume, and the discrepancy between the stimulation of the expression of CD36 and the antiatherogenic effects of the thiazolidinediones also needs further explanation. Long-term clinical trials of thiazolidinediones with cardiovascular endpoints are currently in progress.In conclusion, studying the effects of thiazolidinediones may shed more light on the mechanisms involved in the insulin resistance syndrome. Furthermore, thiazolidinediones could have specific, direct effects on processes involved in the development of vascular abnormalities.

Sympathetic Activation Markedly Reduces Endothelium-Dependent, Flow-Mediated Vasodilation

OBJECTIVESnWe sought to evaluate whether increased sympathetic outflow may interfere with flow-mediated dilation (FMD).nnnBACKGROUNDnEndothelial function, assessed as FMD, is frequently used as an intermediate end point in intervention studies. Many disease states with increased sympathetic tone are also characterized by endothelial dysfunction.nnnMETHODSnSixteen healthy volunteers underwent FMD studies with and without concomitant sympathetic stimulation. Intra-arterial nitroglycerin (NTG) infusion was used to assess endothelium-independent vasodilation. Pathophysiologically relevant sympathetic stimulation was achieved by baroreceptor unloading, using a lower body negative pressure box. In a subset of eight volunteers, this protocol was repeated during loco-regional alpha-adrenergic blockade by intra-arterial infusion of phentolamine (PE). Reactive hyperemic flow was assessed with strain-gauge phlethysmography.nnnRESULTSnOverall, FMD responses (8.3 +/- 3.4%) were significantly attenuated by concomitant sympathetic stimulation (3.6 +/- 3.4%, p < 0.01). Loco-regional alpha-adrenergic blockade had no effect on baseline FMD responses (10.7 +/- 4.7%), whereas the attenuation by sympathetic stimulation was abolished completely during PE co-infusion (11.5 +/- 3.3%). During intra-arterial NTG infusions, arterial diameters relative to baseline were not significantly different between the four possible stages.nnnCONCLUSIONSnSympathetic stimulation, at a clinically relevant range, significantly impairs the FMD response by an alpha-adrenergic mechanism.

Met-RANTES reduces vascular and tubular damage during acute renal transplant rejection: blocking monocyte arrest and recruitment

Chemokines are thought to contribute to the cellular infiltrate characteristic of renal transplant rejection. We show that Met‐RANTES, a chemokine receptor antagonist, suppresses recruitment of inflammatory cells into renal allografts. In a renal transplant model (Fisher RT1lvl rat kidney into Lewis RTI1 rat) where no additional immune suppressant was used, Met‐RANTES‐treated animals showed a significant reduction in vascular injury score (16.10 ± 5.20 vs. 62.67 ± 18.64) and tubular damage score (15.70 ± 5.22 vs. 33.00 ± 6.44) relative to untreated animals. In a more severe rejection model (Brown‐Norway RT1n rat kidney into Lewis RT11 rat), Met‐RANTES significantly augmented low‐dose cyclosporin A treatment to reduce all aspects of renal injury including interstitial inflammation (score 71.00 ± 6.10 vs. 157.30 ± 21.30). The majority of infiltrating cells in these models (60–70%) consisted of monocytes. Potential mechanisms of action of Met‐RANTES were tested using monocyte attachment assays on microvascular endothelium under physiological flow conditions. Preexposure of micro‐vascular endothelium to RANTES resulted in RANTES immobilization and RANTES‐induced firm adhesion of monocytes only after prestimulation of the endothelium with IL‐1β. Met‐RANTES completely inhibited this RANTES‐mediated arrest. Thus, Met‐RANTES may counter acute rejection by blocking leukocyte firm adhesion to inflamed endothelium.—Gröne, H.‐J., Weber, C., Weber, K. S. C., Gröne, E. F., Rabelink, T., Klier, C. M., Wells, T. N. C., Proudfoot, A. E., Schlöndorff, D., Nelson, P. J. Met‐RANTES reduces vascular and tubular damage during acute renal transplant rejection: blocking monocyte arrest and recruitment. FASEB J. 13, 1371–1383 (1999)

Thiazolidinediones and Blood Lipids in Type 2 Diabetes

Abstract—We evaluated study population characteristics and treatment effects on blood lipids between studies in which either rosiglitazone (RSG) or pioglitazone (PIO) was investigated in patients with type 2 diabetes. We performed a summary analysis of all published double-blind, placebo-controlled studies with RSG (4 and 8 mg/d) and PIO (15, 30, and 45 mg/d). Data were analyzed by the random-effects model. Nineteen trials met our inclusion criteria, yielding 5304 patients, 3236 in studies with RSG and 2068 in studies with PIO. Subjects treated with PIO were more obese and showed more pronounced hyperglycemia and dyslipidemia (increased triglycerides and decreased HDL cholesterol) at baseline than did subjects treated with RSG. By weighted linear-regression analysis, studies with PIO showed greater beneficial effects on triglycerides, total cholesterol, and LDL cholesterol, after adjustment for the respective lipid levels at baseline. RSG 8 mg/d showed greater increases in total cholesterol and LDL cholesterol than did RSG 4 mg/d. PIO 30 mg/d showed greater reductions in triglycerides than did PIO 15 mg/d. Studies conducted with PIO showed more beneficial effects on blood lipids, but also different study population characteristics in comparison with studies conducted with RSG. Differences in both pharmacologic properties between agents and study population characteristics are likely to have influenced the results.

Ferric saccharate induces oxygen radical stress and endothelial dysfunction in vivo

Background u2002Intravenous iron supplementation is used widely in haemodialysis patients. However, nontransferrin‐bound iron (NTBI), which increases after intravenous supplementation of ferric saccharate, has been suggested to act as a catalytic agent in oxygen radical formation in vitro and may thus contribute to endothelial impairment in vivo.

Impaired NO-dependent vasodilation in patients with Type II (non-insulin-dependent) diabetes mellitus is restored by acute administration of folate

HeadingAbstractn Aims/hypothesis. Patients with diabetes are characterised by endothelial dysfunction and cardiovascular mortality. In particular endothelium-derived nitric oxide has emerged as a first line mechanism against atherosclerosis. Hyperglycaemia causes oxygen radical stress but has also been associated with endothelial nitric oxide synthase uncoupling, both lead to decreased nitric oxide-availability. We recently showed that folate reverses eNOS uncoupling in vitro. Therefore we hypothesise that folate improves endothelial function in Type II (non-insulin-dependent) diabetes mellitus in vivo.n Methods. Using forearm plethysmography, we evaluated the effect of local, intra-arterial administration of 5-methyltetrahydrofolate (5-MTHF, the active form of folic acid, 1xa0µg/100xa0ml FAV/min) on forearm blood flow in 23 patients with Type II diabetes and 21 control subjects, matched for age, sex, blood pressure, body mass index, weight and smoking habits. Serotonin as a stimulator of nitric oxide-dependent vasodilation and sodium nitroprusside as a stimulator of endothelium-independent vasodilation were infused.n Results. Serotonin-induced vasodilation was blunted (53±30 vs 102±66xa0M/C%, p<0.005) and nitroprusside-induced vasodilation was mildly reduced (275±146 vs 391±203xa0M/C%, p<0.05) in patients with Type II diabetes compared to control subjects. 5-MTHF improved nitric oxide-mediated vasodilation (from 53±30 to 88±59xa0M/C%, p<0.05) in patients with Type II diabetes mellitus. As expected, 5-MTHF had no effect on forearm blood flow in control subjects.n Conclusion/interpretation. These data imply that folate can be used to improve nitric oxide status and to restore endothelial dysfunction in patients with Type II diabetes. Our results provide a strong rationale for the initiation of studies that investigate whether supplementation with folic acid prevents future cardiovascular events in this patient group.

Endothelin: New discoveries and rapid progress in the clinic

This fifth international conference on ET serves to underline the rapid pace of development of our understanding of the very versatile ET system. On the one hand, the body uses ETs at several stages in embryonic development, in normal postnatal growth, and in cardiovascular homeostasis under healthy conditions. On the other hand, overwhelming evidence now exists that ET-1 plays important pathophysiological roles in conditions of decompensated vascular homeostasis. Indeed, in CHF this evidence is sufficient to justify the large-scale studies of morbidity and mortality needed to market ET antagonists as medicines. Other potentially important cardiovascular indications for ET antagonists are still emerging--including hypertension, stroke, subarachnoid haemorrhage and renal failure--and all are likely to be the subject of clinical trials over the next few years. As yet, there has been little work outside the cardiovascular and renal fields, but other areas, such as cancer treatment, may also prove promising. New molecules with increasing selectivity (ETA and ETB) continue to emerge and may be valuable. Inhibition of ECE-1 remains as an alternative approach and nonpeptide ECE inhibitors now exist. There appears to be a consensus that ETA blockade is beneficial in cardiovascular and renal disease. However, several strands of work presented at the meeting--the hypertensive salt-sensitive phenotype of rescued ETB knockout mice, the sustained and progressive hypertensive effects of ETB-selective antagonism in rats, ETB-mediated vasodilatation and natriuresis in dogs, and nitric oxide-dependent ETB-mediated vasodilatation in humans--all suggest that ETB-mediated vascular and renal responses may be protective. The development of selective ETA antagonists, therefore, now seems fully justified. In the future, direct comparisons in animal models, and patients, of ETA and ETA/B antagonists will be important in determining the value of additional ETB receptor blockade in individual diseases.

Comparison of Rosiglitazone and Metformin for Treating HIV Lipodystrophy: A Randomized Trial

Context Antiretroviral therapy sometimes causes peripheral fat loss and central fat accumulation (lipodystrophy) and increases metabolic risk factors in patients with HIV. Contribution The investigators randomly assigned 39 HIV-infected men with lipodystrophy to rosiglitazone or metformin for 6 months. Compared with metformin, rosiglitazone increased abdominal fat and adiponectin levels. Compared with rosiglitazone, metformin reduced fasting lipid levels and improved some measures of brachial artery vasodilation. Both agents decreased glucose and insulin levels similarly. Cautions This open-label trial was small and did not assess clinical outcomes. Implications Available evidence does not establish whether rosiglitazone or metformin better benefits HIV-infected patients with lipodystrophy. The Editors Highly active antiretroviral therapy (HAART) in patients with HIV infection has greatly reduced AIDS-related mortality (1) but is associated with changes in fat distribution (lipodystrophy), including peripheral fat loss and central fat accumulation (2-4). Severe forms of lipodystrophy are a major cosmetic concern and can lead to suboptimal adherence to HAART. In addition, lipodystrophy is associated with metabolic risk factors, including insulin resistance and dyslipidemia (2-6), which have been correlated to surrogate markers of cardiovascular disease (7-9). As survival of patients with HIV infection increases, cardiovascular disease could become an important complication (10). Metformin and rosiglitazone are used in clinical medicine to improve glycemic control in patients with type 2 diabetes (11, 12). However, these agents may also be useful in nondiabetic patients with insulin resistance. Metformin acts mainly by decreasing hepatic insulin resistance and glucose output (11). Rosiglitazone is an agonist for peroxisome proliferatoractivated receptor- activation, thereby directly influencing the transcription of genes that regulate glucose and lipid metabolism (12). Peroxisome proliferatoractivated receptor- is preferentially expressed in adipose tissue, and the improvement of insulin resistance is partly secondary to enhanced fatty acid storage in subcutaneous adipocytes and improved adipocyte function, as reflected by the altered secretion of adiponectin (12). In vitro, rosiglitazone promotes adipogenesis, even in the presence of a protease inhibitor (13). Although metformin and rosiglitazone have been investigated separately (14-19), no studies were available that directly compared the effects of rosiglitazone and metformin for treating HIV lipodystrophy. Endothelial dysfunction is an early marker of atherosclerosis and can be assessed clinically by ultrasonography assessment of brachial artery flow-mediated vasodilation. Flow-mediated vasodilation is correlated with degree of atherosclerosis (20), and impaired flow-mediated vasodilation is an independent predictor of future cardiovascular events (21-23). In HIV-infected patients, the use of protease inhibitors has been linked to endothelial dysfunction (7). However, pravastatin did not improve flow-mediated vasodilation in HIV-infected patients, despite reductions in atherogenic lipoprotein levels (24). Rosiglitazone and metformin improve endothelial function (25-27) in HIV-negative individuals, but the effects of either agent in HIV-infected patients are not known. We conducted a randomized study to compare the effects of rosiglitazone and metformin on insulin sensitivity, body fat distribution, and endothelial function in patients with HIV lipodystrophy. Methods Patients We recruited men between 18 and 70 years of age with documented HIV infection from the Department of Infectious Disease of the University Medical Center, Utrecht, the Netherlands, between March 2003 and January 2004. At the HIV clinic, approximately 600 patients are currently treated for HIV infection, of whom an estimated 25% have clinical signs of lipodystrophy. Inclusion criteria were HIV RNA values less than 10000 copies/mL, the presence of lipodystrophy, and treatment with HAART for at least 18 months with no changes in the treatment regimen during the 6 months before inclusion. Exclusion criteria were the presence of opportunistic infectious disease or malignant conditions; renal disease, thyroid disease, or liver disease; body mass index greater than 30 kg/m2; fasting plasma glucose level greater than 7 mmol/L (>126.13 mg/dL), triglyceride level greater than 10 mmol/L (>884.96 mg/dL), or total cholesterol level greater than 8 mmol/L (>308.89 mg/dL); alcohol intake greater than 3 units (36 g) per day or history of alcohol abuse within the past 5 years; current thiazolidinedione therapy or known sensitivity to thiazolidinediones; clinical evidence of congestive heart failure; use of any other investigational agent within the past 4 weeks; concurrent therapy with glucocorticoids, testosterone, anabolic steroids, or growth hormones; use of any agent that could potentially interfere or interact with rosiglitazone; or history of nonadherence to medical regimens. We also excluded patients whom we considered to be potentially unreliable and those who had any concomitant condition that, in our opinion, could interfere with the interpretation of efficacy and safety data gathered in our trial. We defined the presence of HIV lipodystrophy as self-reported symptoms of loss of subcutaneous fat (face, arms, legs, and buttocks) with or without increased abdominal girth or development of a buffalo hump. An investigator confirmed these findings before enrollment. Clinical criteria for HIV lipodystrophy are controversial, but at the start of the study, no uniformly approved objective criteria were available for diagnosing lipodystrophy (28). We used subjective criteria for lipodystrophy that have been used previously (17, 19). Study Design Most recruited patients were consecutively seen patients with suspected lipodystrophy. At inclusion, we obtained fasting blood specimens and performed anthropometric measurements. We estimated total body fat mass by using bioimpedance analysis (RJL Systems, Clinton Township, Michigan). We recorded concomitant medication use and known cardiovascular risk factors, such as smoking habit, dietary pattern, physical exercise, and hypertension. In addition, we performed a thorough physical examination. Eligible patients underwent an oral glucose tolerance test, single-slice abdominal computed tomography, and flow-mediated vasodilation assessment. Subsequently, we randomly assigned participants in blocks of 4 to receive rosiglitazone (8 mg/d) or metformin (2 g/d) for 26 weeks by using a computer-generated list. The investigators and patients were not blinded to drug assignment. An independent pharmacist ensured allocation concealment. We requested that participants not change their regular diet, physical exercise, and smoking habit during the study. At the end of the period, we performed the same measurements. We also evaluated patients self-reported body fat distribution and physicians impressions of body fat distribution by open-ended questionnaire. Patients visited the hospital after 2 months and 4 months of treatment for safety evaluation, which included an open-ended questionnaire, physical examination, and blood sampling. During these visits, we evaluated adherence to study medication by open-ended questionnaire, although we did not perform pill counting. The participants also underwent a standardized oral fat-loading test before and after treatment with the study drugs. The effects of rosiglitazone and metformin on postprandial lipid and fatty acid metabolism will be published separately. The local research ethics committee of the University Medical Center approved the study protocol, and all participants gave written informed consent. Oral Glucose Tolerance Test Patients visited the hospital after an overnight fast. After insertion of a cannula in the forearm for venous blood sampling, patients rested for 30 minutes before administration of the glucose load (75 g). We obtained peripheral blood samples in sodium EDTA (68.44 mol/L) before (t= 0) and at regular 15-minute intervals up to 2 hours after glucose ingestion. We kept all EDTA blood samples on ice and centrifuged them immediately for 15 minutes at 800 g at 4C and stored them at 80C until assayed. Cross-Sectional Computed Tomography We performed single-slice, cross-sectional computed tomography at the L4L5 level, as described previously (29), to assess distribution of subcutaneous and visceral abdominal fat. Briefly, we obtained a lateral scout image to identify the level of the L4 pedicle, which served as the landmark for the 1-cm single-slice image. Scan variables were 144-cm table height, 80 kV, 70 mA, 2 seconds, 1-cm slice thickness, and a 48-cm field of view. On the computed tomography image, we outlined the border of the intra-abdominal cavity and quantified total and visceral abdominal fat areas by selecting an attenuation range of 250 to 50 Hounsfield units. We calculated subcutaneous abdominal fat as the difference between total and visceral abdominal fat. An independent radiologist who was unaware of the assignment status of the patients read and analyzed the computed tomography images. Endothelial Function We used ultrasonography to measure nitric oxidedependent, flow-mediated vasodilation as percentage diameter change in the brachial artery after reactive hyperemia (30). We measured this at the elbow of the patients right arms by using a Wall Track System (Scanner 200, Pie Medical, Maastricht, the Netherlands), which consists of an ultrasound imager with a 10-MHz linear array transducer connected to a data acquisition system and a personal computer. We averaged 3 measurements to calculate a baseline diameter. By inflation of a blood pressure cuff for 5 minutes at a pressure of 200 mm Hg, ischemia was applied to the forearm distal to the location of the transducer. Ultrasonography continued fo

NFκB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts

Monocyte influx secondary to ischemia‐reperfusion conditions the renal allograft to rejection by presentation of antigens and production of cytokines. Monocyte influx depends on NFicB‐dependent transcription of genes encoding adhesion molecules and chemokines. Here we demonstrate that cationic liposomes containing phosphorothioated oligodeoxynucleotides (ODN) with the kB binding site serving as competitive binding decoy, can prevent TNF‐α‐induced NFκB activity in endothelial cells in vitro. In an allogenic rat kidney transplantation model (BN to LEW), we show that perfusing the renal allograft with this decoy prior to transplantation abolishes nuclear NFκB activity in vivo and inhibits VCAM‐1 expression in the donor endothelium (P<0.05). At 24 h postreperfusion, periarterial infiltration of mono‐cytes/macrophages was significantly reduced in decoy ODN‐treated allografts compared to control allografts (3.7±0.7 vs. 9.2±1.2 macrophages/ves‐sel; P<0.01). At 72 h, there was a reduction of tubulointerstitial macrophage infiltration in decoy ODN‐treated kidneys compared to controls (75.6±13.9 vs. 120.0±11.2 macrophages/tubuloin‐terstitial area; P<0.05). In conclusion, perfusion of the renal allograft with NFκB decoy ODN prior to transplantation decreases the initial inflammatory response in a stringent, nonimmunosup‐pressed allogenic transplantation model. Therefore, the NFκB decoy approach may be useful to explore the role of endothelium and macrophages in graft rejection and may be developed into a graft‐specific immunosuppressive strategy allowing reduction of systemic immunosuppression on organ transplantation.—Vos, I., Govers, R., Groöne, H.‐J., Kleij, L., Schurink, M., de Weger, R., Goldschmeding, R., Rabelink, T. J. NFκB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts FASEB J. 14, 815–822 (2000)

Intensive Lipid Lowering by Statin Therapy Does Not Improve Vasoreactivity in Patients With Type 2 Diabetes

Cardiovascular disease is the most important cause of morbidity and mortality in patients with type 2 diabetes. Endothelial dysfunction predicts cardiovascular outcome. Type 2 diabetes is characterized by endothelial dysfunction, which may be caused by dyslipidemia. Statin therapy restores endothelial function in hyperlipidemic patients. Therefore, we hypothesize a beneficial effect of atorvastatin on NO-dependent vasodilation in patients with type 2 diabetes and mild dyslipidemia (low density lipoproteins >4.0 mmol/L and/or triglycerides >1.8 mmol/L). We evaluated the effect of intensive lipid lowering (4 weeks of 80 mg atorvastatin once daily) on vasoreactivity in 23 patients with type 2 diabetes by using venous occlusion plethysmography. Twenty-one control subjects were matched for age, sex, body mass index, blood pressure, and smoking habits. The ratio of blood flows in the infused (measurement [M]) and noninfused (control [C]) arm was calculated for each recording (M/C ratio), and M/C% indicates the percentage change from the baseline M/C ratio. Serotonin-induced NO-dependent vasodilation was significantly blunted (52±30 versus 102±66 M/C%, P <0.005), and nitroprusside-induced endothelium-independent vasodilation was modestly reduced (275±146 versus 391±203 M/C%, P <0.05) in patients with type 2 diabetes compared with control subjects. Despite significant reduction of total cholesterol, low density lipoproteins, and triglycerides (5.8±1.0 to 3.2±0.6 [P <0.0001], 4.1±1.1 to 1.8±0.7 [P <0.0001], and 2.2±1.3 to 1.4±0.5 [P <0.05] mmol/L, respectively), no effect on NO-dependent (59±44 M/C%) and endothelium-independent (292±202 M/C%) vasodilation was demonstrated. These data suggest that intensive lipid lowering by atorvastatin has no effect on NO availability in forearm resistance arteries in type 2 diabetic patients. Other factors, such as hyperglycemia, may be a more important contributing factor regarding impaired vasoreactivity in this patient group.