In patients with diabetes mellitus, the incidence of cardiovascular disease is increased, and the... more In patients with diabetes mellitus, the incidence of cardiovascular disease is increased, and the outcome following cardiovascular events is worse. The antihyperglycemic drug metformin appears to limit cardiovascular death in patients with type 2 diabetes. Indeed, preclinical studies have demonstrated that metformin limits (myocardial) ischemia and reperfusion injury, independent from its glucoselowering effect. This cardioprotection is mediated by activation of the Reperfusion Injury Salvage Kinase (RISK) pathway, activation of AMPK and by an increased formation of adenosine. In addition, metformin can modulate several cardiovascular risk factors and reduces the development of heart failure in murine models. Consequently, treatment with metformin might potentially improve cardiovascular outcome in patients at risk for myocardial ischemia, even if these patients do not have diabetes. In the current paper, we focus on the direct cardioprotective actions of metformin and the mechanisms that underlie these effects.
Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans Background: Adenosine... more Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans Background: Adenosine receptor stimulation induces several effects that could limit ischemia-reperfusion injury. We hypothesize that treatment with the nucleoside uptake inhibitor dipyridamole increases endogenous adenosine and limits ischemia-reperfusion injury in humans. Methods: Ischemia-reperfusion injury was studied in forearm skeletal muscle by technetium Tc 99m-labeled annexin A5 scintigraphy. Ischemia-reperfusion injury was induced by unilateral forearm ischemic exercise. Immediately on reperfusion, annexin A5 labeled with technetium Tc 99m was administered intravenously, and ischemia-reperfusion injury was expressed as the percentage difference in radioactivity between the experimental arm and the control arm 1 and 4 hours after reperfusion. Targeting was quantified in the region of the thenar muscle and forearm flexor muscles. This approach was used in 9 healthy male volunteers after a 1-week treatment with dipyridamole (200 mg, slow release, twice daily) and in 23 control subjects. Results: Dipyridamole treatment significantly reduced annexin A5 targeting in skeletal muscle compared with the control group (thenar region, 13% ؎ 7% versus 22% ؎ 15% at 1 hour after reperfusion and 9% ؎ 6% versus 27% ؎ 13% at 4 hours for dipyridamole and control groups, respectively [P ؍ .01]; flexor region, 4% ؎ 8% versus 7% ؎ 6% at 1 hour after reperfusion and 1% ؎ 4% versus 10% ؎ 9% at 4 hours for dipyridamole and control groups, respectively [P ؍ .01]). Conclusions: One week of oral treatment with the nucleoside uptake inhibitor dipyridamole (200 mg, slow release, twice daily) significantly limits ischemia-reperfusion injury in humans in vivo, as assessed by technetium Tc 99m-labeled annexin A5 scintigraphy of forearm skeletal muscle.
In patients with type 2 diabetes mellitus, treatment with metformin is associated with a lower ca... more In patients with type 2 diabetes mellitus, treatment with metformin is associated with a lower cardiovascular morbidity and mortality, compared with alternative glucose-lowering drugs. It has been suggested that metformin might exert direct protective effects on the heart. This review appraises recent experimental animal studies on the effect of metformin on myocardial ischaemia-reperfusion injury and remodeling. In murine models of myocardial infarction, the administration of metformin potently limits infarct size. Activation of adenosine monophosphate-activated protein kinase, increased formation of adenosine, and the prevention of opening of the mitochondrial permeability transition pore at reperfusion all contribute to this cardioprotective effect. In addition, metformin therapy attenuates postinfarction cardiac remodeling. There is evidence that activation of adenosine monophosphate-activated protein kinase and endothelial nitric oxide synthase, and a reduced collagen expression are crucial for this effect. The finding that metformin limits myocardial infarct size and remodeling in animal models of myocardial infarction suggests that patients suffering from myocardial ischaemia could benefit from treatment with metformin, even when these patients do not have diabetes. Currently, several clinical trials are being performed to test this hypothesis.
Journal of Cardiovascular Pharmacology, May 1, 2009
Metformin improves cardiovascular outcomes in patients with type 2 diabetes compared with other g... more Metformin improves cardiovascular outcomes in patients with type 2 diabetes compared with other glucose-lowering drugs. Experimental studies have shown that metformin can increase the intracellular concentration of adenosine monophosphate, which is a major determinant of the intracellular formation of adenosine. We hypothesize that metformin, given at reperfusion, can limit myocardial infarct size due to increased adenosine receptor stimulation. Isolated perfused hearts from Sprague-Dawley rats were subjected to 35 minutes of regional ischemia and 120 minutes of reperfusion. Perfusion with metformin (50 mM) for the first 15 minutes of reperfusion reduced infarct size (percent area at risk) from 42% 6 2% to 19% 6 4% (n $ 6; P , 0.01), which was blocked by a concomitant perfusion with the adenosine receptor antagonist 8-p-sulfophenyltheophylline (100 mM; 43% 6 3%) or nitrobenzylthioinosine (a blocker of transmembranous adenosine transport; 1 mM; 45% 6 5%). In addition, intravenous administration of metformin (5 mg/kg) reduced infarct size in a rat in situ model of myocardial infarction (34% 6 6% vs. 62% 6 5%; P , 0.01), which was completely abolished by 8-p-sulfophenyltheophylline (61% 6 3%). We conclude that metformin, given at reperfusion, reduces infarct size in a rat model of myocardial infarction, which is critically dependent on adenosine receptor stimulation, probably via increased intracellular formation of adenosine.
Tyrosine kinase inhibitors targeting angiogenesis have become an important part of the treatment ... more Tyrosine kinase inhibitors targeting angiogenesis have become an important part of the treatment of patients with several types of cancer. One of the most reported side effects of vascular endothelial growth factor receptor (VEGFR)-targeted therapies is hypertension. In this study, we hypothesized that the development of hypertension in patients treated with sunitinib, a multitargeted tyrosine kinase inhibitor, is preceded by reduced endothelium-dependent vasodilation. Moreover, we hypothesized that this endothelial dysfunction is a result of impaired nitric oxide release. In a placebo-controlled experiment, we determined vascular responses in isolated mesenteric arteries of rats (n = 26) after 7 days of sunitinib treatment. Sunitinib reduced endothelium-dependent vasodilation, but not endothelium-independent vasodilation. Moreover, we observed that the difference in endothelium-dependent vasodilation between controls and sunitinib-treated animals disappeared in the presence of N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide antagonist. In patients with metastatic renal cell carcinoma, before and 1 week after start of sunitinib, the endothelium-dependent vasodilator response to intra-arterial acetycholine and the endothelium-independent vasodilator response to intra-arterial sodium nitroprusside was assessed with venous occlusion plethysmography. No changes in forearm blood flow ratios were observed. Mean arterial pressure did significantly increase from 101.9 ± 3.8 to 106.1 ± 2.6 mmHg after 1 week and further to 115.8 (±4.9) mmHg after 2 weeks of treatment. In animals, this study confirms that exposure to high concentrations of sunitinib reduces endothelium-dependent vasodilation by reducing endothelial release of nitric oxide. In humans, however, reduced endothelium-dependent vasodilation does not precede the development of hypertension in patients treated with sunitinib.
Handbook of experimental pharmacology, Aug 19, 2010
In the concentration range that is normally achieved in humans, e.g., after the drinking of coffe... more In the concentration range that is normally achieved in humans, e.g., after the drinking of coffee or in patients treated with theophylline, the cardiovascular effects of methylxanthines are primarily due to antagonism of adenosine A(1) and A(2) receptors. Inhibition of phosphodiesterases or mobilization of intracellular calcium requires much higher concentrations. In conscious humans, acute exposure to caffeine results in an increase in blood pressure by an increased total peripheral resistance, and a slight decrease in heart rate. This overall hemodynamic response is composed of direct effects of caffeine on vascular tone, on myocardial contractility and conduction, and on the sympathetic nervous system. Caffeine is the most widely consumed methylxanthine, mainly derived from coffee intake. Regular coffee consumption can affect various traditional cardiovascular risk factors, including a slight increase in blood pressure, an increase in plasma cholesterol and homocysteine levels, and a reduced incidence of type 2 diabetes mellitus. Although most prospective studies have not reported an association between coffee consumption and coronary heart disease, these findings do not exclude that the acute hemodynamic and neurohumoral effects of coffee consumption could have an adverse effect in selected patient groups who are more vulnerable for these effects, based on their genetic profile or medication use.
a Department of Pharmacology-Toxicology, Radboud University Nijmegen Medical Center, Nijmegen, Th... more a Department of Pharmacology-Toxicology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands b Department of General Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands c Department of Physiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands d Research Institute for Sports and Exercise Sciences, Liverpool John Moores University, United Kingdom
European Journal of Clinical Pharmacology, Mar 15, 2016
Purpose Concomitant treatment with the glucose-lowering drug metformin and the platelet aggregati... more Purpose Concomitant treatment with the glucose-lowering drug metformin and the platelet aggregation inhibitor dipyridamole often occurs in patients with type 2 diabetes mellitus who have suffered a cerebrovascular event. The gastrointestinal uptake of metformin is mediated by the human equilibrative nucleoside transporter 4 (ENT4), which is inhibited by dipyridamole in preclinical studies. We hypothesized that dipyridamole lowers the plasma exposure to metformin. Methods Eighteen healthy volunteers (mean age 23 years; 9 male) were randomized in an open-label crossover study. Subjects were allocated to treatment with metformin 500 mg twice daily in combination with dipyridamole slow-release 200 mg twice daily or to metformin alone for 4 days. After a washout period of 10 days, the volunteers were crossed over to the alternative treatment arm. Blood samples were collected during a 10-h period after intake of the last metformin dose. The primary endpoint was the area under the plasma concentration-time curve (AUC 0-12h) and the maximum plasma metformin concentration (C max). Results In healthy subjects, dipyridamole did not significantly affect C max nor AUC 0-12h of metformin under steady-state conditions. Conclusions Previous in vitro studies report that dipyridamole inhibits the ENT4 transporter that mediates gastrointestinal uptake of metformin. In contrast, coadministration of dipyridamole at therapeutic dosages to healthy volunteers does not have a clinically relevant effect on metformin plasma steady-state exposure. This observation is reassuring for patients who are treated with this combination of drugs.
In this review we discuss the hypothesis, and current evidence, that a decreased concentration of... more In this review we discuss the hypothesis, and current evidence, that a decreased concentration of the endogenous purine-nucleoside adenosine contributes to the increased cardiovascular risk of patients with hyperhomocysteinemia. In hyperhomocysteinemia, the reaction equilibrium of the reaction catalysed by S-adenosylhomocysteine hydrolase will shift towards synthesis of S-adenosylhomocysteine, at the expense of free adenosine. Adenosine receptor stimulation induces several cardiovascular protective effects, such as vasodilation, inhibition of thrombocyte aggregation, of inflammation and of vascular smooth muscle cell proliferation. A decreased adenosine concentration could, therefore, well contribute to the cardiovascular complications of hyperhomocysteinemia. Previous animal studies have shown that administration of homocysteine decreases extracellular adenosine, associated with increased synthesis of S-adenosylhomocysteine. Recently, we showed that in patients with classical homocystinuria, cellular adenosine uptake is enhanced, thus limiting adenosine-induced vasodilation. These observations provide us with additional pharmacological targets, such as adenosine uptake inhibition, to reduce cardiovascular risk in patients with hyperhomocysteinemia.
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Objectives During ischaemia, the extracellular concentration of the endogenous nucleoside adenosi... more Objectives During ischaemia, the extracellular concentration of the endogenous nucleoside adenosine increases rapidly. Subsequent adenosine receptor stimulation induces various effects, including vasodilation, which can protect the tissue against the ischaemic insult. Adenosine deaminase (ADA) is an enzyme that catalyzes the irreversible deamination of adenosine. We hypothesized that the 22G > A polymorphism in the ADA gene inhibits its catalytic activity, and potentiates the protective effects of adenosine.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2005
Objective-Endogenous adenosine has several cardioprotective effects. We postulate that in patient... more Objective-Endogenous adenosine has several cardioprotective effects. We postulate that in patients with hyperhomocysteinemia increased intracellular formation of S-adenosylhomocysteine decreases free intracellular adenosine. Subsequently, facilitated diffusion of extracellular adenosine into cells through dipyridamole-sensitive transporters is enhanced, limiting adenosine receptor stimulation. We tested this hypothesis in patients with classical homocystinuria (nϭ9, plasma homocysteine 93.1Ϯ24.7 mol/L) and matched controls (nϭ8, homocysteine 9.1Ϯ1.0). Methods and Results-Infusion of adenosine (0.5, 1.5, 5.0, and 15.0 g/min/dL forearm) into the brachial artery increased forearm blood flow, as measured with venous occlusion plethysmography, to 2.9Ϯ0.4, 4.3Ϯ0.5, 5.6Ϯ1.1, and 9.6Ϯ2.1 in the patients and to 2.8Ϯ0.6, 4.4Ϯ1.0, 9.0Ϯ1.7, and 17.0Ϯ3.1 mL/min/dL in controls (PϽ0.05). However, adenosine-induced vasodilation in the presence of dipyridamole (100 g/min/dL) was similar in both groups (Pϭ0.9). Additionally, in isolated erythrocytes, adenosine uptake was accelerated by incubation with homocysteine (half-time 6.4Ϯ0.3 versus 8.1Ϯ0.5 minutes, PϽ0.001) associated with increased intracellular formation of S-adenosylhomocysteine (PϽ0.0001). Conclusions-In hyperhomocysteinemia, adenosine-induced vasodilation is impaired but is restored by dipyridamole. Accelerated cellular adenosine uptake probably accounts for these observations. These impaired actions of adenosine could well contribute to the cardiovascular complications of hyperhomocysteinemia.
Despite decades of research, the question as to whether coffee intake increases the risk of coron... more Despite decades of research, the question as to whether coffee intake increases the risk of coronary heart disease (CHD) remains controversial. In the current paper, we discuss the acute and long-term cardiovascular effects of coffee, and its major constituents, which could underlie such an association. Experimental studies have shown that administration of coffee or caffeine acutely raises blood pressure, circulating concentrations of (nor)epinephrine, increases arterial stiffness, impairs endothelium dependent vasodilation and inhibits ischemic preconditioning. The adverse effects of chronic coffee consumption on traditional risk factors for CHD are less consistent: although coffee intake slightly increases blood pressure, and plasma concentrations of homocysteine and cholesterol, there is no association with the incidence of hypertension, and a strong negative association with the incidence of type 2 diabetes mellitus. Moreover, common polymorphisms in genes involved in the metabolism of caffeine, catecholamines, homocysteine, and cholesterol can modulate the effect of coffee intake on cardiovascular parameters. Many epidemiological studies have explored the association between coffee drinking and CHD. Most prospective studies have not shown a positive association, whereas case-control studies in general have reported such an association. This discrepancy could be explained by an acute adverse effect of coffee, rather than a long-term adverse effect. We postulate that coffee drinking may have an acute detrimental effect in triggering coronary events and increasing infarct size in selected patient groups, rather than promoting the development of atherosclerosis in the general population, and we propose an alternative approach to explore such an effect in epidemiological studies.
for moxifloxacin on day 1 and day 24. In treatment group B, placebo for ponesimod was administere... more for moxifloxacin on day 1 and day 24. In treatment group B, placebo for ponesimod was administered once daily for 22 days (days 2-23). In addition, subjects received a single oral dose of moxifloxacin 400 mg either on day 1 or day 24. Replicate ECGs were to be extracted from the continuous digital 12-lead ECG recording over 12 hours. Primary end point was the baseline-adjusted, placebo-corrected effect of individual nonlinear corrected QT intervals (Δ Δ QTcI) on day 12 (40 mg) and on day 23 (100 mg). The relationship between plasma concentrations of ponesimod and metabolites and Δ Δ QTcI was assessed by applying a linear mixed effects modeling approach. Results: Ponesimod caused a small QTc prolongation, with a mean peak effect on Δ Δ QTcI of 6.9 ms (upper bound of the 2-sided 90% CI, 11.3 ms) on 40 mg and 9.1 ms (upper bound of the 2-sided 90% CI, 14.0 ms) on 100 mg. However, upon extrapolation to steady-state concentrations reached with 20-mg ponesimod, the upper bound of the 2-sided 90% CI would be below 10 ms, and should result in a negative study for doses of 20-mg ponesimod or lower. There was no relevant increased incidence of QTcI outliers, either as absolute or change from baseline, and QTcI > 480 ms or QTcI increase > 60 ms from baseline was not observed with ponesimod. Ponesimod did not affect the PR or QRS intervals. Assay sensitivity was demonstrated by moxifloxacin QTcI response with a mean peak effect on Δ Δ QTcI of 11.8 ms (lower bound of the 2-sided 90% CI, 9.0 ms). Conclusion: Ponesimod at doses of 40 mg and 100 mg causes a small QTc prolongation. However, based on the concentration-effect relationship no clinically relevant effect on QTc interval is expected for a dose of 20 mg, the highest selected dose for Phase 3.
British Journal of Clinical Pharmacology, Apr 1, 2007
In patients with coronary artery disease, the 34C. T variant of the adenosine mono-phosphate deam... more In patients with coronary artery disease, the 34C. T variant of the adenosine mono-phosphate deaminase gene (AMPD1), encoding a dysfunctional protein, predicts improved survival. We hypothesized that in subjects with this variant allele, ischaemia-induced intracellular adenosine formation is increased, augmenting reactive hyperaemia and ischaemic tolerance. Methods and results We selected 10 healthy subjects with the CT genotype and 10 CC controls. The forearm vasodilator response to 2 and 5 min of ischaemia (venous occlusion plethysmography, expressed as percentage of maximum blood flow after 13 min of ischaemia) was higher in the CT group 56% (49-74%) and 77% (71-86%) vs. 49% (42-53%) and 60% (55-70%) in the CC group [median (interquartile range), P ¼ 0.01]. Additionally, ischaemia-reperfusion injury was assessed in the thenar muscle using 99m Tc-annexin A5 scintigraphy after forearm ischaemic exercise to detect externalized membrane phosphatidylserines. At reperfusion, 99m Tc-annexin was administered intravenously. The change in annexin targeting between 1 and 4 h post-injection was 22.3% (interquartile range 22.4 to 21.6%) in the CT group vs. 20.3% (20.6 to 1.3%) in controls (n ¼ 7 in both groups, P ¼ 0.03). Conclusion The 34C. T variant of AMPD1 augments vasodilation and reduces tissue injury in response to forearm ischaemia. These mechanisms could contribute to the survival benefit of cardiovascular patients with this variant allele.
Adenosine modulates inflammation and prevents associated organ injury by activation of its recept... more Adenosine modulates inflammation and prevents associated organ injury by activation of its receptors. During sepsis, the extracellular adenosine concentration increases rapidly, but the underlying mechanism in humans is unknown. We aimed to determine the changes in adenosine metabolism and signaling both in vivo during experimental human endotoxemia and in vitro. Design: We studied subjects participating in three different randomized double-blind placebo-controlled trials. In order to prevent confounding by the different pharmacological interventions in these trials, analyses were performed on data of placebo-treated subjects only. Setting: Intensive care research unit at the Radboud University Nijmegen Medical Center. Subjects: In total, we used material of 24 healthy male subjects. Interventions: Subjects received 2 ng/kg Escherichia coli endotoxin (lipopolysaccharide) intravenously. Measurements and Main Results: Following experimental endotoxemia, endogenous adenosine concentrations increased. Expression of 5'ectonucleotidase messenger RNA was upregulated (p = .01), whereas adenosine deaminase messenger RNA was downregulated (p = .02). Further more, both adenosine deaminase and adenosine kinase activity was significantly diminished (both p ≤ .0001). A 2a and A 2b receptor messenger RNA expression was elevated (p = .02 and p = .04, respectively), whereas messenger RNA expression of A 1 and A 3 receptors was reduced (both, p = .03). In vitro, lipopolysaccharide dose-dependently attenuated the activity of both adenosine deaminase and adenosine kinase (both p ≤ .0001). Conclusions: Adenosine metabolism and signaling undergo adaptive changes during human experimental endotoxemia promoting higher levels of adenosine thereby facilitating its inflammatory signaling.
Hexokinase II (HKII) is a glycolytic enzyme that can be bound to mitochondria (mtHKII) or free in... more Hexokinase II (HKII) is a glycolytic enzyme that can be bound to mitochondria (mtHKII) or free in the cytosol. Previous studies showed that increased amounts of mtHKII protect the heart against ischemia/reperfusion injury.We hypothesized that reduced mtHKII binding increases ischemia/reperfusion damage by increased respiration and ROS production. Procedures were in accordance with the Institutional Animal Ethical Committee and conform to National Institutes of Health guidelines. Langendorff-perfused rat hearts were exposed to 20 minutes of saline, 1 μ M TAT-only or 200 nM or 1 μ M TAT-HKII, followed by 15 minutes of ischemia and 30 minutes of reperfusion. TAT-HKII translocates HKII from the mitochondria. During the complete protocol, cardiac oxygen consumption (MVO 2), cardiac function (rate pressure product [RPP]), and ROS (DHE fluorescence) were measured. Mitochondrial oxygen tension was measured by using protoporphyrin IX-delayed fluorescence before and after peptide treatment. During reperfusion, necrosis was measured by lactate dehydrogenase (LDH) activity in effluent.In the 1 μ M TAT-HKII hearts, MV−O/RPP was increased and mitochondrial oxygen tension was reduced during peptide treatment. This was not observed after treatment with 200 nM TAT-HKII nor in the control groups. During reperfusion, MVO 2 /RPP was increased in both treatment groups. ROS production increased in both TAT-HKII groups during ischemia and reperfusion. These effects were accompanied by increased LDH activity during reperfusion in both TAT-HKII treated groups. No LDH activity was observed in control groups.Reduction of mtHKII by TAT-HKII treatment turns reversible ischemia into irreversible ischemia. This might be caused by increased ROS production during ischemia and reperfusion, and increased oxygen consumption.
Journal of Cardiovascular Pharmacology, Apr 1, 2003
In order to investigate the consequences of stress susceptibility on vascular function, the autho... more In order to investigate the consequences of stress susceptibility on vascular function, the authors assessed the respective contributions of nitric oxide (NO), prostanoids, and endothelium-derived hyperpolarizing factor to the vascular tone in rats with a constitutionally determined high and low susceptibility to behavioral stressors. In mesenteric resistance arteries mounted in a small vessel myograph and precontracted with l-phenylephrine hydrochloride (phenylephrine), the NO-synthase inhibitor N omega-nitro-l-arginine (l-NOARG, 100 microM) elicited a smaller increase of vascular tone in apomorphine-susceptible (APO-SUS) rats (P < 0.01). Addition of indomethacin (10 microM), in the presence of l-NOARG, resulted in a smaller decrease of vascular tone in APO-SUS rats (P < 0.01). Although acetylcholine-induced relaxation in phenylephrine-precontracted arteries was not different (P > 0.1), the individual components contributing to this relaxation were. In arteries precontracted with 125 mM K+, and incubated with indomethacin, acetylcholine-induced relaxation was not significantly different (pEC(50) and E(max): P > 0.1). Sensitivity (pEC(50): P < 0.05) and maximum relaxation (E(max): P < 0.001) to sodium nitroprusside, in the presence of 125 mM K+, was more pronounced in APO-SUS rats. In phenylephrine-precontracted arteries, in the presence of l-NOARG and indomethacin, maximum relaxation to ACh was reduced in APO-SUS rats (E(max): P < 0.05). This study showed that in rats with a high susceptibility to stressors, the contribution of NO to vascular tone was decreased as was the ratio of vasoconstrictor and vasodilator cyclooxygenase products in alpha-adrenergic precontracted arteries. End-organ sensitivity to NO was greater in APO-SUS rats, possibly due to up-regulation. Moreover, the contribution of endothelium-derived hyperpolarizing factor to acetylcholine-induced vasodilation was reduced in APO-SUS rat arteries.
In patients with diabetes mellitus, the incidence of cardiovascular disease is increased, and the... more In patients with diabetes mellitus, the incidence of cardiovascular disease is increased, and the outcome following cardiovascular events is worse. The antihyperglycemic drug metformin appears to limit cardiovascular death in patients with type 2 diabetes. Indeed, preclinical studies have demonstrated that metformin limits (myocardial) ischemia and reperfusion injury, independent from its glucoselowering effect. This cardioprotection is mediated by activation of the Reperfusion Injury Salvage Kinase (RISK) pathway, activation of AMPK and by an increased formation of adenosine. In addition, metformin can modulate several cardiovascular risk factors and reduces the development of heart failure in murine models. Consequently, treatment with metformin might potentially improve cardiovascular outcome in patients at risk for myocardial ischemia, even if these patients do not have diabetes. In the current paper, we focus on the direct cardioprotective actions of metformin and the mechanisms that underlie these effects.
Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans Background: Adenosine... more Oral therapy with dipyridamole limits ischemia-reperfusion injury in humans Background: Adenosine receptor stimulation induces several effects that could limit ischemia-reperfusion injury. We hypothesize that treatment with the nucleoside uptake inhibitor dipyridamole increases endogenous adenosine and limits ischemia-reperfusion injury in humans. Methods: Ischemia-reperfusion injury was studied in forearm skeletal muscle by technetium Tc 99m-labeled annexin A5 scintigraphy. Ischemia-reperfusion injury was induced by unilateral forearm ischemic exercise. Immediately on reperfusion, annexin A5 labeled with technetium Tc 99m was administered intravenously, and ischemia-reperfusion injury was expressed as the percentage difference in radioactivity between the experimental arm and the control arm 1 and 4 hours after reperfusion. Targeting was quantified in the region of the thenar muscle and forearm flexor muscles. This approach was used in 9 healthy male volunteers after a 1-week treatment with dipyridamole (200 mg, slow release, twice daily) and in 23 control subjects. Results: Dipyridamole treatment significantly reduced annexin A5 targeting in skeletal muscle compared with the control group (thenar region, 13% ؎ 7% versus 22% ؎ 15% at 1 hour after reperfusion and 9% ؎ 6% versus 27% ؎ 13% at 4 hours for dipyridamole and control groups, respectively [P ؍ .01]; flexor region, 4% ؎ 8% versus 7% ؎ 6% at 1 hour after reperfusion and 1% ؎ 4% versus 10% ؎ 9% at 4 hours for dipyridamole and control groups, respectively [P ؍ .01]). Conclusions: One week of oral treatment with the nucleoside uptake inhibitor dipyridamole (200 mg, slow release, twice daily) significantly limits ischemia-reperfusion injury in humans in vivo, as assessed by technetium Tc 99m-labeled annexin A5 scintigraphy of forearm skeletal muscle.
In patients with type 2 diabetes mellitus, treatment with metformin is associated with a lower ca... more In patients with type 2 diabetes mellitus, treatment with metformin is associated with a lower cardiovascular morbidity and mortality, compared with alternative glucose-lowering drugs. It has been suggested that metformin might exert direct protective effects on the heart. This review appraises recent experimental animal studies on the effect of metformin on myocardial ischaemia-reperfusion injury and remodeling. In murine models of myocardial infarction, the administration of metformin potently limits infarct size. Activation of adenosine monophosphate-activated protein kinase, increased formation of adenosine, and the prevention of opening of the mitochondrial permeability transition pore at reperfusion all contribute to this cardioprotective effect. In addition, metformin therapy attenuates postinfarction cardiac remodeling. There is evidence that activation of adenosine monophosphate-activated protein kinase and endothelial nitric oxide synthase, and a reduced collagen expression are crucial for this effect. The finding that metformin limits myocardial infarct size and remodeling in animal models of myocardial infarction suggests that patients suffering from myocardial ischaemia could benefit from treatment with metformin, even when these patients do not have diabetes. Currently, several clinical trials are being performed to test this hypothesis.
Journal of Cardiovascular Pharmacology, May 1, 2009
Metformin improves cardiovascular outcomes in patients with type 2 diabetes compared with other g... more Metformin improves cardiovascular outcomes in patients with type 2 diabetes compared with other glucose-lowering drugs. Experimental studies have shown that metformin can increase the intracellular concentration of adenosine monophosphate, which is a major determinant of the intracellular formation of adenosine. We hypothesize that metformin, given at reperfusion, can limit myocardial infarct size due to increased adenosine receptor stimulation. Isolated perfused hearts from Sprague-Dawley rats were subjected to 35 minutes of regional ischemia and 120 minutes of reperfusion. Perfusion with metformin (50 mM) for the first 15 minutes of reperfusion reduced infarct size (percent area at risk) from 42% 6 2% to 19% 6 4% (n $ 6; P , 0.01), which was blocked by a concomitant perfusion with the adenosine receptor antagonist 8-p-sulfophenyltheophylline (100 mM; 43% 6 3%) or nitrobenzylthioinosine (a blocker of transmembranous adenosine transport; 1 mM; 45% 6 5%). In addition, intravenous administration of metformin (5 mg/kg) reduced infarct size in a rat in situ model of myocardial infarction (34% 6 6% vs. 62% 6 5%; P , 0.01), which was completely abolished by 8-p-sulfophenyltheophylline (61% 6 3%). We conclude that metformin, given at reperfusion, reduces infarct size in a rat model of myocardial infarction, which is critically dependent on adenosine receptor stimulation, probably via increased intracellular formation of adenosine.
Tyrosine kinase inhibitors targeting angiogenesis have become an important part of the treatment ... more Tyrosine kinase inhibitors targeting angiogenesis have become an important part of the treatment of patients with several types of cancer. One of the most reported side effects of vascular endothelial growth factor receptor (VEGFR)-targeted therapies is hypertension. In this study, we hypothesized that the development of hypertension in patients treated with sunitinib, a multitargeted tyrosine kinase inhibitor, is preceded by reduced endothelium-dependent vasodilation. Moreover, we hypothesized that this endothelial dysfunction is a result of impaired nitric oxide release. In a placebo-controlled experiment, we determined vascular responses in isolated mesenteric arteries of rats (n = 26) after 7 days of sunitinib treatment. Sunitinib reduced endothelium-dependent vasodilation, but not endothelium-independent vasodilation. Moreover, we observed that the difference in endothelium-dependent vasodilation between controls and sunitinib-treated animals disappeared in the presence of N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide antagonist. In patients with metastatic renal cell carcinoma, before and 1 week after start of sunitinib, the endothelium-dependent vasodilator response to intra-arterial acetycholine and the endothelium-independent vasodilator response to intra-arterial sodium nitroprusside was assessed with venous occlusion plethysmography. No changes in forearm blood flow ratios were observed. Mean arterial pressure did significantly increase from 101.9 ± 3.8 to 106.1 ± 2.6 mmHg after 1 week and further to 115.8 (±4.9) mmHg after 2 weeks of treatment. In animals, this study confirms that exposure to high concentrations of sunitinib reduces endothelium-dependent vasodilation by reducing endothelial release of nitric oxide. In humans, however, reduced endothelium-dependent vasodilation does not precede the development of hypertension in patients treated with sunitinib.
Handbook of experimental pharmacology, Aug 19, 2010
In the concentration range that is normally achieved in humans, e.g., after the drinking of coffe... more In the concentration range that is normally achieved in humans, e.g., after the drinking of coffee or in patients treated with theophylline, the cardiovascular effects of methylxanthines are primarily due to antagonism of adenosine A(1) and A(2) receptors. Inhibition of phosphodiesterases or mobilization of intracellular calcium requires much higher concentrations. In conscious humans, acute exposure to caffeine results in an increase in blood pressure by an increased total peripheral resistance, and a slight decrease in heart rate. This overall hemodynamic response is composed of direct effects of caffeine on vascular tone, on myocardial contractility and conduction, and on the sympathetic nervous system. Caffeine is the most widely consumed methylxanthine, mainly derived from coffee intake. Regular coffee consumption can affect various traditional cardiovascular risk factors, including a slight increase in blood pressure, an increase in plasma cholesterol and homocysteine levels, and a reduced incidence of type 2 diabetes mellitus. Although most prospective studies have not reported an association between coffee consumption and coronary heart disease, these findings do not exclude that the acute hemodynamic and neurohumoral effects of coffee consumption could have an adverse effect in selected patient groups who are more vulnerable for these effects, based on their genetic profile or medication use.
a Department of Pharmacology-Toxicology, Radboud University Nijmegen Medical Center, Nijmegen, Th... more a Department of Pharmacology-Toxicology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands b Department of General Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands c Department of Physiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands d Research Institute for Sports and Exercise Sciences, Liverpool John Moores University, United Kingdom
European Journal of Clinical Pharmacology, Mar 15, 2016
Purpose Concomitant treatment with the glucose-lowering drug metformin and the platelet aggregati... more Purpose Concomitant treatment with the glucose-lowering drug metformin and the platelet aggregation inhibitor dipyridamole often occurs in patients with type 2 diabetes mellitus who have suffered a cerebrovascular event. The gastrointestinal uptake of metformin is mediated by the human equilibrative nucleoside transporter 4 (ENT4), which is inhibited by dipyridamole in preclinical studies. We hypothesized that dipyridamole lowers the plasma exposure to metformin. Methods Eighteen healthy volunteers (mean age 23 years; 9 male) were randomized in an open-label crossover study. Subjects were allocated to treatment with metformin 500 mg twice daily in combination with dipyridamole slow-release 200 mg twice daily or to metformin alone for 4 days. After a washout period of 10 days, the volunteers were crossed over to the alternative treatment arm. Blood samples were collected during a 10-h period after intake of the last metformin dose. The primary endpoint was the area under the plasma concentration-time curve (AUC 0-12h) and the maximum plasma metformin concentration (C max). Results In healthy subjects, dipyridamole did not significantly affect C max nor AUC 0-12h of metformin under steady-state conditions. Conclusions Previous in vitro studies report that dipyridamole inhibits the ENT4 transporter that mediates gastrointestinal uptake of metformin. In contrast, coadministration of dipyridamole at therapeutic dosages to healthy volunteers does not have a clinically relevant effect on metformin plasma steady-state exposure. This observation is reassuring for patients who are treated with this combination of drugs.
In this review we discuss the hypothesis, and current evidence, that a decreased concentration of... more In this review we discuss the hypothesis, and current evidence, that a decreased concentration of the endogenous purine-nucleoside adenosine contributes to the increased cardiovascular risk of patients with hyperhomocysteinemia. In hyperhomocysteinemia, the reaction equilibrium of the reaction catalysed by S-adenosylhomocysteine hydrolase will shift towards synthesis of S-adenosylhomocysteine, at the expense of free adenosine. Adenosine receptor stimulation induces several cardiovascular protective effects, such as vasodilation, inhibition of thrombocyte aggregation, of inflammation and of vascular smooth muscle cell proliferation. A decreased adenosine concentration could, therefore, well contribute to the cardiovascular complications of hyperhomocysteinemia. Previous animal studies have shown that administration of homocysteine decreases extracellular adenosine, associated with increased synthesis of S-adenosylhomocysteine. Recently, we showed that in patients with classical homocystinuria, cellular adenosine uptake is enhanced, thus limiting adenosine-induced vasodilation. These observations provide us with additional pharmacological targets, such as adenosine uptake inhibition, to reduce cardiovascular risk in patients with hyperhomocysteinemia.
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Objectives During ischaemia, the extracellular concentration of the endogenous nucleoside adenosi... more Objectives During ischaemia, the extracellular concentration of the endogenous nucleoside adenosine increases rapidly. Subsequent adenosine receptor stimulation induces various effects, including vasodilation, which can protect the tissue against the ischaemic insult. Adenosine deaminase (ADA) is an enzyme that catalyzes the irreversible deamination of adenosine. We hypothesized that the 22G > A polymorphism in the ADA gene inhibits its catalytic activity, and potentiates the protective effects of adenosine.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2005
Objective-Endogenous adenosine has several cardioprotective effects. We postulate that in patient... more Objective-Endogenous adenosine has several cardioprotective effects. We postulate that in patients with hyperhomocysteinemia increased intracellular formation of S-adenosylhomocysteine decreases free intracellular adenosine. Subsequently, facilitated diffusion of extracellular adenosine into cells through dipyridamole-sensitive transporters is enhanced, limiting adenosine receptor stimulation. We tested this hypothesis in patients with classical homocystinuria (nϭ9, plasma homocysteine 93.1Ϯ24.7 mol/L) and matched controls (nϭ8, homocysteine 9.1Ϯ1.0). Methods and Results-Infusion of adenosine (0.5, 1.5, 5.0, and 15.0 g/min/dL forearm) into the brachial artery increased forearm blood flow, as measured with venous occlusion plethysmography, to 2.9Ϯ0.4, 4.3Ϯ0.5, 5.6Ϯ1.1, and 9.6Ϯ2.1 in the patients and to 2.8Ϯ0.6, 4.4Ϯ1.0, 9.0Ϯ1.7, and 17.0Ϯ3.1 mL/min/dL in controls (PϽ0.05). However, adenosine-induced vasodilation in the presence of dipyridamole (100 g/min/dL) was similar in both groups (Pϭ0.9). Additionally, in isolated erythrocytes, adenosine uptake was accelerated by incubation with homocysteine (half-time 6.4Ϯ0.3 versus 8.1Ϯ0.5 minutes, PϽ0.001) associated with increased intracellular formation of S-adenosylhomocysteine (PϽ0.0001). Conclusions-In hyperhomocysteinemia, adenosine-induced vasodilation is impaired but is restored by dipyridamole. Accelerated cellular adenosine uptake probably accounts for these observations. These impaired actions of adenosine could well contribute to the cardiovascular complications of hyperhomocysteinemia.
Despite decades of research, the question as to whether coffee intake increases the risk of coron... more Despite decades of research, the question as to whether coffee intake increases the risk of coronary heart disease (CHD) remains controversial. In the current paper, we discuss the acute and long-term cardiovascular effects of coffee, and its major constituents, which could underlie such an association. Experimental studies have shown that administration of coffee or caffeine acutely raises blood pressure, circulating concentrations of (nor)epinephrine, increases arterial stiffness, impairs endothelium dependent vasodilation and inhibits ischemic preconditioning. The adverse effects of chronic coffee consumption on traditional risk factors for CHD are less consistent: although coffee intake slightly increases blood pressure, and plasma concentrations of homocysteine and cholesterol, there is no association with the incidence of hypertension, and a strong negative association with the incidence of type 2 diabetes mellitus. Moreover, common polymorphisms in genes involved in the metabolism of caffeine, catecholamines, homocysteine, and cholesterol can modulate the effect of coffee intake on cardiovascular parameters. Many epidemiological studies have explored the association between coffee drinking and CHD. Most prospective studies have not shown a positive association, whereas case-control studies in general have reported such an association. This discrepancy could be explained by an acute adverse effect of coffee, rather than a long-term adverse effect. We postulate that coffee drinking may have an acute detrimental effect in triggering coronary events and increasing infarct size in selected patient groups, rather than promoting the development of atherosclerosis in the general population, and we propose an alternative approach to explore such an effect in epidemiological studies.
for moxifloxacin on day 1 and day 24. In treatment group B, placebo for ponesimod was administere... more for moxifloxacin on day 1 and day 24. In treatment group B, placebo for ponesimod was administered once daily for 22 days (days 2-23). In addition, subjects received a single oral dose of moxifloxacin 400 mg either on day 1 or day 24. Replicate ECGs were to be extracted from the continuous digital 12-lead ECG recording over 12 hours. Primary end point was the baseline-adjusted, placebo-corrected effect of individual nonlinear corrected QT intervals (Δ Δ QTcI) on day 12 (40 mg) and on day 23 (100 mg). The relationship between plasma concentrations of ponesimod and metabolites and Δ Δ QTcI was assessed by applying a linear mixed effects modeling approach. Results: Ponesimod caused a small QTc prolongation, with a mean peak effect on Δ Δ QTcI of 6.9 ms (upper bound of the 2-sided 90% CI, 11.3 ms) on 40 mg and 9.1 ms (upper bound of the 2-sided 90% CI, 14.0 ms) on 100 mg. However, upon extrapolation to steady-state concentrations reached with 20-mg ponesimod, the upper bound of the 2-sided 90% CI would be below 10 ms, and should result in a negative study for doses of 20-mg ponesimod or lower. There was no relevant increased incidence of QTcI outliers, either as absolute or change from baseline, and QTcI > 480 ms or QTcI increase > 60 ms from baseline was not observed with ponesimod. Ponesimod did not affect the PR or QRS intervals. Assay sensitivity was demonstrated by moxifloxacin QTcI response with a mean peak effect on Δ Δ QTcI of 11.8 ms (lower bound of the 2-sided 90% CI, 9.0 ms). Conclusion: Ponesimod at doses of 40 mg and 100 mg causes a small QTc prolongation. However, based on the concentration-effect relationship no clinically relevant effect on QTc interval is expected for a dose of 20 mg, the highest selected dose for Phase 3.
British Journal of Clinical Pharmacology, Apr 1, 2007
In patients with coronary artery disease, the 34C. T variant of the adenosine mono-phosphate deam... more In patients with coronary artery disease, the 34C. T variant of the adenosine mono-phosphate deaminase gene (AMPD1), encoding a dysfunctional protein, predicts improved survival. We hypothesized that in subjects with this variant allele, ischaemia-induced intracellular adenosine formation is increased, augmenting reactive hyperaemia and ischaemic tolerance. Methods and results We selected 10 healthy subjects with the CT genotype and 10 CC controls. The forearm vasodilator response to 2 and 5 min of ischaemia (venous occlusion plethysmography, expressed as percentage of maximum blood flow after 13 min of ischaemia) was higher in the CT group 56% (49-74%) and 77% (71-86%) vs. 49% (42-53%) and 60% (55-70%) in the CC group [median (interquartile range), P ¼ 0.01]. Additionally, ischaemia-reperfusion injury was assessed in the thenar muscle using 99m Tc-annexin A5 scintigraphy after forearm ischaemic exercise to detect externalized membrane phosphatidylserines. At reperfusion, 99m Tc-annexin was administered intravenously. The change in annexin targeting between 1 and 4 h post-injection was 22.3% (interquartile range 22.4 to 21.6%) in the CT group vs. 20.3% (20.6 to 1.3%) in controls (n ¼ 7 in both groups, P ¼ 0.03). Conclusion The 34C. T variant of AMPD1 augments vasodilation and reduces tissue injury in response to forearm ischaemia. These mechanisms could contribute to the survival benefit of cardiovascular patients with this variant allele.
Adenosine modulates inflammation and prevents associated organ injury by activation of its recept... more Adenosine modulates inflammation and prevents associated organ injury by activation of its receptors. During sepsis, the extracellular adenosine concentration increases rapidly, but the underlying mechanism in humans is unknown. We aimed to determine the changes in adenosine metabolism and signaling both in vivo during experimental human endotoxemia and in vitro. Design: We studied subjects participating in three different randomized double-blind placebo-controlled trials. In order to prevent confounding by the different pharmacological interventions in these trials, analyses were performed on data of placebo-treated subjects only. Setting: Intensive care research unit at the Radboud University Nijmegen Medical Center. Subjects: In total, we used material of 24 healthy male subjects. Interventions: Subjects received 2 ng/kg Escherichia coli endotoxin (lipopolysaccharide) intravenously. Measurements and Main Results: Following experimental endotoxemia, endogenous adenosine concentrations increased. Expression of 5'ectonucleotidase messenger RNA was upregulated (p = .01), whereas adenosine deaminase messenger RNA was downregulated (p = .02). Further more, both adenosine deaminase and adenosine kinase activity was significantly diminished (both p ≤ .0001). A 2a and A 2b receptor messenger RNA expression was elevated (p = .02 and p = .04, respectively), whereas messenger RNA expression of A 1 and A 3 receptors was reduced (both, p = .03). In vitro, lipopolysaccharide dose-dependently attenuated the activity of both adenosine deaminase and adenosine kinase (both p ≤ .0001). Conclusions: Adenosine metabolism and signaling undergo adaptive changes during human experimental endotoxemia promoting higher levels of adenosine thereby facilitating its inflammatory signaling.
Hexokinase II (HKII) is a glycolytic enzyme that can be bound to mitochondria (mtHKII) or free in... more Hexokinase II (HKII) is a glycolytic enzyme that can be bound to mitochondria (mtHKII) or free in the cytosol. Previous studies showed that increased amounts of mtHKII protect the heart against ischemia/reperfusion injury.We hypothesized that reduced mtHKII binding increases ischemia/reperfusion damage by increased respiration and ROS production. Procedures were in accordance with the Institutional Animal Ethical Committee and conform to National Institutes of Health guidelines. Langendorff-perfused rat hearts were exposed to 20 minutes of saline, 1 μ M TAT-only or 200 nM or 1 μ M TAT-HKII, followed by 15 minutes of ischemia and 30 minutes of reperfusion. TAT-HKII translocates HKII from the mitochondria. During the complete protocol, cardiac oxygen consumption (MVO 2), cardiac function (rate pressure product [RPP]), and ROS (DHE fluorescence) were measured. Mitochondrial oxygen tension was measured by using protoporphyrin IX-delayed fluorescence before and after peptide treatment. During reperfusion, necrosis was measured by lactate dehydrogenase (LDH) activity in effluent.In the 1 μ M TAT-HKII hearts, MV−O/RPP was increased and mitochondrial oxygen tension was reduced during peptide treatment. This was not observed after treatment with 200 nM TAT-HKII nor in the control groups. During reperfusion, MVO 2 /RPP was increased in both treatment groups. ROS production increased in both TAT-HKII groups during ischemia and reperfusion. These effects were accompanied by increased LDH activity during reperfusion in both TAT-HKII treated groups. No LDH activity was observed in control groups.Reduction of mtHKII by TAT-HKII treatment turns reversible ischemia into irreversible ischemia. This might be caused by increased ROS production during ischemia and reperfusion, and increased oxygen consumption.
Journal of Cardiovascular Pharmacology, Apr 1, 2003
In order to investigate the consequences of stress susceptibility on vascular function, the autho... more In order to investigate the consequences of stress susceptibility on vascular function, the authors assessed the respective contributions of nitric oxide (NO), prostanoids, and endothelium-derived hyperpolarizing factor to the vascular tone in rats with a constitutionally determined high and low susceptibility to behavioral stressors. In mesenteric resistance arteries mounted in a small vessel myograph and precontracted with l-phenylephrine hydrochloride (phenylephrine), the NO-synthase inhibitor N omega-nitro-l-arginine (l-NOARG, 100 microM) elicited a smaller increase of vascular tone in apomorphine-susceptible (APO-SUS) rats (P < 0.01). Addition of indomethacin (10 microM), in the presence of l-NOARG, resulted in a smaller decrease of vascular tone in APO-SUS rats (P < 0.01). Although acetylcholine-induced relaxation in phenylephrine-precontracted arteries was not different (P > 0.1), the individual components contributing to this relaxation were. In arteries precontracted with 125 mM K+, and incubated with indomethacin, acetylcholine-induced relaxation was not significantly different (pEC(50) and E(max): P > 0.1). Sensitivity (pEC(50): P < 0.05) and maximum relaxation (E(max): P < 0.001) to sodium nitroprusside, in the presence of 125 mM K+, was more pronounced in APO-SUS rats. In phenylephrine-precontracted arteries, in the presence of l-NOARG and indomethacin, maximum relaxation to ACh was reduced in APO-SUS rats (E(max): P < 0.05). This study showed that in rats with a high susceptibility to stressors, the contribution of NO to vascular tone was decreased as was the ratio of vasoconstrictor and vasodilator cyclooxygenase products in alpha-adrenergic precontracted arteries. End-organ sensitivity to NO was greater in APO-SUS rats, possibly due to up-regulation. Moreover, the contribution of endothelium-derived hyperpolarizing factor to acetylcholine-induced vasodilation was reduced in APO-SUS rat arteries.
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Papers by Gerard Rongen