Luc Rochette

Diabetes, oxidative stress and therapeutic strategies

BACKGROUND Diabetes has emerged as a major threat to health worldwide. SCOPE OF REVIEW The exact mechanisms underlying the disease are unknown; however, there is growing evidence that excess generation of reactive oxygen species (ROS), largely due to hyperglycemia, causes oxidative stress in a variety of tissues. Oxidative stress results from either an increase in free radical production, or a decrease in endogenous antioxidant defenses, or both. ROS and reactive nitrogen species (RNS) are products of cellular metabolism and are well recognized for their dual role as both deleterious and beneficial species. In type 2 diabetic patients, oxidative stress is closely associated with chronic inflammation. Multiple signaling pathways contribute to the adverse effects of glucotoxicity on cellular functions. There are many endogenous factors (antioxidants, vitamins, antioxidant enzymes, metal ion chelators) that can serve as endogenous modulators of the production and action of ROS. Clinical trials that investigated the effect of antioxidant vitamins on the progression of diabetic complications gave negative or inconclusive results. This lack of efficacy might also result from the fact that they were administered at a time when irreversible alterations in the redox status are already under way. Another strategy to modulate oxidative stress is to exploit the pleiotropic properties of drugs directed primarily at other targets and thus acting as indirect antioxidants. MAJOR CONCLUSIONS It appears important to develop new compounds that target key vascular ROS producing enzymes and mimic endogenous antioxidants. GENERAL SIGNIFICANCE This strategy might prove clinically relevant in preventing the development and/or retarding the progression of diabetes associated with vascular diseases.

Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: Possible therapeutic targets?

Nitric oxide (NO) is synthetized enzymatically from l-arginine (l-Arg) by three NO synthase isoforms, iNOS, eNOS and nNOS. The synthesis of NO is selectively inhibited by guanidino-substituted analogs of l-Arg or methylarginines such as asymmetric dimethylarginine (ADMA), which results from protein degradation in cells. Many disease states, including cardiovascular diseases and diabetes, are associated with increased plasma levels of ADMA. The N-terminal catalytic domain of these NOS isoforms binds the heme prosthetic group as well as the redox cofactor, tetrahydrobiopterin (BH(4)) associated with a regulatory protein, calmodulin (CaM). The enzymatic activity of NOS depends on substrate and cofactor availability. The importance of BH(4) as a critical regulator of eNOS function suggests that BH(4) may be a rational therapeutic target in vascular disease states. BH(4) oxidation appears to be a major contributor to vascular dysfunction associated with hypertension, ischemia/reperfusion injury, diabetes and other cardiovascular diseases as it leads to the increased formation of oxygen-derived radicals due to NOS uncoupling rather than NO. Accordingly, abnormalities in vascular NO production and transport result in endothelial dysfunction leading to various cardiovascular disorders. However, some disorders including a wide range of functions in the neuronal, immune and cardiovascular system were associated with the over-production of NO. Inhibition of the enzyme should be a useful approach to treat these pathologies. Therefore, it appears that both a lack and excess of NO production in diseases can have various important pathological implications. In this context, NOS modulators (exogenous and endogenous) and their therapeutic effects are discussed.

Creatine Kinase Is the Main Target of Reactive Oxygen Species in Cardiac Myofibrils

Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100-treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca(2+)-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pre-treated with 1 mmo1/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 mumol/L per 30 mIU/mL) or H2O2. pMgATP-rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the absence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03 +/- 0.17 (n = 21) to 4.22 +/- 0.22 (n = 4) after 25 minutes of incubation in the presence one of 30 mIU/mL. XO and 100 mumol/L xanthine or to 4.04 +/- 0.1 (n = 11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced on increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.

Systemic Free Radical Activation Is a Major Event Involved In Myocardial Oxidative Stress Related to Cardiopulmonary Bypass

Background Cardiopulmonary bypass (CPB) can induce deleterious effects that could be triggered in part by radical oxygen species; however, their involvement in the course of surgery has been elusive. The aim of this study was to evaluate the time course and origin of radical oxygen species release, myocardial or not, in patients undergoing coronary artery surgery involving CPB. Methods Blood samples were taken from periphery and coronary sinus of patients during CPB, and oxidative stress was evaluated by direct and indirect approaches. Direct detection of alkyl and alkoxyl radicals was assessed by electron spin resonance spectroscopy associated with the spin-trapping technique using &agr;-phenyl-N-tert-butylnitrone. Results The authors showed that the spin adduct concentration was not influenced by anesthesia and pre-CPB surgery. A rapid systemic increase of plasma spin adduct concentration occurred after starting CPB, and it stayed at a high concentration until the end of CPB. At the beginning of reperfusion period, radical oxygen species release was accelerated in the coronary sinus; however, it was not significant. A positive correlation was found between &agr;-phenyl-N-tert-butylnitrone adduct concentrations and (1) the duration of CPB and (2) concentration of postoperative creatine phosphokinase of muscle band (CPK MB). Plasma vitamin E and C, ascorbyl radical, uric acid, thiol, plasma antioxidant status, and thiobarbituric acid reacting substances were also measured but did not give relevant indications, except for uric acid, which seemed to be consumed by the heart during reperfusion. Conclusion The results indicate that a systemic production of free radicals occurs during CPB that may overwhelm the production related to reperfusion of the ischemic heart. This systemic oxidative stress is likely to participate in secondary myocardial damage.

Relation Between Body Mass Index, Waist Circumference, and Death After Acute Myocardial Infarction

Background— An elevated body mass index (BMI) has been reported to be associated with a lower rate of death after acute myocardial infarction (AMI). However, waist circumference (WC) may be a better marker of cardiovascular risk than BMI. We used data from a contemporary French population-based cohort of patients with AMI to analyze the impact of WC and BMI on death rates. Methods and Results— We evaluated 2229 consecutive patients with AMI. Patients were classified according to BMI as normal, overweight, obese, and very obese (BMI <25, 25 to 29.9, 30 to 34.5, and >35 kg/m2, respectively) and as increased waistline (WC >88/102 cm for women/men) or normal. Half of the patients were overweight (n=1044), and one quarter were obese (n=397) or very obese (n=128). Increased WC was present in half of the patients (n=1110). Increased BMI was associated with a reduced death rate, with a 5% risk reduction for each unit increase in BMI (hazard ratio, 0.95; 95% CI, 0.93 to 0.98; P<0.001). In contrast, WC as a continuous variable had no impact on all-cause death (P=0.20). After adjustment for baseline predictors of death, BMI was not independently predictive of death. The group of patients with high WC but low BMI had increased 1-year death rate. Conclusions— Neither BMI nor WC independently predicts death after AMI. Much of the inverse relationship between BMI and the rate of death after AMI is due to confounding by characteristics associated with survival. This study emphasizes the need to measure both BMI and WC because patients with a high WC and low BMI are at high risk of death.

Antioxidative properties of pyruvate and protection of the ischemic rat heart during cardioplegia.

Formation of oxygen free radicals during heart transplantation seems to be related to the alterations occurring during ischemia and reperfusion and could explain the short preservation time of donor hearts. The aim of our study was (a) to analyze the protective effects of pyruvate during cold cardioplegia and ischemia/reperfusion sequence, and (b) to investigate in vitro the radical scavenging properties of this compound. After 30 min of perfusion, isolated working rat hearts were arrested by cardioplegic solution, stored 4 h in B21 solutions at 4 degrees C, and reperfused with Krebs-Henseleit buffer for 45 min. Pyruvate (2 mM) was added to Krebs-Henseleit, cardioplegic, and storage solutions, and functional parameters were recorded throughout the experiments. In a second part, control hearts and hearts treated with pyruvate were cannulated via the aorta and perfused for 30 min by the Langendorff method, arrested by cardioplegic solution, stored 4 h in B21 solutions at 4 degrees C, and reperfused for 45 min by the Langendorff method. Malonedialdehyde and alpha-tocopherol levels were determined on heart homogenate. In situ detection of apoptotic cells also was performed on tissue samples (left ventricle) at the end of the ischemia/reperfusion sequence. To demonstrate in vitro the antioxidant effects of pyruvate, we monitored (a) its hydroxyl radical scavenging properties by using electron paramagnetic resonance (EPR) spectroscopy, and (b) the decrease of fluorescence of allophycocyanin, in the presence of a Fenton system (H2O2/Cu2+). Ischemia for 4 h, followed by myocardial reperfusion, resulted in substantially reduced mechanical function. Hearts subjected to this ischemia and pretreated with pyruvate showed a significant improvement in the function recovery. After the ischemia/reperfusion protocol, no significant decrease of malonedialdehyde levels was shown on hearts treated with pyruvate. However, alpha-tocopherol levels were higher in the pyruvate group compared with the control group. At the end of the reperfusion period, levels of apoptotic cells were significantly lower in hearts treated with pyruvate compared with control hearts. EPR studies showed that pyruvate was an efficient hydroxyl scavenger, with a median inhibitory concentration (IC50) of 8 mM. The allophycocyanin assay also showed a dose-dependent effect of pyruvate against hydroxyl radicals. In conclusion, these findings showed that pyruvate could prevent reperfusion injuries in the isolated heart, probably by its antioxidative properties. The application of pyruvate may contribute to the preservation of hearts for organ transplantation.

Direct and indirect antioxidant properties of α-lipoic acid and therapeutic potential

Diabetes has emerged as a major threat to worldwide health. The exact mechanisms underlying the disease are unknown; however, there is growing evidence that the excess generation of reactive oxygen species (ROS) associated with hyperglycemia, causes oxidative stress in a variety of tissues. In this context, various natural compounds with pleiotropic actions like α-lipoic acid (LA) are of interest, especially in metabolic diseases such as diabetes. LA, either as a dietary supplement or a therapeutic agent, modulates redox potential because of its ability to match the redox status between different subcellular compartments as well as extracellularly. Both the oxidized (disulfide) and reduced (di-thiol: dihydro-lipoic acid, DHLA) forms of LA show antioxidant properties. LA exerts antioxidant effects in biological systems through ROS quenching but also via an action on transition metal chelation. Dietary supplementation with LA has been successfully employed in a variety of in vivo models of disease associated with an imbalance of redox status: diabetes and cardiovascular diseases. The complex and intimate association between increased oxidative stress and increased inflammation in related disorders such as diabetes, makes it difficult to establish the temporal sequence of the relationship.

Antioxidant properties of an endogenous thiol: alpha-lipoic acid, useful in the prevention of cardiovascular diseases.

In the past few years, a growing interest has been given to the possible antioxidant functions of a natural acid, synthesized in human tissues: alpha-lipoic acid (ALA). Both the oxidized (disulfide) and reduced (dithiol: dihydrolipoic acid, DHLA) forms of ALA show antioxidant properties. ALA administered in the diet accumulates in tissues, and a substantial part is converted to DHLA via a lipoamide dehydrogenase. Commercial ALA is usually a racemic mixture of the R and S forms. Chemical studies have indicated that ALA scavenges hydroxyl radicals, hypochlorous acid, and singlet oxygen. ALA exerts antioxidant effects in biological systems not only through direct ROS quenching but also via transition metal chelation. ALA has been shown to possess a number of beneficial effects both in the prevention and treatment of diabetes in experimental conditions. ALA presents beneficial effects in the management of symptomatic diabetic neuropathy and has been used in this context in Germany for more than 30 years. In cardiovascular disease, dietary supplementation with ALA has been successfully employed in a variety of in vivo models: ischemia-reperfusion, heart failure, and hypertension. More mechanistic and human in vivo studies are needed to determine whether optimizing the dietary intake of ALA can help to decrease cardiovascular diseases. A more complete understanding of cellular biochemical events that influence oxidative damage is required to guide future therapeutic advances.

Studies by electron paramagnetic resonance of the importance of iron in the hydroxyl scavenging properties of ascorbic acid in plasma : Effects of iron chelators

Summary— Ascorbic acid is considered to be the most important antioxidant of plasma. Its oxidation leads to the ascorbyl free radical (AFR), detected by electron paramagnetic resonance (EPR) spectroscopy. The purpose of this study was to investigate by EPR the interaction of plasma AFR levels in different situations of oxidative stress. Our results showed that plasma AFR remains constant after rat feeding with vitamin C (5 mg or 50 mg per 100 g body weight). We also demonstrated that: (1) the ascorbyl free radical (AFR) level was increased after direct addition of iron Fe3+/EDTA to plasma, the optimal level was reached after addition of 8 μM Fe3+/EDTA (1:2); (2) this AFR production was associated with the formation of hydroxyl radicals. Iron chelators (deferrioxamine, a synthetic iron chelator and apotransferrine, a biological iron chelator) added just before the Fe3+/EDTA complex inhibited the increase of AFR signal induced by this complex. The scavenging effect of plasma was significantly correlated with the AFR production. Therefore, AFR, which is naturally present in plasma, could be used as an index of oxidative stress in which free radicals or adverse iron mobilisation are implicated.

Carbon monoxide: mechanisms of action and potential clinical implications

Small amounts of carbon monoxide (CO) are continuously produced in mammals. The intracellular levels of CO can increase under stressful conditions following the induction of HO-1 (heme oxygnase-1), a ubiquitous enzyme responsible for the catabolism of heme. Unlike nitric oxide, which is a free radical, CO does not contain free electrons but may be involved in oxidative stress. The carbonate radical has been proposed to be a key mediator of oxidative damage resulting from peroxynitrite production, likewise, the precursor of the carbonate radical anion being bicarbonate and carbon dioxide. We report herein some of the transcription factors and protein kinases involved in the regulation of vascular HO-1 expression. Beyond its widely feared toxicity, CO has revealed a very important biological activity as a signaling molecule with marked protective actions namely against apoptosis and endothelial oxidative damage. Abnormal metabolism and function of CO contribute to the pathogenesis and development of cardiovascular diseases. Important results have been reported in which CO and CO-releasing molecules (CO-RMs) prevent intimal hyperplasia by arresting hyperproliferative vascular smooth muscle cells and increased mobilization and recruitment of bone-marrow-derived progenitor cells. Clinical studies have demonstrated beneficial properties of CO-RMs in transplantation. The anti-inflammatory properties of CO and CO-RMs have been demonstrated in a multitude of animal models of inflammation, suggesting a possible therapeutic application for inflammatory diseases. The development of a technology concerning CO-RMs that controls the delivery and action of CO under different pathological conditions represents a major step forward in the development of CO-based pharmaceuticals with therapeutic applications.