Platelet mitochondrial dysfunction is evident in type 2 diabetes in association with modifications of mitochondrial anti-oxidant stress proteins.

OBJECTIVE: Mitochondrial dysfunction and oxidative stress in insulin responsive tissues is implicated in the pathogenesis of type 2 diabetes. Whether these perturbations extend to other tissues and contribute to their pathophysiology is less well established. The objective of this study was to investigate platelet mitochondria to evaluate whether type 2 diabetes associated mitochondrial dysfunction is evident in circulating cells. METHOD: A pilot study of mitochondrial respiratory function and proteomic changes comparing platelets extracted from insulin sensitive (n=8) and type 2 diabetic subjects (n=7). RESULTS: In-situ platelet mitochondria show diminished oxygen consumption and lower oxygen-dependent ATP synthesis in diabetic vs. control subjects. Mass spectrometric identification and confirmatory immunoblot analysis identifies induction of the mitochondrial anti-oxidant enzymes superoxide dismutase 2 and thioredoxin-dependent peroxide reductase 3 in platelets of diabetic subjects. As oxidative stress upregulates anti-oxidant enzymes we assessed mitochondrial protein carbonylation as an index of oxidative-stress. Platelets of diabetic subjects exhibit significantly increased protein carbonylation compared to controls. CONCLUSIONS: As platelets are anuclear fragments of megakaryocytes, our data suggest that the bone marrow compartment in type 2 diabetic subjects is exposed to increased mitochondrial oxidative stress with upregulation of nuclear-encoded antioxidant mitochondrial enzymes. This 'stress-signature' in platelets of diabetic subjects is associated with a diminution of their mitochondrial contribution to energy production and support that mitochondrial perturbations in type 2 diabetes extends beyond the classical insulin responsive tissues. Platelets, as "accessible human tissue", may be useful to measure the mitochondrial modulatory effects of emerging anti-diabetic therapeutics.

[Sphingolipid signaling: a potential pathway for TNF-alpha induced preconditioning]. / TNF alpha mime un effet de préconditionnement ischémique: un rôle pour la voie des sphingolipides.

Although cytokine activation has long been recognized to associate with cardiac ischemia and reperfusion, the concept that these cytokines may enhance some cardioprotective mechanisms has only recently been considered. Ischemic preconditioning is a biologic phenomenon that activates innate cytoprotective programs in the heart. Ischemic preconditioning has been described where a transient non-lethal ischemic "trigger" or endogenous molecules produced/released by ischemia enables the tissue to become more resistant/tolerant to subsequent potentially lethal ischemia. The mechanisms and signalling events involved in this cytoprotective program still remain obscure. Recently, it has been suggested that cytokine activation including tumour necrosis factor (TNF alpha) may play a key role in the preconditioned phenotype. Moreover, new studies have given the evidence that the exploration of cytokine-activated sphingolipid signalling pathways may enhance our understanding of the preconditioning program.

Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling.

The "metabolic cocktail" comprising glucose-insulin-potassium administrated at reperfusion reduces infarct size in the in vivo rat heart. We propose that insulin is the major component mediating this protection and acts via Akt prosurvival signaling. This hypothesis was studied in isolated perfused rat hearts (measuring infarct size to area of risk [%]) subjected to 35 minutes regional myocardial ischemia and 2 hours reperfusion. Insulin administered at the onset of reperfusion attenuated infarct size by >/=45% versus control hearts (P<0.001). Insulin-mediated cardioprotection was found to be independent of the presence of glucose at reperfusion. Moreover, the cell survival benefit of insulin is temporally dependent, in that insulin administration from the onset of reperfusion and maintained for either 15 minutes or for the duration of reperfusion reduced infarct size. In contrast, protection was abrogated if insulin administration was delayed until 15 minutes into reperfusion. Pharmacological inhibition of both upstream and downstream signals in the Akt prosurvival pathway abolished the cardioprotective effects of insulin. Here coadministration of insulin with the tyrosine kinase inhibitor lavendustin A, the phosphatidylinositol3-kinase (PI3-kinase) inhibitor wortmannin, and mTOR/p70s6 kinase inhibitor rapamycin abolished cardioprotection. Steady-state levels of activated/phosphorylated Akt correlated with insulin administration. Finally, downstream prosurvival targets of Akt including p70s6 kinase and BAD were modulated by insulin. In conclusion, insulin administration at reperfusion reduces myocardial infarction, is dependent on early administration during reperfusion, and is mediated via Akt and p70s6 kinase dependent signaling pathway. Moreover, BAD is maintained in its inert phosphorylated state in response to insulin therapy.

Ischemic and pharmacological preconditioning in Girardi cells and C2C12 myotubes induce mitochondrial uncoupling.

Pharmacological uncoupling of mitochondrial oxidation from phosphorylation promotes preconditioning-like cardioprotection in the isolated rat heart. We hypothesized that modest mitochondrial uncoupling may be a critical cellular event in orchestrating preconditioning. Human-derived Girardi cells and murine C2C12 skeletal myotubes were preconditioned using simulated ischemia, adenosine, and diazoxide. Cell viability after 6 hours of simulated ischemia was measured using lactate dehydrogenase release and propidium iodide uptake. Mitochondrial inner membrane potential (DeltaPsim) was investigated by flow cytometry, cellular ATP by recombinant firefly-luciferase bioluminescence, and cellular oxygen consumption using oximetry. Preconditioning enhanced cell viability with attenuation of lactate dehydrogenase release (>/=30%, P<0.05 versus ischemic controls) and a reduction in propidium iodide uptake by >/=26% versus ischemic controls after simulated ischemia in both cell lines. In Girardi cells, preconditioning induced the following phenotype immediately before index ischemia: (1) decreased DeltaPsim (JC-1: simulated ischemia 90+/-3%, adenosine 82+/-7%, diazoxide 87+/-4%, versus control 100%, P<0.05); (2) attenuation in cellular ATP levels (CTL 0.21+/-0.03 nmol/L ATP/microg protein, simulated ischemia 0.12+/-0.02, adenosine 0.15+/-0.02, diazoxide 0.11+/-0.02, P<0.05); and (3) enhanced cellular oxygen consumption (control 2.3+/-0.1 nmol/L oxygen/min/1x10(6) cells, simulated ischemia 3.1+/-0.1, adenosine 3.1+/-0.3, diazoxide 2.6+/-0.2, P<0.05). Cytoprotection, mitochondrial depolarization, and enhanced oxygen consumption were attenuated by the putative mitochondrial K(ATP)-channel antagonist 5-hydroxydecanoate. The uncoupled phenotype in response to preconditioning was similarly observed in C2C12 myotubes. The present study suggests that modest mitochondrial uncoupling represents a unifying cellular response which may be important in directing preconditioning-mediated cytoprotection.

TNF alpha is required for hypoxia-mediated right ventricular hypertrophy.

Hypoxia has been shown to activate the pleiotropic cytokine TNFalpha in the lung. TNFalpha in turn, is known to induce pulmonary vasoconstriction. Additional effects of this cytokine in hypoxia mediated cardiopulmonary remodeling are poorly understood. To further evaluate the role of TNFalpha in chronic hypoxia we exposed TNFalpha null (TNFalpha-/-) and wild-type mice to three weeks of hypobaric hypoxia (10% O2). Equivalent erythocytosis (Hematocrit increased by > 40%) developed in both genetic backgrounds. In contrast, right ventricular systolic pressure increased in response to three weeks of hypoxia in the wild-type mice (> or = 75%), yet was unaltered in the TNFalpha-/- mice. Concomitantly right ventricular hypertrophy was attenuated in the TNFalpha-/- mice (35 +/- 5% increase) when compared to wild-type mice (124 +/- 6% increase p < 0.001, n > or = 20). Interestingly in both strains the lung wet weights increased to a similar degree in response to hypoxia. In conclusion, our data demonstrate that TNFalpha is an integral autocoid in chronic hypoxia mediated right ventricular hypertrophy. Moreover, additional components of cardiopulmonary remodeling may be regulated by TNFalpha signaling as suggested by the negligible right ventricular systolic pressure response to hypoxia in the absence of TNFalpha.

Enhanced angiotensin II activity in heart failure: reevaluation of the counterregulatory hypothesis of receptor subtypes.

There are strong data favoring the pathogenic role of angiotensin II type 1 receptor (AT(1)) activation with subsequent promotion of myocyte growth and cardiac fibrosis in the development of cardiac hypertrophy and heart failure. An emerging hypothesis suggests that the activity of the angiotensin II type 2 receptor (AT(2)) may counterregulate AT(1) receptor effects during cardiac development and during the evolution of cardiac hypertrophy and heart failure. In this review, we examine the potential role of AT(2) activity in the context of this hypothesis. In contrast to the counterregulatory hypothesis, studies in mice with an overabundance of, or a deficiency in, the AT(2) receptor do not suggest that AT(2) signaling is essential for cardiac development. Moreover, the proposed antigrowth effects of AT(2) receptor signaling in pathological cardiac hypertrophy could not be shown in two mice models both deficient in AT(2) receptors. The role of AT(2) receptor signaling in cardiac fibrosis is, however, still debatable because of conflicting data in the same two studies. In angiotensin II-evoked apoptosis in cardiomyocytes, the proposed proapoptotic role of AT(2) activity could not be confirmed. Furthermore, in the progression from the bench to bedside, the results of two large clinical trials in heart failure, namely ELITE II and Val-HeFT, can be explained without ascribing a major protective role to the unopposed activity of the AT(2) receptor in the failing myocardium. In this review, we conclude that the collective evidence does not strongly support a net beneficial effect of AT(2) stimulation in the diseased myocardium.

Delta opioid receptor stimulation mimics ischemic preconditioning in human heart muscle.

OBJECTIVES: The objective of this study was to examine whether the delta (delta) opioid receptor isoform is expressed in the human heart and whether this receptor improves contractile function after hypoxic/reoxygenation injury. BACKGROUND: Delta opioid receptor agonists mimic preconditioning (PC) in rat myocardium, corresponding to known cardiac delta opioid receptor expression in this species. METHODS: The messenger RNA transcript encoding the delta opioid receptor was identified in human atria and ventricles. To evaluate the cardioprotective role of the opioid receptor, human atrial trabeculae from patients undergoing coronary bypass grafting were isolated and superfused with Tyrode's solution. A control group underwent 90 min of simulated ischemia and 120 min of reoxygenation. A second group was preconditioned with 3 min simulated ischemia and 7 min reoxygenation. Additional groups included: superfusion with the delta receptor agonist (DADLE) (10 nM), with the delta receptor antagonist naltrindole (10 nM) and with the mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5HD) (100 microM) either with or without PC, respectively. A final group was superfused with 5HD before DADLE. The end point used was percentage of developed force after 120 min of reoxygenation. RESULTS: Results, expressed as means +/- SEM, were: control = 32.6 + 3.8%; PC = 50.5% + 1.8*; DADLE = 46.0+/-3.9%*; PC + naltrindole = 25.5+/-3.9%; naltrindole alone = 25.5+/-4.3%; 5HD + PC = 28.9+/-7.4%; 5HD alone = 24.1+/-3.0%; 5HD + DADLE = 26.9+/-4.4% (*p < 0.001 vs. controls). CONCLUSIONS: Human myocardium expresses the delta opioid receptor transcript. Stimulation of this receptor appears to protects human muscle from simulated ischemia, similar to PC, and via opening of the mitochondrial K(ATP) channel.

Mechanisms whereby glucose deprivation triggers metabolic preconditioning in the isolated rat heart.

Transient glucose deprivation of the heart [GLU (-)] confers a preconditioning-like protection against subsequent ischemic/reperfusion (I/R). The mechanisms involved remain unclear. We hypothesized that GLU (-) would induce the classic ischemic preconditioning activated signaling cascade. Potential metabolic consequences and putative cell signaling events induced by transient glucose deprivation were evaluated as candidate mediators of this cardioprotection. Isolated glucose-perfused rat hearts were subjected to 30 min global ischemia followed by 30 min reperfusion (index I/R). Cardiac contractile recovery following I/R was used as the functional end-point in these studies. Metabolic preconditioning was stimulated by 15 min GLU (-) followed by 10 min glucose repletion prior to the index I/R. The potential metabolic consequences of GLU (-) were evaluated by using excess octanoate (11 mM OCT Hi) or 11 mM 2-deoxy-D-glucose (2-DG) in place of GLU (-) and by combining GLU (-) with fuels known to inhibit glycolysis supply (20 mM pyruvate or 1 mM octanoate, OCT Lo). The roles of alpha-adrenoceptors, beta-adrenoceptors, adenosine receptors, protein kinase C (PKC) and mitochondrial K(ATP) channels were investigated using inhibitors prazosin (10 microM), propranolol (10 microM), 8-(p-sulfophenyl) theophylline, (SPT 100 microM), chelerythrine (CHEL 10 microM) and 5-hydroxydecanoate (5 HD 100 microM) respectively. GLU (-) increased mechanical recovery (59.8 +/- 4.0 vs. 32.3 +/- 4.7%; p < 0.01). Protection was abolished by pyruvate 26.6 +/- 3.1; SPT 36.6 +/- 3.0; CHEL 35 +/- 4.8 or 5 HD 23.8 +/- 3.3%. In a separate set of experiments, the specificity of SPT in this model was tested by preconditioning with adenosine (100 microM) (34.7 +/- 4 vs. control 16.8 +/- 1.3%, p = 0.01) and blocking this protection with the same dose of SPT (16.3 +/- 1 .5%) used in the GLU (-) studies. Protection was unaltered by prazosin (50.2 +/- 3.3%), propranolol (55.5 +/- 4.0%), or OCT Lo (50.2 +/- 2.5%). Protection was not mimicked by OCT Hi (35.6 +/- 3.8%) or 2-DG (34 +/- 4.3%). Transient glucose deprivation does not seem to achieve preconditioning-like cardioprotection by decreased glycolysis. Rather, the signal system may involve enhanced adenosine release, PKC, and activation of the mitochondrial K(ATP) channel.

Dinitrophenol, cyclosporin A, and trimetazidine modulate preconditioning in the isolated rat heart: support for a mitochondrial role in cardioprotection.

BACKGROUND: Recent studies have postulated that mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel activation may modulate mitochondrial function with the resultant induction of a preconditioning phenotype in the heart. We hypothesized that the modulation of mitochondrial homeostasis might confer preconditioning-like cardioprotection. METHODS: We used a model of regional ischemia in Langendorff-perfused isolated rat hearts. Short-term administration of 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation and cyclosporin A (CSA), an inhibitor of mitochondrial respiration, was used in an attempt to elicit preconditioning-like cardioprotection. The anti-ischemic drug trimetazidine, known to attenuate CSA-induced disruption in mitochondrial function, and the mitoK(ATP) channel blocker 5-hydroxydecanoic acid (5-HD) were used to inhibit the effects of DNP and CSA. Finally, we studied the effect of trimetazidine on adenosine-induced and ischemic preconditioning. Risk zone and infarct size were measured and expressed as a percentage of the risk zone (I/R ratio). RESULTS: DNP, CSA and adenosine pretreatment reduced infarct size (I/R ratio: DNP 9.0+/-2.4%, CSA 12.5+/-1.4%, adenosine 11.9+/-3.6%, all P<0.001 vs. control, 30.2+/-1.3%) similarly to ischemic preconditioning (9.5+/-0.6%, P<0.001 vs. control). Trimetazidine limited the effect of ischemic preconditioning (22.2+/-2.0%, P<0.001 vs. ischemic preconditioning) and completely reversed the DNP, CSA, and the adenosine-mediated reduction in infarct size. 5-HD abolished the effect of ischemic preconditioning and CSA. CONCLUSION: DNP and CSA trigger preconditioning-like cardioprotection in the isolated rat heart. Trimetazidine, a known mitochondrial 'protector', attenuated both drug-induced and ischemic preconditioning. These data support the hypothesis that modulation of mitochondrial homeostasis may be a common downstream cellular event linking different triggers of preconditioning.

Insulin administered at reoxygenation exerts a cardioprotective effect in myocytes by a possible anti-apoptotic mechanism.

The metabolic cocktail of glucose-insulin-potassium (GIK) has been shown to reduce mortality in humans and reduce infarct size in the rat when administered from the onset of reperfusion following an ischemic insult. The mechanisms underlying GIK mediated cardioprotection are, however, still unclear. Recent data implicates insulin "alone" as the major protagonist of cardioprotection when administered at the time of reperfusion. We have therefore begun to investigate an insulin activated signalling pathway and the putative role of apoptosis in this insulin-induced cardioprotection. Simulated ischemia and reoxygenation were induced in rat neonatal cardiocyte experiments. The administration of insulin [0.3 mU/ml] at the moment of reoxygenation (Ins(R)) enhanced myocardial cell viablility as assessed by trypan blue exclusion compared to vehicle alone treated control myocytes (Ins(R)50+/-2%v controls 70+/-1%, P<0.001). This insulin-mediated cardioprotection was due, in part to a reduction in myocyte apoptosis as measured by TUNEL (Ins(R)29+/-2%v controls 49+/-3%, P<0.001) and Annexin V staining (Ins(R)34+/-2%v controls 65+/-3%, P<0.001). These cardioprotective and anti-apoptotic effects of insulin were completely abolished by the tyrosine kinase inhibitor lavendustin A and by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin. Thus, we conclude that the early administration of insulin appears to be an effective modality to reduce reoxgygenation injury in cardiocytes, in part, via the attenuation of ischemia/reoxygenation-induced apoptosis. Moreover, the cardioprotective and anti-apoptotic effects of insulin are mediated via tyrosine kinase and PI3-kinase signalling pathways.

Coordinate regulation of metabolic enzyme encoding genes during cardiac development and following carvedilol therapy in spontaneously hypertensive rats.

Fuel substrate utilization is highly regulated during cardiac development and with the onset of cardiac hypertrophy. Glucose and lactate are the predominant fuel substrates utilized during cardiac development. Postnatally, a switch occurs so that fatty acids become the chief energy substrate in the nonfed adult mammalian heart. A reversion back towards fetal energy metabolism occurs with the development of cardiac hypertrophy. To evaluate the role of this substrate preference switch in the development of cardiac hypertrophy, the molecular regulation directing these switches is being explored. Thus, we have begun by defining the temporal expression patterns of genes encoding key rate-controlling enzymes directing major fuel substrate metabolism during cardiac development, with pressure-overload-induced cardiac hypertrophy, and following antihypertensive therapy in spontaneously hypertensive rats. The genes encoding the fatty acid and adult enriched rate-controlling glycolytic enzymes are expressed at low levels in the fetal and neonatal rat heart. The genes encoding these enzymes are significantly and coordinately upregulated (> or = 70%) in adult rat hearts compared to the fetal expression patterns. A reciprocal and coordinate downregulation (> or = 40% reduction) of the fatty acid and adult enriched glycolytic enzyme encoding genes are observed with the induction of pressure-overload-induced hypertrophy in spontaneously hypertensive rats compared to Wistar-Furth normotensive control rats. Antihypertensive therapy with carvedilol, a vasodilating alpha- and beta-adrenoreceptor antagonist, attenuates this reversion of the metabolic gene expression pattern towards fetal levels compared to placebo-treated littermate controls. This coordinate developmental and hypertrophy-induced regulation of genes that encode enzymes controlling both fatty acid and glycolytic catabolic pathways in the heart implicates potential mutual/overlapping regulatory signaling proteins within their gene regulatory programs. These gene regulatory pathways need to be identified and modulated in order to characterize the functional role of fuel substrate metabolism in cardiac development and with the induction of cardiac hypertrophy.

The energy substrate switch during development of heart failure: gene regulatory mechanisms (Review).

During cardiac hypertrophy and in the failing heart, the chief myocardial energy substrate switches from fatty acids to glucose. In this review, we describe recent progress in the elucidation of the molecular regulatory events involved in the dramatic downregulation of the expression of fatty acid utilization enzymes during development of cardiac hypertrophy and failure. Much of this work has focused on the gene encoding medium-chain acyl-CoA dehydrogenase (MCAD), which catalyzes a pivotal step in the mitochondrial fatty acid -oxidation (FAO) cycle. In vivo ventricular pressure overload studies performed in mice transgenic for human MCAD promoter fragments linked to reporter genes have shown that transcription is markedly downregulated within seven days of pressure overload. The temporal pattern of this alteration in MCAD gene expression has also been characterized in a rat model of progressive pressure overload-induced left ventricular hypertrophy (LVH) and heart failure (HF) [SHHF/Mcc-facp (SHHF) rat]. MCAD mRNA levels are downregulated (>70%) during both the LVH and HF stages in the SHHF rats compared with controls. In contrast, the activity and immunodetectable levels of MCAD enzyme were not significantly reduced until the HF stage, indicating additional compensatory control at the translational or post-translational levels in the hypertrophied but non-failing ventricle. FAO enzyme expression was also shown to be downregulated in human subjects with dilated cardiomyopathy compared to age-matched controls. Taken together, these results have identified a gene regulatory program that is involved in the alterations in myocardial energy substrate utilization in the failing heart. The temporal correlation of diminished enzyme expression with onset of heart failure suggests that this alteration in lipid metabolism may play a role in the pathogenesis of pressure-overload induced heart failure. This gene regulatory pathway should be a useful target for experimental studies aimed at the molecular pathogenesis of the transition from stable cardiac hypertrophy to overt heart failure.

A role for Sp and nuclear receptor transcription factors in a cardiac hypertrophic growth program.

During cardiac hypertrophy, the chief myocardial energy source switches from fatty acid beta-oxidation (FAO) to glycolysis-a reversion to fetal metabolism. The expression of genes encoding myocardial FAO enzymes was delineated in a murine ventricular pressure overload preparation to characterize the molecular regulatory events involved in the alteration of energy substrate utilization during cardiac hypertrophy. Expression of genes involved in the thioesterification, mitochondrial import, and beta-oxidation of fatty acids was coordinately down-regulated after 7 days of right ventricular (RV) pressure overload. Results of RV pressure overload studies in mice transgenic for the promoter region of the gene encoding human medium-chain acyl-CoA dehydrogenase (MCAD, which catalyzes a rate-limiting step in the FAO cycle) fused to a chloramphenicol acetyltransferase reporter confirmed that repression of MCAD gene expression in the hypertrophied ventricle occurred at the transcriptional level. Electrophoretic mobility-shift assays performed with MCAD promoter fragments and nuclear protein extracts prepared from hypertrophied and control RV identified pressure overload-induced protein/DNA interactions at a regulatory unit shown previously to confer control of MCAD gene transcription during cardiac development. Antibody "supershift" studies demonstrated that members of the Sp (Sp1, Sp3) and nuclear hormone receptor [chicken ovalbumin upstream promoter transcription factor (COUP-TF)/erbA-related protein 3] families interact with the pressure overload-responsive unit. Cardiomyocyte transfection studies confirmed that COUP-TF repressed the transcriptional activity of the MCAD promoter. The DNA binding activities and nuclear expression of Sp1/3 and COUP-TF in normal fetal mouse heart were similar to those in the hypertrophied adult heart. These results identify a transcriptional regulatory mechanism involved in the reinduction of a fetal metabolic program during pressure overload-induced cardiac hypertrophy.

Fatty acid oxidation enzyme gene expression is downregulated in the failing heart.

BACKGROUND: During the development of heart failure (HF), the chief myocardial energy substrate switches from fatty acids to glucose. This metabolic switch, which recapitulates fetal cardiac energy substrate preferences, is thought to maintain aerobic energetic balance. The regulatory mechanisms involved in this metabolic response are unknown. METHODS AND RESULTS: To characterize the expression of genes involved in mitochondrial fatty acid beta-oxidation (FAO) in the failing heart, levels of mRNA encoding enzymes that catalyze the first and third steps of the FAO cycle were delineated in the left ventricles (LVs) of human cardiac transplant recipients. FAO enzyme and mRNA levels were coordinately downregulated (> 40%) in failing human LVs compared with controls. The temporal pattern of this alteration in FAO enzyme gene expression was characterized in a rat model of progressive LV hypertrophy (LVH) and HF [SHHF/Mcc-facp (SHHF) rat]. FAO enzyme mRNA levels were coordinately downregulated (> 70%) during both the LVH and HF stages in the SHHF rats compared with controls. In contrast, the activity and steady-state levels of medium-chain acyl-CoA dehydrogenase, which catalyzes a rate-limiting step in FAO, were not significantly reduced until the HF stage, indicating additional control at the translational or post-translational levels in the hypertrophied but nonfailing ventricle. CONCLUSIONS: These findings identify a gene regulatory pathway involved in the control of cardiac energy production during the development of HF.

Autoantibody titers to oxidized low-density lipoprotein in patients with coronary atherosclerosis.

Oxidation of low-density lipoprotein (LDL) is considered to be the initial step in the atherosclerotic process. Autoantibodies to oxidized LDL (ox-LDL) have been detected in human serum. We used an enzyme-linked immunosorbent assay technique to measure autoantibody titers in 63 normal subjects and patients with coronary artery disease. Thirty-five patients underwent coronary angiography for suspected coronary artery disease. Patients were divided into the following categories: group 1, 20 healthy young volunteers; group 2, 8 patients age-matched to the catheterization patients; group 3, 10 patients with normal coronary angiograms; and group 4, 25 patients with angiographic coronary artery disease. Autoantibody titers to ox-LDL were group 1, 0.142 +/- 0.023; group 2, 0.197 +/- 0.039; group 3, 0.183 +/- 0.038; and group 4, 0.340 +/- 0.026. There was no statistical difference among groups 1, 2, and 3, but the difference between these groups and group 4 was highly significant (p < 0.05). This study demonstrates that (1) autoantibodies to ox-LDL can be detected in normal subjects and in patients with abnormal coronary angiograms and (2) significantly higher titers of autoantibodies to ox-LDL were seen in patients with angiographic evidence of coronary artery disease.

Effect of antioxidant vitamins on low density lipoprotein oxidation and impaired endothelium-dependent vasodilation in patients with hypercholesterolemia.

OBJECTIVES: The aims of this study were to determine whether antioxidant vitamins could reduce the susceptibility of low density lipoprotein (LDL) to oxidation and improve endothelium-dependent vasodilator responsiveness in patients with hypercholesterolemia. BACKGROUND: Animals and humans with hypercholesterolemia have exhibited impaired endothelium-dependent vasodilation. In vitro studies suggest that oxidatively modified LDL can impair nitric oxide production. METHODS: Forearm blood flow was measured with strain gauge plethysmography and brachial artery drug infusions in 19 patients, aged 52 +/- 9 years, with hypercholesterolemia (mean +/- SD total cholesterol 283 +/- 22 mg/dl, LDL 197 +/- 31 mg/dl) and in 14 subjects, aged 48 +/- 8 years, with normal cholesterol levels (total cholesterol 169 +/- 20 mg/dl, LDL 102 +/- 25 mg/dl). Acetylcholine (7.5, 15 and 30 micrograms/min) was utilized as an endothelium-dependent vasodilator, and sodium nitroprusside (0.8, 1.6 and 3.2 micrograms/min) was used to test endothelium-independent vasodilation. Oxidative susceptibility of LDL was measured by a spectrophotometric assay of conjugated diene production after the addition of copper chloride. Hypercholesterolemic patients then received daily antioxidant vitamin supplements (beta-carotene [30 mg], ascorbic acid [vitamin C] [1,000 mg], vitamin E [800 IU]) for 1 month, with repeat measurement of both forearm blood flow responsiveness to the same agonists and LDL oxidizability. RESULTS: The maximal flow in response to acetylcholine was impaired in patients compared with that in normal subjects (9.8 +/- 7.8 vs. 15.9 +/- 8.1 ml/min per 100 ml, p = 0.03), with similar maximal flow responses to sodium nitroprusside (9.5 +/- 4.2 vs. 9.0 +/- 2.8 ml/min per 100 ml, p = 0.72). After 1 month of vitamin therapy, the onset of LDL oxidation was prolonged over baseline measurements by 71 +/- 67%, and the maximal rate of oxidation was decreased by 26 +/- 25% (both p < 0.001). However, the maximal forearm blood flow response to acetylcholine remained unchanged from baseline values (maximal flow after acetylcholine 9.0 +/- 6.2 vs. 9.8 +/- 7.8 ml/min per 100 ml, p = 0.57). This study had 80% power (alpha = 0.05) to exclude a 45% increase over baseline value in acetylcholine-stimulated flow during vitamin therapy. CONCLUSIONS: Although 1 month of administration of antioxidant vitamin supplements in hypercholesterolemic patients reduced the susceptibility of LDL to oxidation, impairment in endothelial function remained unaltered. The use of nonvitamin antioxidants or concomitant reduction in LDL levels, as well as more sensitive techniques for measuring vascular responsiveness, may be required to show a beneficial effect on endothelial vasodilator function.

Oestrogen and inhibition of oxidation of low-density lipoproteins in postmenopausal women.

Oxidative modification of low-density lipoprotein (LDL) may be atherogenic. We studied the time of onset of LDL oxidation (lag) in 18 postmenopausal women before and after intraarterial infusion of 17 beta-oestradiol, after 3 weeks' patch administration in 12 of these women, and 1 month after discontinuation in 10. The lag increased from baseline after acute infusion (from 134 [SD41] to 167 [36] min, p = 0.01) and after the patch (132 [31] to 178 [45] min, p = 0.009). After discontinuation of oestradiol, the lag returned to baseline. This study shows an antioxidant effect of physiological levels of 17 beta-oestradiol, which may contribute to an anti-atherogenic action.