Antioxidant therapy attenuates JNK activation and apoptosis in the remote noninfarcted myocardium after large myocardial infarction.

We hypothesized that the concomitant occurrence of increased oxidative stress, JNK activation, and myocyte apoptosis in the remote myocardium (RM) following a large myocardial infarction (MI) are causally related. Three days following coronary ligation, rats were randomized to treatment with probucol and PDTC (MI-T) or vehicle (MI). Control rats (C) underwent sham operation. At 7 weeks, TBARS assay showed increased level of lipid-peroxidation within the RM in the MI group vs C, which was completely inhibited in the MI-T group. Similarly, Western blot analysis showed a twofold increase in p-JNK in the MI group, vs C, which was attenuated in MI-T, a result confirmed by a JNK-kinase activity. Furthermore, apoptosis was increased within the RM in MI vs C, while this was inhibited in MI-T. We conclude that long-term antioxidant therapy with probucol and PDTC attenuates oxidative stress, JNK activation, and myocyte apoptosis within the RM after large MI.

Antioxidants attenuate myocyte apoptosis in the remote non-infarcted myocardium following large myocardial infarction.

OBJECTIVE: Increased oxidative stress and myocyte apoptosis co-exist in the remote non-infarcted myocardium (RM) following a large myocardial infarction. We proposed that these phenomena are causally related. METHODS AND RESULTS: On day 3 after induction of myocardial infarction, Sprague-Dawley rats were randomized to receive probucol and pyrrolidine dithiocarbamate (MI-T), or vehicle only (MI) for 7 weeks. Control rats (C) received vehicle. At 7 weeks, lipidperoxidation within the RM was assessed by measuring thiobarbituric acid reactive substances, which were significantly increased in MI vs. C, while MI-T was not different from C. There was a significant increase in cardiac myocytes positive for in situ TdT-UTP nick-end labeling within the RM in MI vs. C, which was inhibited in MI-T. Furthermore, internucleosomal DNA fragmentation was clearly demonstrated on agarose gels from RM in the MI group, while it was much less apparent on gels from RM in the C and MI-T groups. Western blot analysis showed a significant increase in p53, Bax and caspase-3 protein expression within the RM of MI vs. C, all of which were inhibited in the MI-T group. Furthermore, there was evidence for an increase in caspase-3 activity within the RM from MI vs. C, which was normalized in the MI-T group. CONCLUSIONS: Long-term treatment with the antioxidants probucol and pyrrolidine dithiocarbamate attenuates oxidative stress, myocyte apoptosis, caspase-3 like activity and the expression of p53, bax and caspase-3 within RM in rats after a large myocardial infarction.

Effect of protein kinase C and phospholipase A2 inhibitors on the impaired ability of human diabetic platelets to cause vasodilation.

1. The aim of this study was to examine the mechanism of impaired platelet-mediated endothelium-dependent vasodilation in diabetes. Exposure of human platelets to high glucose in vivo or in vitro impairs their ability to cause endothelium-dependent vasodilation. While previous data suggest that the mechanism for this involves increased activity of the cyclo-oxygenase pathway, the signal transduction pathway mediating this effect is unknown. 2. Platelets from diabetic patients as well as normal platelets and normal platelets exposed to high glucose concentrations were used to determine the role of the polyol pathway, diacylglycerol (DAG) production, protein kinase C (PKC) activity and phospholipase A2 (PLA2) activity on vasodilation in rabbit carotid arteries. 3. We found that two aldose-reductase inhibitors, tolrestat and sorbinil, caused only a modest improvement in the impairment of vasodilation by glucose exposed platelets. However, sorbitol and fructose could not be detected in the platelets, at either normal or hyperglycaemic conditions. We found that incubation in 17 mM glucose caused a significant increase in DAG levels in platelets. Furthermore, the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused significant impairment of platelet-mediated vasodilation. The PKC inhibitors calphostin C and H7 as well as inhibitors of PLA2 activity normalized the ability of platelets from diabetic patients to cause vasodilation and prevented glucose-induced impairment of platelet-mediated vasodilation in vitro. 4. These results suggest that the impairment of platelet-mediated vasodilation caused by high glucose concentrations is mediated by increased DAG levels and stimulation of PKC and PLA2 activity.

Activation of JNK in the remote myocardium after large myocardial infarction in rats.

A large myocardial infarction (MI) causes a chronic hemodynamic load on the uninjured remote myocardium (RM). This may lead to oxidative stress, activation of stress-induced cell signaling and increase in myocyte apoptosis. MI was produced in 6 rats (INF) while 4 rats underwent sham operation (CON). At four weeks, there was 128% increase in right ventricular hypertrophy in the hearts from INF vs. CON. Western blot analysis showed 3.8 fold increase in JNK phosphorylation within the RM from INF vs. CON, confirmed by a 4.2 fold increase in JNK kinase activity. There was a 52% increase in TBARS within the RM from INF vs. CON, suggesting increased lipid peroxidation. Furthermore, there was a twofold increase in myocyte apoptosis within the RM in INF vs. CON. We conclude that the RM from INF is associated with activation of JNK, increased oxidative stress and enhanced myocyte apoptosis.

Diabetic human platelets release a substance that inhibits platelet-mediated vasodilation.

This study was performed to investigate the mechanism for impaired vasodilation in response to activated diabetic human platelets. As observed previously, diabetic platelets failed to cause vasorelaxation, whereas normal platelets produced normal vasodilation. However, when activated and perfused through quiescent, NG-nitro-L-arginine-pretreated arteries, diabetic and normal platelets caused similar degrees of vasoconstriction. Inhibition of serotonergic and thromboxane A2 receptors in preconstricted normal arteries also failed to improve vasodilatory responses to diabetic platelets. The amount of ADP released into the supernatant from activated diabetic and normal platelets was similar. Concomitant perfusion of activated diabetic platelets impaired vasodilation produced by abluminally applied acetylcholine but perfusion of normal platelets did not. Whereas activated diabetic platelets failed to produce vasodilation, supernatant from the same platelets caused normal vasorelaxation. Dimethylthiourea and Tiron, intracellular free radical scavengers, normalized the vasodilatory response to diabetic platelets, whereas superoxide dismutase, catalase, and mannitol did not. We conclude that the impaired vasorelaxation in response to activated diabetic platelets is caused by an unidentified, short-acting, platelet-derived substance(s) that interferes with the normal dilatory response.

Cyclosporine impairs the ability of human platelets to mediate vasodilation.

Cyclosporine causes various platelet abnormalities. Whether it affects the ability of platelets to mediate vasodilation is unknown. Platelets were isolated from healthy volunteers and 13 heart transplant patients on cyclosporine. When perfused through preconstricted normal rabbit carotid arteries, activated platelets from transplant patients failed to cause vasorelaxation, whereas normal platelets produced significant vasodilation (-4.0 +/- 1.9% versus 30 +/- 3% [P < .0001] change in vessel diameter, respectively). When normal platelets were exposed to cyclosporine in vitro, they lost their ability to cause vasodilation in a dose- and time-dependent fashion. However, when activated and perfused through quiescent, N omega-nitro-L-arginine-pretreated arteries, platelets from transplant patients and normal platelets caused similar degrees of vasoconstriction. The amount of adenosine triphosphate in the supernatant from activated cyclosporine-exposed and control platelets was similar (1.7 +/- 0.4 versus 1.5 +/- 0.3 mumol/L [P = NS], respectively). However, concomitant perfusion of activated platelets from transplant patients impaired acetylcholine-mediated, endothelium-dependent vasodilation but perfusion of normal platelets did not. Although cyclosporine-exposed platelets showed an impaired ability to produce vasorelaxation, supernatant from the same platelets caused near normal vasodilation. Human platelets exposed to cyclosporine have an impaired ability to mediated vasodilation. This is not due to increased platelet-mediated vasoconstriction or a decrease in the release of platelet-derived nucleotides but rather to a short-acting compound released by cyclosporine-exposed platelets that interferes with endothelium-dependent vasodilation.

Platelets from patients with diabetes mellitus have impaired ability to mediate vasodilation.

OBJECTIVES: The purpose of this study was to examine vasomotor responses mediated by platelets from patients with diabetes mellitus. BACKGROUND: Diabetes mellitus is associated with increased cardiovascular morbidity and mortality, which in part may be due to a variety of abnormalities reported in diabetic platelets. However, the effects of diabetic platelets on vasomotor tone have not been characterized. METHODS: We compared platelet-mediated vasodilation elicited by platelets isolated from 30 healthy volunteers and 29 patients with diabetes mellitus as they were perfused through a preconstricted normal rabbit carotid artery. RESULTS: Platelets from the diabetic patients mediated an impaired dilatory response in comparison with normal platelets: 2.7 +/- 2% versus 15.8 +/- 3.4% (p < 0.001) and 4.1 +/- 2.7% versus 32.7 +/- 3.3% (p < 0.001) (mean +/- SEM) increase in vessel diameter, for 5 X 10(7) and 1 X 10(8) platelets per milliliter perfused, respectively. The degree of impairment was similar for type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus. Normal platelets incubated in high D-glucose concentrations lost their ability to mediate dilation in a concentration-dependent and time-dependent manner. This was not true for incubation of normal platelets in high concentrations of L-glucose or insulin. However, there was not a significant correlation between glucose control in the diabetic patients and the ability of their platelets to mediate vasodilation. CONCLUSIONS: Platelets from patients with diabetes mellitus have an impaired ability to mediate vasodilation. This impairment appears to be mediated by high glucose concentration. Further work is needed to elucidate the mechanisms for this abnormality in diabetic platelets.

Acute effect of cigarette smoking on the coronary circulation: constriction of epicardial and resistance vessels.

OBJECTIVES: This study was performed to determine the acute effect of cigarette smoking on proximal and distal epicardial conduit and coronary resistance vessels. BACKGROUND: Cigarette smoking causes constriction of epicardial arteries and a decrease in coronary blood flow in patients with coronary artery disease, despite an increase in myocardial oxygen demand. The role of changes in resistance vessel tone in the acute coronary hemodynamic effect of smoking has not been examined. METHODS: Twenty-four long-term smokers were studied during cardiac catheterization after vasoactive medications had been discontinued. The effect of smoking one cigarette 10 to 15 mm long on proximal and distal conduit vessel segments was assessed before and immediately after smoking and at 5, 15 and 30 min after smoking (n = 8). To determine the effect of smoking on resistance vessels, coronary flow velocity was measured in a nonobstructed artery with a 3F intracoronary Doppler catheter before and for 5 min after smoking (n = 8). Eight patients were studied without smoking to control for spontaneous changes in conduit arterial diameter (n = 5) and resistance vessel tone (n = 3). RESULTS: The average diameter of proximal coronary artery segments decreased from 2.56 +/- 0.12 mm (mean +/- SEM) before smoking to 2.41 +/- 0.09 mm 5 min after smoking (-5 +/- 2%, p < 0.05). Distal coronary diameter decreased from 1.51 +/- 0.07 to 1.39 +/- 0.06 mm (-8 +/- 2%, p < 0.01). Marked focal vasoconstriction after smoking was observed in two patients. Coronary diameter returned to baseline by 30 min after smoking. There was no change in vessel diameter in control patients. Despite a significant increase in the heart rate-mean arterial pressure product, coronary flow velocity decreased by 7 +/- 4% (p < 0.05) and coronary vascular resistance increased by 21 +/- 4% (p < 0.01) 5 min after smoking. There was no change in these variables in the control subjects. CONCLUSIONS: Smoking causes immediate constriction of proximal and distal epicardial coronary arteries and an increase in coronary resistance vessel tone, despite an increase in myocardial oxygen demand. These acute coronary hemodynamic effects may contribute to the adverse cardiovascular consequences of cigarette smoking.

Dietary fish oil supplementation reduces myocardial infarct size in a canine model of ischemia and reperfusion.

OBJECTIVES: This study was conducted to determine whether the long-term administration of fish oil attenuates myocardial necrosis in an occlusion-reperfusion model of myocardial ischemia. BACKGROUND: Omega-3 fatty acids found in fish oil have various biologic properties that may modify myocardial injury caused by severe ischemia and reperfusion. METHODS: Of 21 dogs fed an identical diet, 10 were given supplemental fish oil containing 0.06 g/kg per day of eicosapentaenoic acid for 6 weeks. Under anesthesia and open chest conditions, the left circumflex coronary artery was occluded for 90 min, followed by 6 h of reperfusion. Regional myocardial blood flow was measured with 15-microns spheres before and during occlusion and during reperfusion. The area at risk and infarct size were measured using standard staining techniques. RESULTS: In the dogs receiving supplemental fish oil, the platelet cell membrane content of eicosapentaenoic acid increased from 0.9 +/- 0.56% to 7.1 +/- 4.0% (p < 0.001). Infarct size was 29 +/- 7% in the control group and 13 +/- 3% in the fish oil group (p < 0.05). There was no significant difference in the myocardial area at risk or rate-pressure product between the control and fish oil groups. There was no difference in regional myocardial blood flow between the groups at baseline study or during coronary occlusion and reperfusion. CONCLUSIONS: Dietary fish oil supplementation significantly reduced myocardial infarct size in this model. The difference in infarct size did not appear to be related to dissimilarities in regional myocardial blood flow or determinants of oxygen consumption. Further investigation is needed to determine the nature of the protective mechanisms of omega-3 fatty acids on myocardial infarct size.

Interaction of lysophosphatidylcholine with aortic endothelial cells.

To better understand the vascular actions of lysophosphatidylcholine (lysoPC), we studied the interaction of [1-14C]palmitate-labeled lysoPC with bovine aortic endothelial cells. These cells took up lysoPC from media containing albumin, low-density lipoproteins (LDL), or acetyl-LDL. Uptake occurred faster than conversion to phosphatidylcholine (PC), leading to some lysoPC accumulation in endothelial lipids. Endothelial cell monolayers grown on micropore filters took up lysoPC from both apical and basolateral surfaces, preventing substantial amounts from passage across the endothelial monolayer. However, lysoPC present in the interstitial medium of an endothelial-smooth muscle coculture was incorporated primarily by the smooth muscle cells. Endothelial cells grown on filters released lysoPC into both the apical and basolateral medium in the presence of albumin or lipoproteins. Exposure to 50 microM lysoPC produced no evidence of endothelial cytotoxicity, but prostaglandin (PG)I2 production was reduced. These studies suggest that the endothelium can participate in the processing of circulating lysoPC and, through basolateral uptake, can facilitate the removal of lysoPC formed within the arterial wall. By decreasing PGI2 output, however, exposure to high concentrations of lysoPC may reduce the antithrombotic and vasodilator capacity of the endothelium.