P.L. da Luz

Vascular free radical release. Ex vivo and in vivo evidence for a flow-dependent endothelial mechanism.

Mechanisms underlying production of vascular free radicals are unclear. We hypothesized that changes in blood flow might serve as a physiological stimulus for endothelial free radical release. Intact isolated aortas from 45 rabbits were perfused with the spin trap alpha-phenyl-N-tert-butylnitrone (PBN, 20 mmol/L) and formed radical adducts detected by electron paramagnetic resonance spectroscopy (EPR). Sequential perfusion at 2, 7.5, and 12 mL/min changed cumulative vascular PBN radical adduct yields, respectively, from 3.2 +/- 0.9 to 4.1 +/- 0.7 (P < .05) and 7.0 +/- 1.5 (P < .005) pmol/mg with endothelium and from 3.6 +/- 1.6 to 3.8 +/- 1.4 and 2.2 +/- 0.8 pmol/mg without endothelium (P = NS). In endothelialized aortas, superoxide dismutase (SOD) completely blocked flow-induced free radical production, whereas inactivated SOD, indomethacin, and the nitric oxide synthetase antagonist nitro-L-arginine methyl ester (L-NAME) had no effect; relaxations to acetylcholine remained unchanged with higher flows. To assess the role of flow on in vivo radical production, femoral arterial plasma levels of the ascorbyl radical, a stable ascorbate oxidation product, were measured by direct EPR in 56 other rabbits. Ascorbyl levels were assessed at baseline (30.2 +/- 0.7 nmol/L) and at peak-induced iliac flow changes. Flow increases from 25% to 100% due to saline injections through an extracorporeal aortic loop induced significant dose-dependent increases in ascorbyl levels (n = 5). In addition, after papaverine bolus injections, flow increased by 114 +/- 8% versus baseline, and ascorbyl levels increased by 5.4 +/- 0.7 nmol/L (n = 31, P < .001); similar results occurred with adenosine, isoproterenol, or hyperemia after 30-second occlusions (P < .05, n = 4 or 5 in each group). Active SOD completely blocked papaverine-induced ascorbyl radical increase, despite preserved flow response (delta ascorbyl = 0.02 +/- 1.6 nmol/L, P = NS); inactivated SOD, catalase, indomethacin, and L-NAME had no effect. Blood flow decreases of 65% to 100% due to phenylephrine or 60-second balloon occlusions were accompanied by an average decrease of 4.4 nmol/L (P < .05) in ascorbyl levels. No change in ascorbyl signal was observed when rabbit blood alone was submitted to in vitro flow increases through a tubing circuit. Thus, increases in blood flow trigger vascular free radical generation; such a response seems to involve endothelium-derived superoxide radicals unrelated to cyclooxygenase or nitric oxide synthetase activities. This mechanism may contribute to explain vascular free radical generation in physiological or pathological circumstances.

Evidence for superoxide radical-dependent coronary vasospasm after angioplasty in intact dogs.

BackgroundActive oxygen species can influence vascular tone and platelet activation through a variety of mechanisms. This study assessed the role of the superoxide anion, the hydroxyl radical, and hydrogen peroxide in vasoconstriction and mural thrombosis after coronary artery angioplasty in intact dogs. Methods and ResultsInjury was induced by inflation of a balloon catheter 50±6% above baseline arterial diameter; dogs were followed for 2 hours before death. Epicardial coronary diameters at arteriography and extent of thrombus deposition at serial histological sections were analyzed in controls (n =20) and in dogs pretreated with superoxide dismutase (SOD, a superoxide radical scavenger, n = 10); other dogs were pretreated with the hydrogen peroxide scavenger catalase (n =8), the iron chelator deferoxamine (n =6), or the hydroxyl radical scavenger 1,3-dimethyl-2-thiourea (n =9). Angioplasty-induced injury was similar among groups. After angioplasty, control dogs exhibited localized and persistent vessel constriction, which was maximal at the initial 5 minutes (28.9 + 6.3% diameter decrease versus baseline). Corresponding arterial diameters of SOD-treated dogs were 24–69% larger (95% confidence interval, p < 0.001) than controls at 5 minutes and, on average, 32% larger than controls thereafter (p < 0.01). Vasoconstriction was not prevented by the other treatments. The SOD dose used accounted for inhibition of zymosan-stimulated blood cytochrome c reduction versus baseline (7 + 3 versus 30±6 nmol/min/106 cells, respectively, p = 0.003); such inhibition occurred in no other group. Prevalence of mural thrombosis was similar among all groups, but large thrombi (>15% of lumen area) were absent in SOD-treated dogs, contrary to control group (p = 0.028); other groups were similar to control. In the absence of injury, SOD alone induced no change in coronary diameter, coronary blood flow, or platelet aggregation. ConclusionsThese data provide evidence implicating the superoxide radical in the genesis of vasoconstriction after coronary angioplasty in vivo. Such effects seem to be independent of its conversion to hydroxyl radicals and availability of hydrogen peroxide or catalytic iron complexes.

Functional significance of regional ischemic contraction abnormalities.

To evaluate the progression of segment function following induction of ischemia, the left anterior descending coronary artery was ligated (eight dogs) or cannulated and perfused at various pressures via a bypass-oxygenator (six dogs). Mercury-in-silastic length gauges were sutured to the anterior left ventricle, and pressure was recorded by a catheter-tipped transducer. Segment function was determined from the area of the pressure-length loop by plotting instantaneous left ventricular pressure against segment length and by evaluation of the degree of systolic shortening. Segment function decreased linearly as flow in the left anterior descending artery was decreased in a stepwise fashion by reduction in perfusion pressure from 100 to 20 mm Hg. With both left anterior descending coronary artery ligation and stepwise flow reduction, the pressure-length loop invariably showed four clearly identifiable morphologic patterns which relate conceptually to the specific left ventricular contraction patterns: dyssynchrony, hypokinesis, akinesis, and paradoxic systolic expansion. Re-oxygenation following occlusion invariably revealed return to a normal pattern in reverse order. This study demonstrates that a consistent and predictable progression of segmental contraction abnormalities occurs with ischemia.

The action of red wine and purple grape juice on vascular reactivity is independent of plasma lipids in hypercholesterolemic patients

Although red wine (RW) reduces cardiovascular risk, the mechanisms underlying the effect have not been identified. Correction of endothelial dysfunction by RW flavonoids could be one mechanism. We measured brachial artery reactivity by high-resolution ultrasonography, plasma lipids, glucose, adhesion molecules (ICAM-1 and VCAM), and platelet function in 16 hypercholesterolemic individuals (8 men and 8 women; mean age 51.6 +/- 8.1 years) without other risk factors. Twenty-four normal subjects were used as controls for vascular reactivity. Subjects randomly received RW, 250 ml/day, or purple grape juice (GJ), 500 ml/day, for 14 days with an equal wash-out period. At baseline, all 16 subjects were hypercholesterolemic (mean LDL = 181.0 +/- 28.7 mg/dl) but HDL, triglycerides, glucose, adhesion molecules, and platelet function were within normal limits. Brachial artery flow-mediated dilation was significantly decreased compared to controls (9.0 +/- 7.1 vs 12.1 +/- 4.5%; P < 0.05) and increased with both GJ (10.1 +/- 7.1 before vs 16.9 +/- 6.7% after: P < 0.05) and RW (10.1 +/- 6.4 before vs 15.6 +/- 4.6% after; P < 0.05). RW, but not GJ, also significantly increased endothelium-independent vasodilation (17.0 +/- 8.6 before vs 23.0 +/- 12.0% after; P < 0.01). GJ reduced ICAM-1 but not VCAM and RW had no effect on either molecule. No significant alterations were observed in plasma lipids, glucose or platelet aggregability with RW or GJ. Both RW and GJ similarly improved flow-mediated dilation, but RW also enhanced endothelium-independent vasodilation in hypercholesterolemic patients despite the increased plasma cholesterol. Thus, we conclude that GJ may protect against coronary artery disease without the additional negative effects of alcohol despite the gender.

Wine, alcohol and atherosclerosis: clinical evidences and mechanisms

Atherosclerosis is a chronic inflammatory disease which may cause obstructions of the coronary, cerebral and peripheral arteries. It is typically multifactorial, most often dependent on risk factors such as hypercholesterolemia, diabetes, smoking, hypertension, sedentarism, and obesity. It is the single main cause of death in most developed countries due to myocardial infarction, angina, sudden death, and heart failure. Several epidemiological studies suggest that moderate alcohol intake, especially red wine, decrease cardiac mortality due to atherosclerosis. The alcohol effect is described by a J curve, suggesting that moderate drinkers may benefit while abstainers and heavy drinkers are at higher risk. Experimental studies indicate that most beneficial effects of drinking are attributable to flavonoids that are present in red wine, purple grape juice and several fruits and vegetables. The mechanisms include antiplatelet actions, increases in high-density lipoprotein, antioxidation, reduced endothelin-1 production, and increased endothelial nitric oxide synthase expression which causes augmented nitric oxide production by endothelial cells. These findings lead to the concept that moderate red wine drinking, in the absence of contraindications, may be beneficial to patients who are at risk of atherosclerotic cardiovascular events. Moreover, a diet based on fruits and vegetables containing flavonoids may be even more beneficial.

Contrasting influences of alterations in ventricular preload and afterload upon systemic hemodynamics, function, and metabolism of ischemic myocardium.

This study of anesthetized, open-chest dogs compares the effects of primary increases in left ventricular preload and afterload upon global and regional myocardial function and metabolism in the presence of a left anterior descending coronary artery stenosis (LAD). When LAD flow was reduced to 40–50% of control, regional systolic shortening declined by 20 to 25% and regional lactate extraction changed to production. In seven control dogs the mechanical abnormalities persisted during the 30 min of observation, but lactate production was reduced spontaneously. In ten dogs, increases in left ventricular end-diastolic pressure (LVEDP) during dextran infusion were associated with increases in cardiac output and regional systolic shortening; however, regional lactate production also increased (P < 0.05) despite an augmentation in LAD flow. In seven dogs mean arterial pressure increased by an average of 32 mm Hg during angiotensin infusion (0.2 to 0.4 Asg/kg/min); LVEDP did not change but cardiac output decreased significantly. LAD artery flow improved markedly and lactate production shifted to extraction (P < 0.05) while systolic shortening remained unchanged. When angiotensin was discontinued, lactate extraction worsened again.Thus, in the presence of a severe coronary stenosis, a primary increase in preload improves cardiac output but at the expense of aggravated ischemia. In contrast, a primary increase in afterload reduces cardiac output but may improve perfusion and lactate uptake of the ischemic myocardium.

Expression of inducible nitric oxide synthase is increased in patients with heart failure due to ischemic disease

The objective of the present study was to determine the relationship between nitric oxide synthases (NOS) and heart failure in cardiac tissue from patients with and without cardiac decompensation. Right atrial tissue was excised from patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) <35% (N = 10), and from patients with CAD and LVEF >60% (N = 10) during cardiac surgery. NOS activity was measured by the conversion of L-[H(3)]-arginine to L-[H(3)]-citrulline. Gene expression was quantified by the competitive reverse transcription-polymerase chain reaction. Both endothelial NOS (eNOS) activity and expression were significantly reduced in failing hearts compared to non-failing hearts: 0.36 +/- 0.18 vs 1.51 +/- 0.31 pmol mg-1 min-1 (P < 0.0001) and 0.37 +/- 0.08 vs 0.78 +/- 0.09 relative cDNA absorbance at 320 nm (P < 0.0001), respectively. In contrast, inducible NOS (iNOS) activity and expression were significantly higher in failing hearts than in non-failing hearts: 4.00 +/- 0.90 vs 1.54 +/- 0.65 pmol mg-1 min-1 (P < 0.0001) and 2.19 +/- 0.27 vs 1.43 +/- 0.13 cDNA absorbance at 320 nm (P < 0.0001), respectively. We conclude that heart failure down-regulates both eNOS activity and expression in cardiac tissue from patients with LVEF <35%. In contrast, iNOS activity and expression are increased in failing hearts and may represent an alternative mechanism for nitric oxide production in heart failure due to ischemic disease.

Myocardial reperfusion in acute experimental ischemia. Beneficial effects of prior treatment with steroids.

To test the hypothesis that prior steroid administration may enhance the mechanical and metabolic response to myocardial reperfusion, regional myocardial function (Hg-in-silastic length gauges), transmyocardial lactate balance and K+ difference were measured in 12 control and 13 treated (30 mg/kg methylprednisolone, 30 to 60 min postocclusion) dogs. At three hours of ischemia, systolic shortening in the ischemic segment was greater in treated dogs (40.6% vs. 12%, P < 0.05), while both lactate balance and K+ arteriovenous difference became positive. Lactate balance and K+ difference remained negative in the untreated animals. After three hours of occlusion and one hour of reperfusion, recovery of shortening was significantly greater in the treated animals (75.9 vs. 31.6%, P < 0.05). In addition, while lactate balance remained negative among the control dogs, it further improved in the treated dogs.Thus, steroid administration during experimental coronary occlusion impedes the progression of ischemia and is additive to reperfusionin reversing ischemic dysfunction.

Hyperhomocystinemia in patients with coronary artery disease

Hyperhomocystinemia has been related to an increased risk of cardiovascular disease in several studies. The C677T polymorphism for the gene that encodes the methylenetetrahydrofolate reductase enzyme (MTHFR) and low plasma folate levels are common causes of hyperhomocystinemia. Due to differences in nutritional patterns and genetic background among different countries, we evaluated the role of hyperhomocystinemia as a coronary artery disease (CAD) risk factor in a Brazilian population. The relation between homocysteine (Hcy) and the extent of CAD, measured by an angiographic score, was determined. A total of 236 patients referred for coronary angiography for clinical reasons were included. CAD was found in 148 (62.7%) patients and 88 subjects had normal or near normal arteries. Patients with CAD had higher Hcy levels [mean (SD)] than those without disease (14 (6.8) vs 12.5 (4.0) microM; P = 0.04). Hyperhomocystinemia (Hcy >17.8 microM) prevalence was higher in the CAD group: 31.1 vs 12.2% (P = 0.01). After adjustment for major risk factors, we found an independent association between hyperhomocystinemia and CAD (OR = 2.48; 95% CI = 1.02-6.14). Patients with a more advanced coronary score had a higher frequency of hyperhomocystinemia and tended to have higher mean Hcy levels. An inverse relation between plasma folate and Hcy levels was found (r = -0.14; P = 0.04). Individuals with the MTHFR C677T polymorphism had a higher prevalence of hyperhomocystinemia than those without the mutated allele. We conclude that hyperhomocystinemia is independently associated with CAD, with a positive association between Hcy level and disease severity.

Hormone replacement therapy increases levels of antibodies against heat shock protein 65 and certain species of oxidized low density lipoprotein

Hormone replacement therapy (HRT) reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp) and oxidized low density lipoprotein (LDL) have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 +/- 0.03 vs 0.558 +/- 0.11) and against LDL with a low degree of oxidative modification (0.100 +/- 0.01 vs 0.217 +/- 0.02) (P<0.05 and P<0.001, respectively, ANOVA). The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.