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Background: Despite the well-established association between T-cell–mediated inflammation and nonischemic heart failure, the specific mechanisms triggering T-cell activation during the progression of heart failure and the antigens involved are poorly understood. We hypothesized that myocardial oxidative stress induces the formation of isolevuglandin (IsoLG)–modified proteins that function as cardiac neoantigens to elicit CD4+ T-cell receptor (TCR) activation and promote heart failure. Methods: We used transverse aortic constriction in mice to trigger myocardial oxidative stress and T-cell infiltration. We profiled the TCR repertoire by mRNA sequencing of intramyocardial activated CD4+ T cells in Nur77 GFP reporter mice, which transiently express GFP on TCR engagement. We assessed the role of antigen presentation and TCR specificity in the development of cardiac dysfunction using antigen presentation–deficient MhcII −/− mice and TCR transgenic OTII mice that lack specificity for endogenous antigens. We detected IsoLG protein adducts in failing human hearts. We also evaluated the role of reactive oxygen species and IsoLGs in eliciting T-cell immune responses in vivo by treating mice with the antioxidant TEMPOL and the IsoLG scavenger 2-hydroxybenzylamine during transverse aortic constriction, and ex vivo in mechanistic studies of CD4+ T-cell proliferation in response to IsoLG-modified cardiac proteins. Results: We discovered that TCR antigen recognition increases in the left ventricle as cardiac dysfunction progresses and identified a limited repertoire of activated CD4+ T-cell clonotypes in the left ventricle. Antigen presentation of endogenous antigens was required to develop cardiac dysfunction because MhcII −/− mice reconstituted with CD4+ T cells and OTII mice immunized with their cognate antigen were protected from transverse aortic constriction–induced cardiac dysfunction despite the presence of left ventricle–infiltrated CD4+ T cells. Scavenging IsoLGs with 2-hydroxybenzylamine reduced TCR activation and prevented cardiac dysfunction. Mechanistically, cardiac pressure overload resulted in reactive oxygen species–dependent dendritic cell accumulation of IsoLG protein adducts, which induced robust CD4+ T-cell proliferation. Conclusions: Our study demonstrates an important role of reactive oxygen species–induced formation of IsoLG-modified cardiac neoantigens that lead to TCR-dependent CD4+ T-cell activation within the heart.

Article, see p 1353 Hypertension affects 30% of the population and 70% of elderly adults. Despite its frequency and decades of research, the etiology of most cases of adult hypertension remains undefined. Perturbations of the kidney, the vasculature, and the central nervous system have all been implicated in the pathogenesis of hypertension. As examples, in most cases of adult hypertension, systemic vascular resistance is elevated and vasodilators lower blood pressure, supporting a vascular etiology. Alternatively, renal cross-transplantation studies have shown that hypertension follows the kidney, and in most cases of experimental hypertension, pressure natriuresis is impaired. Likewise, most single-gene mutations that cause hypertension affect sodium transport in the distal nephron. In keeping with a renal etiology, diuretics and sodium restriction are effective in lowering blood pressure. There is ample evidence to suggest a central-neural cause of hypertension. Sympathetic outflow is almost uniformly increased in experimental models of hypertension and in humans with this disease, and renal denervation has been variably reported to reduce blood pressure. Adrenergic receptor antagonists are commonly used to treat hypertension, further supporting a neural etiology. Is there a unifying mechanism that underlies abnormalities of the brain, kidneys, and the vasculature in hypertension? Accumulating evidence shows that immune cells infiltrate these organs in response to hypertensive stimuli, causing their dysfunction and leading to elevated blood pressure and subsequent end-organ damage. Both innate and adaptive immune cells play a key role in this process. Monocyte/macrophages are increased in the kidney and vasculature of animal models of experimental hypertension and in humans with hypertension. Wenzel et al1 showed that deletion of monocytes/macrophages completely eliminates angiotensin II-induced hypertension and markedly improves vascular function. Monocytes have several fates on tissue entry. One is to become inflammatory macrophages that can generate reactive oxygen species, release cytokines, and produce matrix metalloproteinases. A …

Background— The pathogenesis of hypertension remains poorly understood, and treatment is often unsuccessful. Recent evidence suggests that the adaptive immune response plays an important role in this disease. Various hypertensive stimuli cause T-cell activation and infiltration into target organs such as the vessel and the kidney, which promotes vascular dysfunction and blood pressure elevation. Classically, T-cell activation requires T-cell receptor ligation and costimulation. The latter often involves interaction between B7 ligands (CD80 and CD86) on antigen-presenting cells with the T-cell coreceptor CD28. This study was therefore performed to examine the role of this pathway in hypertension. Methods and Results— Angiotensin II–induced hypertension increased the presence of activated (CD86 + ) dendritic cells in secondary lymphatic tissues. Blockade of B7-dependent costimulation with CTLA4-Ig reduced both angiotensin II– and deoxycorticosterone acetate (DOCA)–salt–induced hypertension. Activation of circulating T cells, T-cell cytokine production, and vascular T-cell accumulation caused by these hypertensive stimuli were abrogated by CTLA4-Ig. Furthermore, in mice lacking B7 ligands, angiotensin II caused minimal blood pressure elevation and vascular inflammation, and these effects were restored by transplantation with wild-type bone marrow. Conclusions— T-cell costimulation via B7 ligands is essential for development of experimental hypertension, and inhibition of this process could have therapeutic benefit in the treatment of this disease.

Background— Increased inactivation of nitric oxide by superoxide (O 2 ·− ) contributes to endothelial dysfunction in patients with coronary disease (CAD). We therefore characterized the vascular activities of xanthine oxidase and NAD(P)H oxidase, 2 major O 2 ·− -producing enzyme systems, and their relationship with flow-dependent, endothelium-mediated vasodilation (FDD) in patients with CAD. Methods and Results— Xanthine- and NAD(P)H-mediated O 2 ·− formation was determined in coronary arteries from 10 patients with CAD and 10 controls by using electron spin resonance spectroscopy. Furthermore, activity of endothelium-bound xanthine oxidase in vivo and FDD of the radial artery were determined in 21 patients with CAD and 10 controls. FDD was measured before and after infusion of the antioxidant vitamin C (25 mg/min i.a.) to determine the portion of FDD inhibited by radicals. In coronary arteries from patients with CAD, xanthine- and NAD(P)H-mediated O 2 ·− formation was increased compared with controls (xanthine: 12±2 versus 7±1 nmol O 2 ·− /μg protein; NADH: 11±1 versus 7±1 nmol O 2 ·− /μg protein; and NADPH: 12±2 versus 9±1 nmol O 2 ·− /μg protein; each P 200% in patients with CAD (25±4 versus 9±1 nmol O 2 ·− /μL plasma per min; P r =−0.55; P r =0.54; P Conclusions— The present study represents the first electron spin resonance measurements of xanthine and NAD(P)H oxidase activity in human coronary arteries and supports the concept that increased activities of both enzymes contribute to increased vascular oxidant stress in patients with CAD. Furthermore, the present study suggests that increased xanthine oxidase activity contributes to endothelial dysfunction in patients with CAD and may thereby promote the atherosclerotic process.

Background— In the arterial endothelium, laminar flow and cyclical stretch induce expression of NO synthase (NOS). We hypothesized that atrial fibrillation (AF) causes a downregulation of atrial endocardial NOS expression and NO · production. Because NO · has antithrombotic properties, this may contribute to thromboembolism in AF. Methods and Results— In pigs, AF was produced with rapid atrial pacing at 600 bpm for 1 week, whereas controls had atrial pacing at 100 bpm. All animals had catheter ablation of the AV junction and ventricular pacing at 100 bpm. NO · production from freshly isolated tissue was measured by a NO · -specific microelectrode. Left atrial basal and stimulated NO · production was decreased in AF by 73% and 71% ( P · and was increased in the left atrium by 1.8-fold in AF ( P · concentration was decreased in the left atrial appendage, although NOS expression was not affected. Neither NOS concentration, NO · levels, nor PAI-1 expression were altered in the aortas or right atria of animals with AF. Conclusions— AF is associated with a marked decrease in endocardial NOS expression and NO · bioavailability and an increase in PAI-1 expression in the left atrium. These data suggest that organized atrial contraction is needed to maintain normal endocardial expression of NOS. It is likely that loss of this antithrombotic enzyme contributes to the thromboembolic phenomena commonly observed in AF.

Despite intense investigation in the last decade, many questions concerning the etiology of atherosclerosis remain unanswered. Hypercholesterolemia and hypertension are clearly risk factors for the development of vascular lesions, although the precise molecular steps between elevated lipid or blood pressure levels and the development of atherosclerotic lesions are not completely defined.1,2⇓ Hypertension is frequently associated with additional risk factors such as hypercholesterolemia, estrogen deficiency, or hyperinsulinemia. This clustering of risk factors greatly enhances the probability to develop atherosclerosis.3 Nevertheless, the cellular events responsible for the mutual appearance of several risk factors are poorly understood. A series of recent studies have addressed the hypothesis that enhanced AT1 receptor activation could explain the association of various hormonal and metabolic disorders with hypertension, and ultimately, with accelerated progression of vascular lesions. As early as 1980, Alexander and colleagues discovered that the vasoconstriction caused by angiotensin II in resistance vessels was variable.4 Further investigations revealed that AT1 receptor activation is subject to a negative feedback, in that increased levels of angiotensin II diminish and decreased angiotensin II concentrations enhance AT1 receptor activation.5–8⇓⇓⇓ More recently, it has been shown that multiple agonists other than angiotensin II modulate AT1 receptor expression. This phenomenon, referred to as heterologous AT1 receptor regulation, is induced by various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or fibroblast growth factor (FGF), all which downregulate AT1 receptor expression.9 Numerous other factors, including glucocorticoids, aldosterone, forskolin, TNFα, cytokines, nitric oxide, insulin, LDL, estrogen, progesterone, sodium, free radicals, IGF-1, and isoprenaline are known to influence AT1 receptor expression (Table). 10–38⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ …

Background —Previous data suggest that 1 endothelial NO synthase (eNOS) gene is sufficient to allow normal expression and function of eNOS under basal conditions. We hypothesized that this might not hold true for conditions known to increase eNOS gene expression, such as exercise. Methods and Results —Male mice heterozygous for a disruption of the eNOS gene (eNOS +/ − ) and normal C56Bl/6J mice (eNOS +/+ ), 3 to 4 months of age, underwent exercise training for 3 weeks. Nontrained mice were exposed to the exercise environment (noise and vibration of the treadmill) without exercise for an identical period. In eNOS +/+ mice (n=7), exercise increased aortic eNOS protein expression by 3.4±0.4-fold ( P P P +/ − mice ( P >0.05). Thus, although eNOS expression appears to be normal in eNOS +/ − mice under basal conditions, these mice are unable to increase eNOS expression during exercise. Conclusions —These findings show that regulation of eNOS expression during exercise requires the presence of both alleles of the gene and may have implications for conditions in which polymorphisms of eNOS are present in only 1 allele in humans. These individuals may have a normal vascular reactivity under basal conditions but may be unable to adapt their vascular reactivity in response to exercise training.

Background —Altered endothelial cell nitric oxide (NO · ) production in atherosclerosis may be due to a reduction of intracellular tetrahydrobiopterin, which is a critical cofactor for NO synthase (NOS). In addition, previous literature suggests that inactivation of NO · by increased vascular production superoxide (O 2 ·− ) also reduces NO · bioactivity in several disease states. We sought to determine whether these 2 seemingly disparate mechanisms were related. Methods and Results —Endothelium-dependent vasodilation was abnormal in aortas of apoE-deficient (apoE −/− ) mice, whereas vascular superoxide production (assessed by 5 μmol/L lucigenin) was markedly increased. Treatment with either liposome-entrapped superoxide dismutase or sepiapterin, a precursor to tetrahydrobiopterin, improved endothelium-dependent vasodilation in aortas from apoE −/− mice. Hydrogen peroxide had no effect on the decay of tetrahydrobiopterin, as monitored spectrophotometrically. In contrast, superoxide modestly and peroxynitrite strikingly increased the decay of tetrahydrobiopterin over 500 seconds. Luminol chemiluminescence, inhibitable by the peroxynitrite scavengers ebselen and uric acid, was markedly increased in apoE −/− aortic rings. In vessels from apoE −/− mice, uric acid improved endothelium-dependent relaxation while having no effect in vessels from control mice. Treatment of normal aortas with exogenous peroxynitrite dramatically increased vascular O 2 ·− production, seemingly from eNOS, because this effect was absent in vessels lacking endothelium, was blocked by NOS inhibition, and did not occur in vessels from mice lacking eNOS. Conclusions —Reactive oxygen species may alter endothelium-dependent vascular relaxation not only by the interaction of O 2 ·− with NO · but also through interactions between peroxynitrite and tetrahydrobiopterin. Peroxynitrite oxidation of tetrahydrobiopterin may represent a pathogenic cause of “uncoupling” of NO synthase.

Background —Angiotensin II–induced hypertension is associated with increased vascular superoxide production, which contributes to hypertension caused by the octapeptide. In cell culture, stretch increases endothelial and vascular smooth muscle production of reactive oxygen species (ROS). In perfused isolated vessels, elevations of pressure can increase vessel angiotensin II production. The effects of low-renin hypertension on vascular ROS production remain unclear. Furthermore, the role of ROS in vascular function and hypertension in low-renin hypertension is undefined. Methods and Results —Rats were treated with DOCA and saline drinking water for 3 weeks. Both systolic blood pressure (189±4 versus 126±2 mm Hg) and aortic superoxide production (3972±257 versus 852±287, P P P −1 · d −1 PO) for 7 days did not correct the hypertension or endothelium-dependent vessel relaxation in DOCA-salt rats, excluding a role of a local renin/angiotensin II system. Conclusions —These findings indicate that increased vascular superoxide production occurs not only in angiotensin II–induced hypertension but also in hypertension known to be associated with low-renin states. Increased superoxide production alters large-vessel endothelium-dependent vascular relaxation but does not modulate blood pressure in low-renin hypertension.

Background —Cardiovascular ischemic events may occur more frequently in hypertensive patients with activated renin-angiotensin systems. We tested the hypothesis that angiotensin II (Ang II) may contribute to atherosclerosis by increasing expression of vascular inflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1). Methods and Results —Rats infused with norepinephrine or Ang II for 6 days developed similar hypertensive responses, but only Ang II-treated rats exhibited significant increases in aortic VCAM-1 protein and mRNA expression. Oral losartan treatment (50 mg · kg −1 · d −1 ) inhibited Ang II-induced hypertension and aortic VCAM-1 mRNA expression. Ang II treatment significantly increased VCAM-1 mRNA expression in cultured rat aortic smooth muscle cells (RASMCs). Ang II also induced nuclear NF-κB-like binding activity and transactivated an NF-κB–driven VCAM-1 promoter. Losartan and proteasome inhibitors blocked Ang II-induced NF-κB activation and VCAM-1 mRNA accumulation. IκB-α overexpression in RASMCs inhibited Ang II-induced VCAM-1 promoter transactivation. Conclusions —Ang II may contribute to atherogenesis by activation of VCAM-1 through proteasome dependent, NF-κB-like transcriptional mechanisms.

Background —We have previously shown that nitroglycerin (NTG) therapy increases vascular expression of endothelin 1 (ET-1) and stimulates vascular superoxide (O 2 ·− ) production via activation of NADH/NADPH oxidases. Both phenomena are stimulated by angiotensin II in vitro, and the renin-angiotensin system is activated during early nitrate therapy. We hypothesized that either angiotensin II or ET-1 may increase vascular O 2 ·− production during nitrate therapy. Methods and Results —In New Zealand White rabbits, 3 days of treatment with NTG patches increased plasma renin activity for the entire treatment period. After 24 hours of NTG treatment, angiotensin II type 1 (AT 1 ) receptor expression and vascular ACE activity were significantly decreased. At this time, constrictions to angiotensin I and II were depressed, but there was no loss of NTG vasodilator potency. Within 3 days of continuous NTG treatment, relaxations to NTG were markedly blunted. This was associated with an increase in AT 1 receptor mRNA expression, a return of ACE activity back to baseline, and a marked increase in constrictions to angiotensin I and II despite continuously increased plasma renin activity. Tolerance was associated with a 2-fold increase in vascular O 2 ·− , as estimated by lucigenin-enhanced chemiluminescence. Concomitant treatment with the AT 1 receptor antagonist losartan (5 to 25 mg · kg −1 · d −1 ) dose-dependently normalized vascular O 2 ·− and prevented tolerance to NTG and cross-tolerance to endogenous nitric oxide released by acetylcholine. The nonselective ET-1 receptor blocker bosentan (100 mg · kg −1 · d −1 ) had similar but less pronounced effects. Conclusions —The positive effects of AT 1 and ET-1 receptor blockade on tolerance and O 2 ·− production imply a pathophysiological role for angiotensin II and to some extent for ET-1 in the development of nitrate tolerance.

Background —Angiotensin II activates NAD(P)H-dependent oxidases via AT 1 -receptor stimulation, the most important vascular source of superoxide (O 2 · − ). The AT 1 receptor is upregulated in vitro by low-density lipoprotein. The present study was designed to test whether hypercholesterolemia is associated with increased NAD(P)H-dependent vascular O 2 · − production and whether AT 1 -receptor blockade may inhibit this oxidase and in parallel improve endothelial dysfunction. Methods and Results —Vascular responses were determined by isometric tension studies, and relative rates of vascular O 2 · − production were determined by use of chemiluminescence with lucigenin, a cypridina luciferin analogue, and electron spin resonance studies. AT 1 -receptor mRNA was quantified by Northern analysis, and AT 1 -receptor density was measured by radioligand binding assays. Hypercholesterolemia was associated with impaired endothelium-dependent vasodilation and increased O 2 · − production in intact vessels. In vessel homogenates, we found a significant activation of NADH-driven O 2 · − production in both models of hyperlipidemia. Treatment of cholesterol-fed animals with the AT 1 -receptor antagonist Bay 10-6734 improved endothelial dysfunction, normalized vascular O 2 · − and NADH-oxidase activity, decreased macrophage infiltration, and reduced early plaque formation. In the setting of hypercholesterolemia, the aortic AT 1 receptor mRNA was upregulated to 166±11%, accompanied by a comparable increase in AT 1 -receptor density. Conclusions —Hypercholesterolemia is associated with AT 1 -receptor upregulation, endothelial dysfunction, and increased NADH-dependent vascular O 2 · − production. The improvement of endothelial dysfunction, inhibition of the oxidase, and reduction of early plaque formation by an AT 1 -receptor antagonist suggests a crucial role of angiotensin II–mediated O 2 · − production in the early stage of atherosclerosis.

Background —In a variety of disease states, endothelium-dependent vasodilation is abnormal. Reduced nitric oxide (NO) production, increased destruction of NO by superoxide, diminished cellular levels of l -arginine or tetrahydrobiopterin, and alterations in membrane signaling have been implicated. We examined these potential mechanisms in human vessels. Methods and Results —Relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin, as well as superoxide production and NO synthase expression, were examined in vascular segments from patients with identified cardiovascular risk factors. Endothelium-dependent relaxations were also studied after incubation with l -arginine, l -sepiapterin, and liposome-entrapped superoxide dismutase (SOD) and after organoid culture with cis -vaccenic acid. Relaxations to acetylcholine and to a lesser extent the calcium ionophore A23187 were highly variable and correlated with the number of risk factors present among the subjects studied. Treatment of vessels with l -arginine, l -sepiapterin, liposome-entrapped SOD, or cis -vaccenic acid did not augment endothelium-dependent relaxations. Hypercholesterolemia was the only risk factor associated with high levels of superoxide; however, there was no correlation between superoxide production and the response to either endothelium-dependent vasodilator used. Conclusions —In human internal mammary arteries, depressed endothelium-dependent relaxations could not be attributed to increases in vascular superoxide production, deficiencies in either l -arginine or tetrahydrobiopterin, or reduced membrane fluidity. Variability in signaling mechanisms may contribute to the differences in responses to acetylcholine and the calcium ionophore A23187.

Background The major source of superoxide (·O 2 − ) in vascular tissues is an NADH/NADPH-dependent, membrane-bound oxidase. We have previously shown that this oxidase is activated in angiotensin II– but not norepinephrine-induced hypertension. We hypothesized that hypertension associated with chronically elevated angiotensin II might be caused in part by vascular ·O 2 − production. Methods and Results We produced hypertension in rats by a 5-day infusion of angiotensin II or norepinephrine. Rats were also treated with liposome-encapsulated superoxide dismutase (SOD) or empty liposomes. Arterial pressure was measured in conscious rats under baseline conditions and during bolus injections of either acetylcholine or nitroprusside. Vascular ·O 2 − production was assessed by lucigenin chemiluminescence. In vitro vascular relaxations were examined in organ chambers. Norepinephrine infusion increased blood pressure to a similar extent as angiotensin II infusion (179±5 and 189±4 mm Hg, respectively). In contrast, angiotensin II–induced hypertension was associated with increased vascular ·O 2 − production, whereas norepinephrine-induced hypertension was not. Treatment with liposome-encapsulated SOD reduced blood pressure by 50 mm Hg in angiotensin II–infused rats while having no effect on blood pressure in control rats or rats with norepinephrine-induced hypertension. Similarly, liposome-encapsulated SOD enhanced in vivo hypotensive responses to acetylcholine and in vitro responses to endothelium-dependent vasodilators in angiotensin II–treated rats. Conclusions Hypertension caused by chronically elevated angiotensin II is mediated in part by ·O 2 − , likely via degradation of endothelium-derived NO·. Increased vascular ·O 2 − may contribute to vascular disease in high renin/angiotensin II states.

It has become apparent that major sources of reactive oxygen species (ROS) in mammalian cells are the NADPH oxidases. These multimeric enzymes are composed of membrane-bound catalytic subunits, or Nox enzymes, and a small docking subunit called p22phox. Depending on the Nox isoform, cytosolic regulatory subunits also play a role in their activation. There are 7 Nox isoforms. The first identified was Nox2, also called gp91 phox , which is present in phagocytic cells and is responsible for the oxidative burst. Unlike Nox2, the other Nox enzymes exhibit sustained production of ROS at somewhat lower levels. Their modes of activation differ, as do their cellular distributions. Nox1 through Nox4 are expressed in vascular cells in rodents. Nox5 has been identified in atherosclerotic lesions of humans but is not expressed in rodents. Stimulation of cells with angiotensin II, cytokines, catecholamines, high glucose, or mechanical stretch promotes NADPH oxidase activation, in large part as a result of recruitment of cytosolic subunits to the membrane subunit, leading to the formation of the functional enzyme complex that can transfer electrons to molecular oxygen and the formation of superoxide (O2·−).1 O2·−serves as a progenitor for other ROS, including hydrogen peroxide, peroxynitrite, and hypochlorous acid. Enhanced activity of the NADPH oxidases and increased expression of its subunits occur in many pathological conditions, including hypertension, diabetes mellitus, atherosclerosis, cardiac hypertrophy, and heart failure. Studies of genetically altered mice have shown that the deletion of various NADPH oxidase subunits protects against these diseases and their overexpression promotes pathology.2−4 Article see p 1888 A consequence of excessive ROS formation is loss of endothelial nitric oxide (NO), which can occur by rapid radical-radical reactions with O2·− and other radicals or as a result of dysfunction of the endothelial NO synthase. …

During the past 15 years, it has become apparent that the vascular endothelium plays a crucial role in modulating vasomotor tone. The endothelium accomplishes this by synthesizing and releasing several vasoactive substances, including prostacyclin, adenine nucleotides, kinins, NO·, an as-yet-unidentified hyperpolarizing factor, and vasoconstrictor substances such as endothelin, vasoconstrictor prostanoids such as PGH2, and the superoxide anion (·O2−).1 2 3 Among the best characterized and likely the most important of these is NO·, which was previously known as EDRF.4 NO is produced by the endothelium when a variety of neurohumoral, paracrine, and pharmacological stimuli act on endothelial cell receptors to increase intracellular calcium, which ultimately activates endothelial cell NO synthase. This enzyme catalyzes the complex oxidation of one of the guanidino nitrogens of arginine to NO·.5 There is substantial evidence that NO· may be transported attached to other molecules, such as nitrosothiols, both between cells and in the plasma.6 7 Vascular smooth muscle cells possess receptors to many of the same agonists that activate NO· release from the endothelium. Thus, the ultimate effect of any given stimulus for NO· release is a balance between the vasodilator effect of NO· versus the direct vasoconstrictive effect of the stimulus on the vascular smooth muscle. A clinically important aspect of this important role of the endothelium is that it is impaired in a variety of diseases and conditions. These include hypertension, hypercholesterolemia, diabetes, transplant atherosclerosis, congestive heart failure, and cigarette smoking. NO· has a variety of beneficial, potentially antiatherogenic effects in the vessel wall. These include inhibition of platelet aggregation, leukocyte adhesion, and inhibition of adhesion molecule expression. It is conceivable, therefore, that loss of an NO· effect in various disease conditions could contribute to the atherosclerotic process. On the basis of these considerations, …

Background We have shown that hypercholesterolemia increases vascular superoxide anion (O 2 − ) production, which could be responsible for augmented inactivation of endothelium-derived vascular relaxing factor. We sought to determine whether this increased vascular O 2 − production is due to infiltration of macrophages into the intima and whether dietary treatment of hypercholesterolemia normalizes O 2 − production. Methods and Results A specific and sensitive assay for O 2 − based on chemiluminescence of lucigenin was used; the amount of O 2 − produced by vascular ring segments was quantified based on known quantities of O 2 − produced by xanthine–xanthine oxidase standards. O 2 − production of aortic segments from normal rabbits (n=9), cholesterol-fed rabbits (1% cholesterol diet for 1 month, n=7), and rabbits fed a 1% cholesterol diet for 1 month followed by a normal diet for 1 month (regression rabbits, n=5) was measured. At the end of these diets, serum cholesterol levels were 1.5±0.2, 26.0±3.9, and 1.8±0.5 mmol/L (58±6, 1000±150, and 71±19 mg/dL) in the normal, cholesterol-fed, and regression animals, respectively. Vessels from normal rabbits with endothelium produced 0.32±0.06 nmol O 2 − /mg dry wt per minute, whereas those without endothelium produced approximately twice as much O 2 − (0.66±0.12 nmol O 2 − mg dry wt per minute. Vessels with endothelium from cholesterol-fed rabbits produced 4.5-fold more O 2 − than vessels from normal animals. This increased production of O 2 − was normalized by endothelial removal. This increased production of O 2 − was not due to infiltration of macrophages in the intima, because there was no correlation between vascular O 2 − production and macrophage infiltration assessed by immunohistochemistry with use of a specific antibody against rabbit macrophage. O 2 − production by vessels from regression rabbits was similar to that observed in normal animals, and as in the normal rabbits, endothelial removal increased O 2 − production. Aortic rings from these animals also were studied in organ chambers. Dietary lowering of cholesterol dramatically improved vasodilator responses to acetylcholine and A23187 ( P Conclusions Dietary lowering of cholesterol not only improves endothelium-dependent vascular relaxation but also normalizes endothelial O 2 − production. Decreases of O 2 − production by dietary lowering of cholesterol not only may improve vasomotor control but also may improve other aspects of vascular integrity in atherosclerosis.

The nitrovasodilators are a diverse group of pharmacological agents that produce vascular relaxation by releasing nitric oxide. The mechanisms by which these compounds release nitric oxide vary, depending on their chemical structure. Compounds with lower oxidation states of nitrogen such as nitroprusside, nitrosamines, and nitrosothiols release nitric oxide nonenzymatically. In the case of nitroprusside, this involves a one-electron reduction that may occur upon exposure to a variety of reducing agents and tissues such as vascular smooth muscle membranes. In the case of the organic nitrates, which have higher oxidation states of nitrogen, the release of nitric oxide in vascular tissue occurs predominantly by a poorly understood enzymatic process. This interesting property of nitroglycerin is important because it "targets" its effect to vascular tissues that are capable of this enzymatic process. In the case of the coronary circulation, nitroglycerin predominantly dilates the larger coronary arteries while having a minimal effect on coronary resistance vessels < 100 microns in diameter. This prevents the development of coronary steal, which is often encountered with agents that produce intense vasodilation of the coronary resistance vessels. In this review, the mechanisms by which the nitrovasodilators (particularly nitroglycerin) release nitric oxide will be considered, and recent studies of nitroglycerin bioconversion in various-sized coronary vessels will be discussed in detail.

During the past decade, numerous experimental and clinical studies have demonstrated that many common conditions predisposing to atherosclerosis, such as hypercholesterolemia, hypertension, diabetes, and smoking, are associated with a reduced vascular availability of nitric oxide (NO•). Nitric oxide not only produces vasodilation but also has potent antiatherogenic properties. These properties include inhibition of platelet aggregation, prevention of smooth muscle cell proliferation, reduction of lipid peroxidation, and inhibition of adhesion molecule expression. Thus, the loss of NO• observed in these various conditions not only alters vascular tone but also may explain in part why these conditions are risk factors for atherosclerosis. See p 2673 Given this apparent link between loss of nitric oxide and atherosclerosis, several groups have been interested in the concept that endothelium-dependent vasodilation, a surrogate for NO• bioavailability, may predict cardiovascular events. Indeed, Suwaidi et al1 followed 157 patients with mildly diseased coronary arteries for an average of 28 months and observed cardiac events only in the patients with the lowest tertile of coronary vasodilation to acetylcholine. Similarly, in a study of 147 patients, Schachinger et al2 used 3 different stimuli for endothelial release of NO: acetylcholine, cold pressor testing, and increased blood flow. The authors showed that responses to each of these stimuli were independent predictors of cardiovascular events during a follow-up period of ≈8 years. Perticone et al3 also demonstrated that endothelial dysfunction in the forearm circulation predicts cardiovascular events in hypertensive subjects. There have been several explanations for why the various risk factors impair endothelial function. One that has received substantial attention is increased production of reactive oxygen species within the vessel.4 In particular, superoxide (O2•-) reacts rapidly with NO•, resulting in the formation of the peroxynitrite anion and loss of NO• …

We agree with the proposal by Antoniades et al that folate supplementation may be an effective way of restoring cardiovascular tetrahydrobiopterin (BH4) concentrations. On the other hand, a pilot study from our group demonstrated that oral BH4 improves endothelial nitric oxide bioavailability in hypertensive patients. …

BACKGROUND L-Methionine potentiates systemic hemodynamic effects of intravenous glyceryl trinitrate (GTN) in tolerant and nontolerant patients to a similar extent as N-acetylcysteine (NAC). This potentiation of GTN action by L-methionine has been attributed to enhanced intracellular formation of nitrosothiols, known to be potent stimulators of soluble guanylyl cyclase. This study was performed to analyze directly the effects of L-methionine on GTN-induced dilation of large epicardial arteries and the venous capacitance system of the dog in the tolerant and nontolerant states. Cultured rat aortic vascular smooth muscle cells and purified guanylyl cyclase were used to study potential intracellular and extracellular mechanisms responsible for this interaction. METHODS AND RESULTS In awake nontolerant dogs, L-methionine (100 mg/kg) potentiated the tachycardic response to GTN (5.0 and 15 micrograms/kg/min) and enhanced the hypotensive action of GTN (1.5 and 5.0 micrograms/kg/min) in anesthetized, nonreflexic dogs. In nontolerant and tolerant dogs, however, L-methionine did not alter the dose-response of large epicardial artery dilation to intravenous GTN challenges and did not modify nitrate tolerance of the low pressure system of the dog. The infusion of L-methionine (100 mg/kg) significantly increased plasma methionine levels (from 52 +/- 12 to 1,141 +/- 239 microM), cystine levels (from 12 +/- 4 to 26 +/- 7 microM), but not homocystine levels. In vitro, the L-methionine conversion product L-cysteine (0.1-1.0 mM) but not homocysteine significantly enhanced the augmentation of purified guanylyl cyclase activity by GTN (100 microM). Incubation of cultured rat aortic smooth muscle cells with L-methionine (10 microM or 1 mM) did not result in a significant increase of free intracellular sulfhydryl group content. CONCLUSIONS The L-methionine conversion product L-cysteine mediates tolerance independent the potentiation of GTN action. This may result from an L-cysteine-induced formation of a vasoactive metabolite of GTN (nitric oxide) or nitrosothiol. This effect occurs primarily in the resistance vessel circulation, not in large epicardial arteries and veins. The lack of effect of L-methionine on sulfhydryl group content in large conductance vessels indicates that hepatic L-methionine metabolism constitutes the significant source of L-cysteine. These findings strongly suggest that administration of sulfhydryl-group precursor L-methionine does not represent a therapeutic alternative to a nitrate-free interval to restore nitrate sensitivity in tolerant large epicardial arteries and veins.

Increased peripheral vascular resistance is a hallmark of advanced chronic congestive heart failure (CHF) and contributes to the phenomenon of “increased afterload” that complicates this condition. Multiple factors probably underlie this phenomenon, including increased water and sodium content of the vasculature, increased neurohormonal activation, and intrinsic abnormalities of the vasculature.1 During the past decade, it has also been shown that endothelium-dependent vasodilation is strikingly abnormal in both experimental animals and humans with compromised cardiac function.2 3 Given that endothelial regulation of vasomotion plays a major role in the control of systemic hemodynamics, this phenomenon is probably a major cause of increased systemic vascular resistance and afterload in heart failure. Because endothelial regulation of vascular tone is mediated predominantly by endothelium-derived nitric oxide (NO), numerous studies have examined abnormalities of the l-arginine/NO pathway in heart failure. Two major abnormalities have surfaced as important. Several studies have suggested that CHF leads to a decline in expression of endothelial cell NO synthase (eNOS), which is ultimately responsible for endothelial production of NO. Wang et al4 produced heart failure in dogs by cardiac pacing at a rapid rate for 4 weeks. Microvascular endothelial cells released markedly less nitrite (the stable degradation product of NO) than cells from hearts of normal animals. Using the same heart failure model, Smith and colleagues5 observed a decrease in the expression of both eNOS and cyclooxygenase 1, the enzyme responsible for production of prostacyclin. CHF is also associated with increased circulating levels of the cytokine TNF-α.6 In vitro, TNF-α increases degradation of eNOS mRNA, most likely via …

Oxidative stress is strongly implicated in the pathogenesis of hypertension; however, the role of mitochondrial oxidative stress is not clear. We have investigated the role of mitochondria in endothelial dysfunction and hypetention using cultured bovine endothelial cells (BAECs) and mice infused with angiotensin II (AngII). Production of O 2 •− was measured by dihydroethidium and HPLC. Nitric oxide and H 2 O 2 were detected by ESR. Sytolic blood presure was measured in mice infused with saline, AngII (0.7mg/kg/day), AngII plus mitochondria targeted antioxidant mitoTEMPO or non-targeted antioxidant TEMPOL (50 μg/kg/day). AngII significantly increased mitochondrial H 2 O 2 . MitoTEMPO blocked AngII-induced oxidative stress and restored NO • production in BAECs and mouse aorta. Supplementation of BAECs with malonate, inhibitor of complex II, or rotenone abolished AngII induced oxidative stress. Interestingly, inhibition of mitochondrial oxidative stress decreased the activity of NADPH oxidases. Co-infusion of mitoTEMPO with AngII significantly attenuated the increase in the blood pressure by 30-mm Hg (Fig. 1 ), while i.p. injection to hypertensive mice decreased the blood presure by 20-mm Hg. We suggest that mitoTEMPO can be used as an effective inhibitor of the oxidative stress induced by Ang II as well as an antihypertensive agent and, thereby, can be effective in ameliorating hypertension-related nephrosclerosis, diabetic nephropathy, or atherosclerosis. Figure 1. (A) Attenuation of hypertensive effect of Ang II in C57Blk/6 mice by co-infusion of mitoTEMPO. (B) Dose dependent effect of mitoTEMPO (50, 150 and 500 μg/kg/day) after 14-days of Ang II and drug administration. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Previous in vivo studies have shown that vasopressin, which releases the endothelium-derived relaxing factor and constricts coronary smooth muscle, produces augmented constriction of coronary microvessels perfused by mature collaterals. We hypothesized that chronic perfusion through collaterals produces endothelial dysfunction in the recipient vasculature. Mature collaterals were stimulated in mongrel dogs by the ameroid constrictor technique. After 3-6 months, rings of conduit vessels (obtuse marginals) were studied in organ chambers, and coronary microvessels (100-220 microns) were studied in a pressurized, no-flow state with a microvessel imaging apparatus. Eleven dogs were used as controls. Large vessels were preconstricted with prostaglandin F2 alpha to 30-70% of the maximum potassium chloride tension, and microvessels were preconstricted to 20-60% of the baseline diameter with the thromboxane mimetic U46619. Relaxations to the receptor-mediated agents acetylcholine and ADP were markedly impaired in collateral-dependent coronary microvessels, whereas relaxations to nitroglycerin were enhanced compared with microvessels from control dogs. Relaxation to the calcium ionophore A23187, which releases the endothelium-derived relaxing factor through nonreceptor-mediated mechanisms, were similar in control and ameroid microvessels. Constriction to vasopressin was augmented in collateral-dependent microvessels compared with controls. Responses to all agonists were similar between control and collateral-dependent large vascular rings. In conclusion, chronic perfusion through collateral vessels selectively impairs receptor-mediated endothelium-dependent relaxations and augments constriction to vasopressin in the coronary microcirculation. These findings may have important implications regarding neurohumoral regulation of perfusion to collateral-dependent myocardium.

In this issue of Circulation ,1 Zhang and coworkers provide convincing evidence that ACE inhibitors reduce myocardial oxygen consumption by preventing the degradation of bradykinin, which subsequently stimulates the release of nitric oxide (NO). They propose that NO inhibits mitochondrial respiration, thus reducing myocyte oxygen use. This is an exciting observation because it provides a new mechanism whereby ACE inhibitors might benefit ischemia beyond simply inhibiting the formation of angiotensin II, thus reducing the vasoconstrictor effect of this octapeptide. These results are further evidence for a role of bradykinin in the response of tissues to ACE inhibitors. The mechanism enabling these interactions to occur is coming to light on the basis of studies such as the current one from Zhang et al. Interest in bradykinin and related kinins as cardiovascular mediators has grown tremendously in the past several years, largely because it has been recognized that they potently stimulate release of NO and prostacyclin from endothelial cells. Bradykinin and Lys-bradykinin, respectively, are formed when one of a family of enzymes, known as kallikreins, act on either heavy- or low-molecular-weight kininogens.2 This action involves cleavage of amino acids from both the C- and N-terminal ends of the kininogen peptides and takes place both in plasma (for heavy-molecular-weight kininogen) and tissues (for low-molecular-weight kininogen). The kininogens were previously thought to be expressed primarily by hepatocytes; however, it is now clear that many tissues, including vascular cells3 4 and the heart,5 also express kininogens. The kallikreins as well are made by many cell types and are produced by vascular cells in a continuous fashion.6 Thus, many tissues, including the heart and vascular cells, have an intact kallikrein/kininogen system capable of producing bradykinin. The mechanisms whereby bradykinin production is regulated remain unclear; however, there is some evidence that flow …

Background— Atrial fibrillation (AF) is associated with an increased risk of stroke due almost exclusively to emboli from left atrial appendage (LAA) thrombi. Recently, we reported that AF was associated with endocardial dysfunction, limited to the left atrium (LA) and LAA and manifest as reduced nitric oxide (NO · ) production and increased expression of plasminogen activator inhibitor-1. We hypothesized that reduced LAA NO · levels observed in AF may be associated with increased superoxide (O 2 ·− ) production. Methods and Results— After a week of AF induced by rapid atrial pacing in pigs, O 2 ·− production from acutely isolated heart tissue was measured by 2 independent techniques, electron spin resonance and superoxide dismutase–inhibitable cytochrome C reduction assays. Compared with control animals with equivalent ventricular heart rates, basal O 2 ·− production was increased 2.7-fold ( P P P 2 ·− production was observed using a cytochrome C reduction assay. The increases could not be explained by changes in atrial total superoxide dismutase activity. Addition of either apocyanin or oxypurinol reduced LAA O 2 ·− , implying that NADPH and xanthine oxidases both contributed to increased O 2 ·− production in AF. Enzyme assays of atrial tissue homogenates confirmed increases in LAA NAD(P)H oxidase ( P =0.04) and xanthine oxidase ( P =0.01) activities. Although there were no changes in expression of the NADPH oxidase subunits, the increase in superoxide production was accompanied by an increase in GTP-loaded Rac1, an activator of the NADPH oxidase. Conclusions— AF increased O 2 ·− production in both the LA and LAA. Increased NAD(P)H oxidase and xanthine oxidase activities contributed to the observed increase in LAA O 2 ·− production. This increase in O 2 ·− and its reactive metabolites may contribute to the pathological consequences of AF such as thrombosis, inflammation, and tissue remodeling.

In the current Adult Treatment Panel guidelines for cardiovascular risk detection,1 the plasma-based markers recommended for use in global risk assessment or in the definition of the metabolic syndrome are low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, and triglycerides. It is widely recognized, however, that more than half of all future vascular events occur in individuals without overt hyperlipidemia. For example, in a recent large-scale analysis of >27 000 healthy American women, 77% of all future events occurred in those with LDL-C levels

Background— Impaired flow-dependent, endothelium-mediated vasodilation (FDD) in patients with chronic heart failure (CHF) results, at least in part, from accelerated degradation of nitric oxide by oxygen radicals. The mechanisms leading to increased vascular radical formation, however, remain unclear. Therefore, we determined endothelium-bound activities of extracellular superoxide dismutase (ecSOD), a major vascular antioxidant enzyme, and xanthine-oxidase, a potent radical producing enzyme, and their relation to FDD in patients with CHF. Methods and Results— ecSOD and xanthine-oxidase activities, released from endothelium into plasma by heparin bolus injection, were determined in 14 patients with CHF and 10 control subjects. FDD of the radial artery was measured using high-resolution ultrasound and was assessed before and after administration of the antioxidant vitamin C (25 mg/min; IA). In patients with CHF, endothelium-bound ecSOD activity was substantially reduced (5.0±0.7 versus 14.4±2.6 U · mL −1 · min −1 ; P r =0.61). Endothelium-bound xanthine-oxidase activity was increased by >200% (38±10 versus 12±4 nmol O 2 ·− · μL −1 ; P r =−0.35) in patients with CHF. In patients with low ecSOD and high xanthine-oxidase activity, a greater benefit of vitamin C on FDD was observed, ie, the portion of FDD inhibited by radicals correlated negatively with ecSOD ( r =−0.71) but positively with xanthine-oxidase ( r =0.75). Conclusions— These results demonstrate that both increased xanthine-oxidase and reduced ecSOD activity are closely associated with increased vascular oxidative stress in patients with CHF. This loss of vascular oxidative balance likely represents a novel mechanism contributing to endothelial dysfunction in CHF.

One of the most effective approaches in the treatment of atherosclerosis has been the use of 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitors (statins) to treat hypercholesterolemia. During the past decade, numerous studies involving >20 000 individuals have shown that these drugs dramatically reduce cardiovascular death, myocardial infarction, unstable angina, and stroke. Statin therapy prevents events in individuals with established cardiovascular disease and is also effective in primary prevention.1 A major mechanism by which lipid lowering is thought to improve outcome is by preventing the development of new atherosclerotic lesions and by depleting lipids from established plaques (ie, plaque stabilization).2 3 A striking finding is that statins seem to decrease clinical events within a few months of the onset of therapy.4 This suggests that they may have beneficial effects beyond those of plaque stabilization and lesion prevention. One such beneficial effect might be the restoration of nitric oxide production by the endothelium. Endothelium-derived nitric oxide, previously known as the endothelium-derived relaxing factor, modulates vasodilatation and prevents platelet adhesion, the expression of adhesion molecules, and smooth muscle cell proliferation.5 Nitric oxide has also been shown to have antioxidant effects by enhancing the expression of superoxide dismutase,6 preventing lipid-chain reactions,7 and reacting with the superoxide anion.5 Thus, nitric oxide seems to play a major role as an endogenous protective factor against atherosclerosis. Indeed, virtually every atherosclerosis risk factor is associated with decreased endothelial cell nitric oxide production, and it is likely that this loss of nitric oxide is a major reason why these conditions predispose to vascular lesion development. …

Background Heart rate variability is an important prognostic indicator for sudden death. An increased risk of sudden death and arrhythmia is associated with reduced heart rate variability in heart failure. In heart failure, there is also dilatation of the atria, which raises the prospect that there could be some physiological basis to possibly link heart rate variability with atrial dilatation. We therefore investigated whether sustained atrial stretch could modulate heart rate variability directly. Methods and Results Pigs were anesthetized and their hearts exposed. A specially built device stretched the sinoatrial node before and after vagal section and then after administration of propranolol. Stretch of the sinoatrial node decreases heart rate variability in the following ways: The standard deviation of the beat-to-beat interval decreases (4.2 to 2.6 ms; P =.004), and the high-frequency components are reduced (control, 6.5±2.2 ms 2 ; during stretch, 1.4±0.3 ms 2 ; P =.003). After section of both vagi, the high-frequency components are reduced by stretch of the sinoatrial node (2.8±0.9 ms 2 for control versus 1.2±0.3 ms 2 during stretch; P =.05). Similarly, after both vagal section and β-blockade, stretch of the sinoatrial node reduces the high-frequency components (10.6±3.5 ms 2 for control versus 3.0±1.5 ms 2 during stretch; P =.01). Conclusions We conclude that stretch of the sinoatrial node reduces high-frequency heart rate variability. This may account in part for the reduced heart rate variability seen in clinical conditions in which the right atrium is dilated, such as congestive cardiac failure.

Background This experiment sought to determine the effects of fish oil on superoxide anion production in the myocardium and coronary arteries of atherosclerotic monkeys. Recent evidence indicates that dietary supplementation with fish oil preserves normal vasomotion of atherosclerotic coronary arteries and reduces damage to the myocardium after ischemia and reperfusion, although the mechanisms remain unclear. Methods and Results Adult male cynomolgus monkeys were fed an atherogenic diet with (n=15) or without (n=15) half the fat calories from fish oil. After 12 months, chemiluminescence of lucigenin was used to measure superoxide anion production in coronary arteries and myocardium after 1 hour of ischemia and 2 hours of reperfusion. The signals were calibrated with known quantities of xanthine and xanthine oxidase. Superoxide anion production in ischemic myocardium was (mean±SEM, nmol/mg dry wt per minute) 1±1 and 4±1 in monkeys fed fish oil and not fed fish oil, respectively ( P P P Conclusions Dietary supplementation with fish oil reduced vascular superoxide anion production and prevented the increase in vascular and myocardial superoxide anion production that accompanied ischemia and reperfusion. These phenomena may underlie some of the beneficial cardiovascular effects of fish oil.

Chronic and acute hypertension have multiple untoward effects on the coronary circulation, several of which may either mimic or markedly worsen the clinical manifestations of coronary artery disease. Early after the onset of left ventricular hypertrophy secondary to hypertension, coronary vasodilator reserve is significantly impaired. During cardiac hypertrophy secondary to hypertension, the coronary arteries fail to enlarge in concert with ventricular enlargement. This failure results in a relative decrease by approximately 50% in the ratio of epicardial vessel diameter to the mass of myocardium perfused. The lower range of coronary subendocardial autoregulation is altered by chronic renovascular hypertension. A variety of vascular smooth muscle homeostatic mechanisms are abnormal in genetic models of hypertension, as is endothelium-dependent vascular relaxation. Acute hypertension may enhance constriction to serotonin, most likely through the release of potent vasoconstrictor substances from leukocytes and platelets that adhere to the endothelium as a result of endothelial damage. Finally, many of the consequences of myocardial infarction are worsened in the setting of hypertension and left ventricular hypertrophy.

Mature coronary collaterals, which develop during chronic coronary occlusion, are not simply passive conduits but are capable of active vasomotion. Collateral perfusion must traverse not only these vessels but also proximal and distal coronary vessels. This series of resistances significantly modulates perfusion to collateral-dependent and potentially ischemic myocardium. The collateral vessels themselves possess unique vasomotor characteristics, particularly markedly augmented constriction to vasopressin. The recipient coronary microcirculation develops endothelial dysfunction during collateral development, a phenomenon that may markedly alter neurohumoral regulation of perfusion to collateral-dependent myocardium. Finally, the resistances proximal to the origin of the collateral vasculature, which are negligible at rest, become significant when flow to nonischemic regions (non-collateral-dependent) is increased, predisposing to the collateral steal phenomenon. Although collateral vessels play a crucial role in preventing myocardial infarction and often restore both resting and exercise perfusion to normal, the need to use these vessels is associated with important alterations of regulatory mechanisms in the coronary circulation.

BACKGROUND The endothelium-derived relaxing factor has been shown to be nitric oxide or a related nitroso compound, synthesized by the enzyme nitric oxide synthetase, which oxidizes the guanidono nitrogens of arginine. This enzyme is activated by increases in cytosolic calcium. The effect of the clinically used calcium channel antagonists on this process is controversial. The present study was performed to determine whether calcium channel blockade with these pharmacologic agents would alter the activity of nitric acid synthetase in intact endothelial cells. METHODS AND RESULTS A specific and sensitive chemiluminescence assay was used to measure the release of nitrogen oxides (nitric oxide and one-electron oxidation products of nitric oxide) from bovine aortic endothelial cells grown in culture. Under basal conditions, the release of nitrogen oxides was about 0.2 nmol/100 micrograms protein/hr. Bradykinin doubled this response. Removal of extracellular calcium abolished basal and bradykinin-stimulated release of nitrogen oxides. Neither diltiazem, verapamil, nor nifedipine in concentrations that are encountered clinically altered the release of nitrogen oxides. CONCLUSIONS These experiments show that although the production of nitrogen oxides is dependent on extracellular calcium, the clinically used calcium channel antagonists do not inhibit the release of the endothelium-derived relaxing factor.

We examined the effects of ischemia with and without reperfusion on endothelium-dependent and -independent vascular relaxation in both conduit and resistance coronary arteries. Studies were performed on dogs under control conditions (n = 13) or after 1 hour of circumflex coronary artery occlusion with (n = 10) or without (n = 8) 1 hour of reperfusion. Rings of obtuse marginal branches of the left circumflex coronary artery (conduit arteries) were studied in organ chambers. Coronary microvessels (110-220-microns diameter) were studied in a pressurized state with an in vitro microvessel imaging apparatus. Relaxation was evaluated after preconstriction with prostaglandin F2 alpha and U46619 (a thromboxane A2 analogue) in conduit and resistance vessels, respectively. Conduit vessel function was not altered by ischemia with or without reperfusion. Endothelium-dependent microvascular relaxation was depressed in response to acetylcholine, ADP, and calcium ionophore A23187 after ischemia with reperfusion compared with control relaxation (ED50 as -log[M]: 6.0 +/- 0.2 [p less than 0.05], 5.1 +/- 0.4 [p less than 0.05], and 5.8 +/- 0.1 versus 6.8 +/- 0.2, 6.8 +/- 0.2, and 6.6 +/- 0.2, respectively). Ischemia without reperfusion modestly altered microvascular endothelium-dependent relaxation. Microvascular relaxation to nitroglycerin was not altered by ischemia with reperfusion. We conclude that 1) endothelium-dependent relaxation in large epicardial coronary arteries is relatively refractory to ischemia with or without reperfusion, 2) ischemia alone produces mild alterations of coronary microvascular reactivity, 3) ischemia followed by reperfusion produces a marked and selective impairment of endothelium-dependent responses in the coronary microcirculation.

Atherosclerosis impairs endothelium-dependent relaxation of large conduit arteries. Because coronary resistance vessels are spared from the development of overt atherosclerosis, endothelium-dependent responses were examined in these vascular segments. Malaysian cynomolgus monkeys (n = 6) were made atherosclerotic by being fed a 0.7% cholesterol diet for 18 months. Control monkeys (n = 6) were fed a standard diet. Coronary microvessels (122-220 microns) were studied in a pressurized (20 mm Hg), no-flow state using a video-imaging apparatus. Relaxations of microvessels, preconstricted with the thromboxane analogue U46619, were determined in response to acetylcholine, bradykinin, the calcium ionophore A23187, adenosine, and sodium nitroprusside. Microvascular relaxations to bradykinin and A23187 were reduced in atherosclerotic monkeys compared with controls, whereas acetylcholine produced additional contraction in atherosclerotic monkeys. Responses of preconstricted microvessels to adenosine and sodium nitroprusside were identical in atherosclerotic and control animals. Indomethacin did not alter responses in control or atherosclerotic animals. Histologic examination revealed neither intimal thickening nor plaque formation in microvessels of this size class despite marked changes in conduit arteries. Electron microscopy showed minor alterations of endothelial cell morphology in microvessels of atherosclerotic animals. In conclusion, long-term hypercholesterolemia markedly impairs endothelium-dependent vascular relaxation in the coronary microcirculation where overt atherosclerosis does not develop. These changes in endothelial cell function may significantly alter regulation of myocardial perfusion by neurohumoral stimuli.

The results of previous work from this laboratory have shown a poor correlation between percent stenosis (determined visually with calipers) and the coronary reactive hyperemic response (an index of maximal coronary vasodilator capacity) determined during cardiac surgery. This study was performed to determine whether other parameters of lesion severity could predict the reactive hyperemic response and thus the hemodynamic significance of coronary stenoses in human beings. Twenty-three patients with lesions in the proximal left anterior descending coronary artery were studied. To account for differences in expected vessel size, patients with large diagonal branches (greater than one-half the diameter of the left anterior descending artery) arising before the lesion were excluded. Computer-assisted quantitative coronary angiography was used to measure percent diameter stenosis, percent area stenosis, and minimal stenosis cross-sectional area. With a pulsed Doppler velocity probe, reactive hyperemic responses were recorded after a 20 sec coronary occlusion of the left anterior descending artery at cardiac surgery before cardiopulmonary bypass and were quantified by the peak/resting velocity ratio (normal greater than 3.5:1). Percent area stenosis ranged from 7% to 54% for vessels with normal reactive hyperemic responses and from 27% to 94% for vessels with abnormal reactive hyperemic responses. With both percent diameter stenosis and percent area stenosis there was substantial overlap between vessels with normal and abnormal reactive hyperemic responses. In contrast, nine of nine vessels with normal reactive hyperemic responses had lesion minimal cross-sectional areas of greater than 3.5 mm2 and 13 of 14 vessels with abnormal reactive hyperemic responses had minimal cross-sectional areas of less than 3.5 mm2.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown that dogs with renal hypertension and left ventricular hypertrophy have larger infarcts (per risk area size) than do control animals. A potential explanation for this is that collateral resistance is higher in these dogs. Paradoxically, previous postmortem studies in human hearts with left ventricular hypertrophy have suggested that coronary collaterals are actually increased in this condition. To test the hypothesis that left ventricular hypertrophy is associated with alterations in coronary collateral resistance, studies were performed in dogs with renal hypertension and left ventricular hypertrophy and in patients with aortic valvular disease at the time of cardiac surgery. With an isolated, adenosine-vasodilated, blood-perfused cardiac preparation, collateral and normal zone pressure-flow relationships were established by means of radioactive microspheres in nine dogs with renal hypertension and left ventricular hypertrophy and in 17 controls. Collateral resistance calculated from these pressure-flow relationships were similar in both groups (4.0 +/- 0.7 in dogs with renal hypertension and left ventricular hypertrophy and 3.9 +/- 0.4 mm Hg/ml/min/100 g in controls). In addition, normal zone resistance was not different between groups (transmural resistances 0.17 +/- 0.01 in controls and 0.18 +/- 0.02 in dogs with renal hypertension and left ventricular hypertrophy. In five patients with aortic valve disease, left ventricular hypertrophy, and normal coronary arteries and in six patients without left ventricular hypertrophy who had normal left anterior descending coronary arteries, a 7 MHz suction-mounted echo transducer was used to monitor systolic wall thickening during transient occlusions of the left anterior descending artery at the time of cardiac surgery. Because noncollateralized myocardium ceases to contract promptly after coronary occlusion, this approach provides an indirect index of collateral perfusion. Twenty seconds after the onset of coronary occlusion, systolic thickening had markedly decreased in both groups (15 +/- 10% of control values in nonhypertrophied hearts and 10 +/- 10% in hearts with left ventricular hypertrophy; p = NS between groups). Thus the severity of contraction abnormality induced during transient coronary occlusion in these two groups of patients was similar, suggesting that the degree of severity of ischemia was comparable between the two groups. We conclude that collateral resistance is not altered by hypertension and left ventricular hypertrophy and that left ventricular hypertrophy in patients is not associated with functional evidence of an enhanced collateral circulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Although immediate and late changes in coronary stenoses after percutaneous transluminal coronary angioplasty (PTCA) have been reported, most investigators have employed qualitative or semiquantitative techniques to analyze the angiograms. Such data is not optimal because of considerable interobserver variability and the use of relative instead of absolute changes in lesion geometry. Analysis is further compounded by the indistinct edges that characterize coronary lesions immediately after angioplasty. To quantify the changes in minimal cross-sectional area (MCSA) of the coronary lumen that occur during and after PTCA, we analyzed the angiograms of 23 patients before PTCA, immediately after PTCA, and at 7.2 +/- 3.0 (mean +/- SD) months follow-up using two computer-assisted methods of angiographic analysis--quantitative coronary angiography (QCA) and videodensitometry (VID). QCA provides an absolute measure of the area of the lumen; VID is a nongeometric method that is not dependent on exact border recognition. Based on these quantitative methods, we found that successful angioplasty is associated with about a three-fold increase in the MCSA of the lesion (from 1.0 to 3.2 mm2). This area is, however, well below normal and is less than half of the average MCSA of the inflated dilating balloon. Analysis of follow-up angiograms demonstrated that eight of 23 patients had a substantial late increase in the MCSA of the lesion (from 2.7 to 4.1 mm2) after the angioplasty procedure. Clinical, hemodynamic, and angiographic characteristics immediately after PTCA were not predictive of MCSA of the lumen at follow-up.(ABSTRACT TRUNCATED AT 250 WORDS)

These studies were performed to test the hypothesis that left ventricular hypertrophy arising as a complication of chronic hypertension is associated with impaired coronary autoregulation. Twelve dogs with hypertension and left ventricular hypertrophy (one-kidney, one-clip model) and 11 normal dogs were instrumented and subsequently studied while conscious. Circumflex pressure, measured with an intracoronary catheter, was adjusted to 100, 75, and 40 mm Hg with a hydraulic occluder that was placed proximally. At each circumflex pressure, myocardial perfusion was measured with radioactive microspheres. Reduction of circumflex pressure over this range did not significantly alter heart rate, left atrial pressure, or arterial pressure. In normal dogs, reduction of circumflex pressure did not alter total myocardial perfusion or the transmural distribution of perfusion. In contrast, in dogs with hypertension and left ventricular hypertrophy, circumflex subendocardial perfusion decreased 46% when pressure was decreased from 100 to 40 mm Hg (p less than .05 compared with normal). Autoregulation was quantified for each third of myocardium with the use of autoregulatory gain values (1 = perfect autoregulation; 0 = the absence of autoregulation). For pressure changes of 100 to 75 mmHg, values for autoregulatory gain were near unity for all layers of myocardium in both groups of animals. When pressure was decreased from 75 to 40 mm Hg, values for autoregulatory gain among the normal and hypertensive groups were, respectively: for subepicardium 1 +/- 0.2 (mean +/- SE) vs 0.9 +/- 0.2 (p = NS), for the midwall 0.8 +/- 0.2 vs 0.5 +/- 0.2 (p = NS), and for the subendocardium 0.8 +/- 0.1 vs 0.1 +/- 0.2 (p less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

The absent pulmonary valve syndrome consists of a severely hypoplastic pulmonary valve with anular stenosis, aneurysmal dilatation of the main pulmonary artery with one or both pulmonary branches also dilated, and a ventricular septal defect. From 1955 to 1975, 15 patients were seen at Texas Children's Hospital with this syndrome. Although anatomically similar to tetralogy of Fallot, the most significant symptoms during early infancy were secondary to bronchial compression resulting from the dilated pulmonary arteries and enlarged left atrium. Intracardiac repair performed on two infants at four months of age failed to ameliorate the respiratory symptoms and both died. Seven patients from 2.3 to 16 years of age were operated upon, with one late postoperative death. Five other patients have not yet received surgical intervention. We recommend vigorous continual respiratory therapy for for infants and small children and intracardiac repair in older children since repair in infancy does not relieve respiratory symptoms. We do not recommend a prosthetic pulmonary valve unless there is persistently elevated main pulmonary artery pressure.

Myocardial infarct extension, defined as reelevation or reappearance of creatine phosphokinase-MB (CK-MB) 48 hours after the onset of symptoms, was evaluated prospectively in 56 consecutive patients with acute myocardial infarction. Myocardial infarct extension occurred in eight patients (14%). The sensitivity, specificity and predictive accuracy in the diagnosis of myocardial infarct extension were 63%, 85% and 42%, respectively, for recurrent chest pain requiring morphine; 50%, 65% and 19% for recurrent ST-segment elevation on routine 12-lead ECGs; and 88%, 63% and 28% for reelevation of total CK. Three of the eight episodes of extension were clinically silent. Four of eight patients (50%) with extension died, compared with one of 46 patients (2%) without extension (p = 0.0009). CK-MB persisted for 72 hours or longer in 16 patients and identified seven of eight patients who subsequently had infarct extension. We conclude that myocardial infarct extension is an infrequent complication of acute myocardial infarction and is associated with a very high mortality rate. Persistence of CK-MB for 72 hours or more identifies a subgroup of patients at high risk for subsequent infarct extension and death.

During the past decade our understanding of the complex interaction between cardiac muscle and coronary vascular growth has increased substantially. Some types of cardiac hypertrophy, for example, left ventricular hypertrophy secondary to hyperthyroidism, are associated with increased coronary vascular growth. However, in most animal preparations of hypertrophy and in several clinical types of hypertrophy of the left and/or right ventricles, pathologic cardiac enlargement impairs the ability of the coronary circulation to allow normal increases and perfusion in response to intense dilator stimuli. In general, clinical studies have demonstrated far more profound abnormalities than studies in experimental animals. These observations provide a plausible explanation of why patients with hypertrophied ventricles often exhibit signs and symptoms of myocardial ischemia in the absence of coronary obstructive disease. The recent observation that experimentally produced left ventricular hypertrophy secondary to renal hypertension augments infarct size and the incidence of sudden lethal arrhythmias has additional implications relevant to the interaction between cardiac hypertrophy and myocardial perfusion. Although coronary reserve is impaired in many types of pathologic hypertrophy, the anatomic or biochemical basis for these observations remains elusive.

In isolated vascular rings, we have shown that mature coronary collateral vessels are highly responsive to the vasoconstrictor effects of vasopressin. The purpose of the present study was to determine the effect of concentrations of vasopressin encountered in pathophysiologic states on the collateral circulation in vivo. We studied eight open-chest anesthetized dogs with mature coronary collateral vessels 3-6 months after placement of an ameroid constrictor on the left circumflex coronary artery. The left anterior descending coronary artery was perfused at constant pressure, and peripheral coronary pressure was monitored continuously throughout each experiment. At baseline and during intracoronary infusion of vasopressin, which resulted in concentrations ranging from 8 +/- 3 to 1,340 +/- 327 microM/ml, we measured regional myocardial perfusion with radiolabeled microspheres. At baseline, regional myocardial perfusion to the collateral-dependent myocardium and to the normally perfused myocardium was similar; however, during vasopressin infusion, collateral-dependent zone flow decreased by 49 +/- 14% whereas normal zone flow decreased by only 9 +/- 9% (p less than 0.0005, normal zone perfusion vs. collateral perfusion). Vasopressin increased transcollateral resistance by 242 +/- 95% above baseline but produced a more modest increase in normal zone resistance (15 +/- 10%). The subendocardial to subepicardial perfusion ratio increased by 28 +/- 12% in the normal zone in response to vasopressin but decreased by 18 +/- 11% in the collateral-dependent zone. These data show that mature coronary collateral vessels are responsive to the vasoconstrictor effects of vasopressin at concentrations encountered in various pathophysiologic states.(ABSTRACT TRUNCATED AT 250 WORDS)

Bilateral determinations of the blood pressures were made nonsimultaneously and simultaneously by the indirect method under basal conditions on 447 patients. In this group 26.6 per cent of the paired measurements by the nonsimultaneous indirect method exhibited systolic differences of 10 mm. Hg or greater and 15 per cent exhibited diastolic differences of this magnitude, whereas only 5.3 per cent of the measurements by the simultaneous indirect method had systolic differences and only 4 per cent had diastolic differences of this degree. This indicates that bilateral determinations of blood pressure must be performed simultaneously on patients being examined for possible inequalities of blood pressure. Bilateral differences in blood pressure obtained by the simultaneous indirect and simultaneous direct methods in 14 normal subjects and 53 selected patients were compared. In the normal subjects, 3 of the 42 paired indirect measurements and none of the paired direct measurements in either the radial or brachial arteries had systolic differences of 10 mm. Hg or greater, and none of the diastolic differences were of this level. In the 53 selected patients, 10 per cent of the indirect and 6 per cent of the direct measurements of systolic blood pressure differed by 10 mm. Hg or more and 8 per cent of the paired indirect measurements of diastolic blood pressure and less than 1 per cent of the paired direct measurements differed by this amount. These differences in bilateral blood pressures were characterized by their inconstancy and lack of agreement with subsequent measurements when studied by both the indirect and direct methods, which apparently separates them from those due to altered hemodynamics from pathologic conditions of the aortic arch or its tributaries. Bilateral differences of blood pressure are of clinical importance when they are great and are reproducible by the direct as well as by the indirect methods, as illustrated by a patient encountered in this study. In addition, 1 case of extreme obesity and 1 of advanced arteriosclerosis were reported. Both patients were found to have pseudohypertension, which was detected in this study. A slight increase in the incidence of bilateral differences in indirect blood pressures was found in a group of patients whose blood pressures were measured while they were in the supine and then in the sitting position. A slight increase in the incidence of bilateral differences was found at higher levels of blood pressure by comparing the blood pressures of nonhypertensive and hypertensive patients and of hypertensive patients before and during treatment with antihypertensive drugs. Bilateral inequalities of blood pressure did not appear to be related to the age or sex of the subjects. Likewise, differences in circumferences of the arm or right or left-handedness did not appear to influence these inequalities, and there was no marked side dominance for differences found. Bilaterally simultaneous, indirect measurements of blood pressure should be carried out on patients with hypertension who will be treated with antihypertensive drugs. Bilateral direct measurements may be used to verify the existence of inequality of blood pressures detected by the indirect method and to determine the correct pressure to be followed. This is particularly important in patients being screened for pheochromocytomas, since such differences may produce false-positive results.