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(Circulation. 1997;95:588-593.)
© 1997 American Heart Association, Inc.

Articles

Role of Superoxide in Angiotensin II–Induced but Not Catecholamine-Induced Hypertension

Jørn Bech Laursen, MD; Sanjay Rajagopalan, MD; Zorina Galis, PhD; Margaret Tarpey, MD; Bruce A. Freeman, PhD; David G. Harrison, MD

the Department of Medicine, Cardiology Division, Emory University School of Medicine (J.B.L., S.R., Z.G., D.G.H.), Atlanta, Ga; the Atlanta (Ga) Veterans Administration Medical Center (D.G.H.); the Department of Anesthesiology (M.T., B.A.F.), University of Alabama at Birmingham; and Medical Department B, Division of Cardiology (J.B.L.), Rigshospitalet, Denmark.

Correspondence to David G. Harrison, MD, PO Box LL, Cardiology Division, Emory University School of Medicine, Atlanta, GA 30322.

	Abstract

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Background The major source of superoxide (·O₂^-) 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 ·O₂^- 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 ·O₂^- 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 ·O₂^- 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 ·O₂^-, likely via degradation of endothelium-derived NO·. Increased vascular ·O₂^- may contribute to vascular disease in high renin/angiotensin II states.

Key Words: hypertension • angiotensin • endothelium-derived factors

	Introduction

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It has become clear that both endothelial and vascular smooth muscle cells are capable of producing superoxide anions (·O₂^-) and other oxygen-derived radical metabolites of ·O₂.^{1 2 3 4 5 6} A major source of ·O₂^- in vascular tissues is a membrane-bound oxidase that uses NADH predominantly as a substrate for electron donation to molecular oxygen.^{1 4 5 6} In cultured rat aortic vascular smooth muscle cells, the activity of this oxidase is increased by relatively low (0.1 to 100 nmol/L) concentrations of angiotensin II.⁷ More recently, we found that treatment of rats with angiotensin II for 5 days increases ·O₂^- production from aortic segments and increases NADH oxidase activity in aortic homogenates.⁸ This increase in vascular ·O₂^- production was associated with impaired endothelium-dependent relaxations to acetylcholine and the calcium ionophore A23187. In those studies, we found that norepinephrine infusion, which produced a similar degree of hypertension, did not increase vascular ·O₂^- production or vascular relaxations. These previous studies were performed in segments of conduit vessels. If angiotensin II had a similar effect in the resistance circulation, an increase in vascular ·O₂^- production might contribute to the hypertension caused by the peptide. We performed the present study to examine this hypothesis. Vascular superoxide dismutase (SOD) levels were increased by daily injections of liposome-entrapped SOD. The effect of this treatment on blood pressure in control rats and rats with either norepinephrine- or angiotensin II–induced hypertension was examined.

	Methods

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Liposome-Entrapped SOD
Liposomes were composed of dioleoyl-phosphatidylethanolamine and dioleoyl-glycero-3-succinate (1:1; Avanti Polar Lipids). Lipids were dried under N₂ and hydrated 36 hours in 210 mmol/L sucrose and 7 mmol/L HEPES. During hydration, pH 8.5 was maintained with tetraethylammonium hydroxide (Sigma Chemical Co). Lipids were added to CuZn SOD (Diagnostic Data Inc) and dissolved in sucrose-HEPES buffer, and the mixture was extruded through a 600-nm filter under N₂· pressure (Extruder, Lipex Biomembranes); mean liposome diameter was 217 nm.

Animal Preparation
Catheters were implanted for drug administration and blood pressure measurements in male Sprague-Dawley rats as described before.⁹ The rats were treated with daily injections of either empty liposomes or liposome-entrapped SOD (1000 U).

Three days after the start of treatment with liposome-entrapped SOD or empty liposomes (day 3), osmotic minipumps containing either angiotensin II (0.7 mg·kg^-1·d^-1, treatment group 1), norepinephrine (2.8 mg·kg^-1·d^-1, treatment group 2), or vehicle (NaCl, treatment group 3) were implanted as previously described.⁸

Arterial Pressure Measurements
The animals were handled daily and exposed to the environment eventually used for blood pressure measurement. On the fifth day of osmotic minipump treatment (day 8), the mean arterial blood pressure (MAP) was measured while the animals were conscious. In addition, blood pressure responses to either acetylcholine (40 µg/kg) or sodium nitroprusside (80 µg/kg) were measured as described previously.⁹ These doses were selected on the basis of preliminary dose-response curves to elicit a fall in MAP of 35 mm Hg (30 to 40 mm Hg) in normal untreated rats. In preliminary experiments, the effect of acetylcholine was completely inhibited by pretreatment with 120 mg/kg L-N-monomethyl arginine, indicating that the hypotensive effect of a bolus of acetylcholine was due to the activation of NO synthase.

In Vitro Vessel Studies
Vessels were studied in organ chambers by the use of methods described previously.⁸ Superoxide (·O₂^-) production was measured by the use of lucigenin chemiluminescence. The details of this assay have been published previously.^{10 11}

Determination of SOD Enzyme Activity
Aortas were homogenized with a Dounce homogenizer in a 50 mmol/L potassium phosphate buffer. SOD activity was determined spectrophotometrically by the ability of the homogenate (50 µg total protein) to inhibit reduction of ferricytochrome c by the superoxide anion generated by the addition of xanthine and xanthine oxidase. Values obtained were represented as units of SOD per milligram of protein. Calibrations were performed with the use of known amounts of purified bovine SOD.

Detection of Macrophages by Immunocytochemistry
Rat macrophages were identified by immunofluorescent staining with the use of a purified monoclonal antibody (PharMingen) recognizing an ED-2–like antigen on rat tissue macrophages.¹² Rat aortas were harvested from three control and three 5-day angiotensin II–treated rats. Specimens of rat spleen and lungs were also collected and processed as positive controls. Tissues were embedded in OCT (Miles) and frozen in liquid nitrogen. Primary and secondary antibodies were diluted with 50 mmol/L Tris-HCl, pH 7.2, containing 1 mg/mL bovine serum albumin (wash buffer) supplemented with 10% horse serum. Frozen tissue sections (8 µm) were fixed for 5 minutes with cold acetone (-20°C). Nonspecific staining was blocked by incubating the sections for 5 minutes with 10% horse serum, then sections were incubated with anti-rat macrophage antibody (1/200) for 1 hour. After rinsing and washing with wash buffer, sections were incubated for 30 minutes with Affinipure donkey anti-mouse IgG antibodies (affinity adsorbed with rat serum proteins to prevent cross-reaction; Jackson ImmunoResearch) coupled to Texas Red or fluorescein isothiocyanate (FITC). Some of the specimens were processed without the primary antibody as negative controls. Cell nuclei were stained with 0.5 µg/mL bis-benzimide (Calbiochem) in PBS for 2 minutes. Specimens were observed and photographed with a Zeiss Axioskop microscope equipped with a triple-fluorescence filter.

Data Analysis
Data are expressed as mean±SEM. Comparisons between groups were made by use of one-way ANOVA. When significance was indicated, a Student-Newman-Keuls post hoc analysis was used. To examine interactions between angiotensin II or sham treatment and liposome-encapsulated SOD treatment, a two-way ANOVA was used, in which either sham or angiotensin II treatment was one independent variable and either empty liposomes or liposome-encapsulated SOD was the other independent variable. Significance was considered present when the probability value was <.05.

	Results

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Effect of Liposome-Encapsulated SOD Treatment on Vascular SOD Activity
In all three groups of rats, liposome-entrapped SOD increased total SOD activity by 30% (Fig 1).

View larger version (17K): [in this window] [in a new window]   Figure 1. Superoxide dismutase (SOD) activity in vascular homogenates from control rats and rats made hypertensive by infusion of either angiotensin II or norepinephrine. Animals were treated with either norepinephrine (2.8 mg·kg-1·d-1) or angiotensin II (0.7 mg·kg-1·d-1) or served as a control. Half of the animals received daily injections (x8 days) of liposome-encapsulated SOD (4000 U·kg-1·d-1) and half received liposomes lacking SOD (empty liposomes). Aortas were homogenized, and SOD activity was measured by use of inhibition of cytochrome c reduction by xanthine and xanthine oxidase as previously described.13

Vascular Superoxide Production
Lucigenin counts from aortic segments of control rats receiving empty liposomes averaged 3.0±0.5·10³ counts·min^-1·mg^-1. Treatment with liposome-encapsulated SOD did not alter this value in controls (Fig 2). Superoxide production, as assessed by lucigenin-enhanced chemiluminescence, was increased threefold in vascular segments from angiotensin II–infused rats treated with empty liposomes (P<.05 versus control). Liposome-entrapped SOD normalized ·O₂^- production in vessels from angiotensin II–treated rats. In contrast to angiotensin II treatment, and in agreement with our previous study, norepinephrine infusion did not affect vascular ·O₂^- production (Fig 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Vascular superoxide anion production assessed by lucigenin chemiluminescence in aortic segments from control, angiotensin II–infused, and norepinephrine-infused animals. The effect of concomitant treatment with liposome-encapsulated superoxide dismutase (SOD) or empty liposomes (EmpLip) was also examined.

Effect of Liposome-Encapsulated SOD on MAP in Angiotensin II– and Norepinephrine-Treated Animals
MAP was not different between animals receiving either liposome-encapsulated SOD or empty liposomes in either controls or norepinephrine-infused rats (Fig 3; P>.05). In contrast, treatment with liposome-entrapped SOD for 8 consecutive days markedly decreased the hypertension caused by angiotensin II (P<.05; Fig 3).

View larger version (15K): [in this window] [in a new window]   Figure 3. Effect of either liposome-encapsulated superoxide dismutase (LIPOSOME-SOD) or empty liposomes on mean arterial blood pressure in control, angiotensin II (AII)–treated, and norepinephrine (NE)-treated rats. Liposome-encapsulated SOD or empty liposomes were given as an intravenous bolus (4000 U·kg-1·d-1) for 8 days. Both AII (0.7 mg·kg-1·d-1) and NE (2.8 mg·kg-1·d-1) were infused subcutaneously for the final 5 days by means of an osmotic minipump implanted in the abdominal region. Values are mean±SE of blood pressures obtained on day 5 after onset of AII infusion.

Effect of Encapsulated SOD on In Vivo Responses to Acetylcholine and Sodium Nitroprusside
In control and angiotensin II–infused animals, liposome-entrapped treatment increased the hypotensive responses to acetylcholine, whether expressed as an absolute change in pressure or as a percent change in arterial pressure (Table 1). In contrast, treatment with liposome-encapsulated SOD had no effect on the hypotensive response to acetylcholine in the rats treated with norepinephrine.

View this table: [in this window] [in a new window]   Table 1. Changes in Blood Pressure Evoked by Intravenous Acetylcholine

Responses to sodium nitroprusside were not altered by treatment with liposome-encapsulated SOD (Table 2).

View this table: [in this window] [in a new window]   Table 2. Changes in Blood Pressure Evoked by Intravenous Sodium Nitroprusside

In Vitro Vascular Relaxations
In normal vessels from animals treated with empty liposomes, relaxations to acetylcholine were 86±3%. Treatment with liposome-entrapped SOD increased these to 98±2%. In angiotensin II–treated rats that had received empty liposomes, maximal relaxations to acetylcholine were markedly reduced compared with control rings (37±6%). Treatment of angiotensin II–infused animals with liposome-entrapped SOD substantially enhanced maximal relaxations to acetylcholine (Fig 4a; Table 3). Sensitivity to acetylcholine, as reflected by the ED₅₀s, was likewise increased in both control and angiotensin II–treated animals by long-term administration of liposome-encapsulated SOD. In control rats, the calcium ionophore A23187 produced relaxations averaging 86±7% and 100±1% of the preconstricted tension in the empty liposome and liposome-encapsulated SOD groups, respectively. In vessels from angiotensin II–treated animals, relaxations to the calcium ionophore were significantly impaired in the empty-liposome group (58±7%). In the angiotensin II–treated animals, administration of liposome-encapsulated SOD markedly enhanced relaxations to A23187 (Table 3; Fig 4b).

View larger version (24K): [in this window] [in a new window]   Figure 4. In vitro vascular relaxations to acetylcholine (Ach), calcium ionophore A23187, and nitroglycerin (Ntg) in control and angiotensin II (Ang II)–infused rats treated with either liposome-encapsulated superoxide dismutase (SOD) or empty liposomes were measured. Vessels were studied as ring segments in organ chambers. Relaxations were studied after the vessels had been preconstricted with phenylephrine (0.1 µmol/L).

View this table: [in this window] [in a new window]   Table 3. Peak Relaxations and ED50s for Acetylcholine, A23187, and Nitroglycerin

In control vessels, nitroglycerin produced maximal relaxations of 99±1% and 97±3% in the empty-liposome and liposome-encapsulated SOD groups, respectively. In vessels from angiotensin II–infused rats treated with empty liposomes, relaxations evoked by nitroglycerin were significantly impaired compared with relaxations in the control vessels (68±8%). These were substantially enhanced by treatment with liposome-encapsulated SOD. ED₅₀s to nitroglycerin were similar in the angiotensin II and control animals. In both groups, treatment with liposome-encapsulated SOD lowered the ED₅₀ (Table 3; Fig 4c).

Two-way ANOVA indicated that the effect of liposome-encapsulated SOD on peak relaxations in response to acetylcholine, calcium ionophore A23187, and nitroglycerin was greater in the rats with angiotensin II–induced hypertension than in controls.

Macrophage Content in Aortas of Control and Angiotensin II–Treated Rats
Anti-macrophage antibodies stained isolated cells in the adventitia in both the control and the angiotensin II–treated rats. There was no obvious difference between adventitial staining in the two groups. Positive control specimens for the anti-rat macrophage antibody showed staining of white pulp macrophages in the spleen and alveolar macrophages in the lungs. Only background fluorescence was observed in sections in which the primary antibody was not added (data not shown).

	Discussion

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In the present study, we found that increasing vascular SOD levels by daily injection of liposome-entrapped SOD attenuated the rise in blood pressure by 50 mm Hg in rats with angiotensin II–induced hypertension. Liposome-entrapped SOD treatment also enhanced the in vivo hypotensive effect of intravenous acetylcholine and increased in vitro relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin. The blood pressure–lowering effect of liposome-encapsulated SOD was not observed in norepinephrine-induced hypertension. In this latter model, ·O₂^- production by larger conduit vessels was not increased.

These findings confirm a recent study from our laboratory⁸ showing that angiotensin II–induced but not norepinephrine-induced hypertension was associated with an increase in vascular ·O₂^- production and an alteration in endothelium-dependent vascular relaxation. The present findings suggest that the effect of angiotensin II to increase vascular ·O₂^- production extends to the resistance circulation, which is involved in blood pressure control. These findings are also in keeping with a previously published report¹⁴ showing that a recombinant form of SOD that would bind to vascular glycosaminoglycans could acutely lower blood pressure in spontaneously hypertensive rats when injected intravenously. In the report by Nakazono et al,¹⁴ the effect of the recombinant SOD was short-lived. In contrast, we found that long-term treatment with liposome-encapsulated SOD produced a sustained decrease in blood pressure. Our findings also extend those of Nakazono et al¹⁴ in that we demonstrated that this effect of SOD did not occur in catecholamine-induced hypertension. We hypothesize that forms of hypertension associated with elevated levels of either circulating or local angiotensin II (which can activate vascular membrane oxidases) would more likely be susceptible to treatment with SOD.

Treatment with liposome-entrapped SOD did not completely correct hypertension in the angiotensin II–treated rats. Whether a higher dose of SOD might have proven more beneficial remains unknown; however, it is almost certain that mechanisms independent of ·O₂^- are important in this form of hypertension. Furthermore, changes in the morphology of resistance vessels may contribute importantly to hypertension in the setting of elevations of angiotensin II.

In our previous study,⁸ we found that the source of the increase in ·O₂^- in rats with angiotensin II–induced hypertension was likely an NADH-dependent oxidase. This oxidase has been shown⁷ to be the predominant source of ·O₂^- in the vessel wall and recently was found to be activated by angiotensin II in cultured vascular smooth muscle cells. In the present study, because of the small size of the resistance vessels involved, we could not characterize the oxidase involved, but we assume it is similar to that activated in the larger vessels.

One consideration is that macrophages in the vessel wall contribute to the increase in vascular ·O₂^- production. This seems unlikely for two reasons. First, in our previous study,⁸ we found the predominant oxidase in aortas of these animals was NADH dependent, whereas the macrophage oxidase is NADPH dependent. Second, in the present study, we found a small number of macrophages were scattered about the adventitia in both groups of vessels, and no macrophages were observed in the media or subintimal space. Although this immunohistochemical approach is only semiquantitative, we were unable to detect a difference in the number of macrophages present in the two vessel types.

It is likely that the increase in ·O₂^- production in the setting of angiotensin II–induced hypertension results in diversion of locally released NO· to nitrite, nitrate, and the peroxynitrite anion. Inorganic nitrite and nitrate are not vasoactive, except in very high (>100 µmol/L) concentrations. Peroxynitrite is a highly reactive molecule implicated in lipid peroxidation and cellular injury.^{15 16 17 18} Peroxynitrite is capable of producing vasodilation,¹⁹ likely via formation of nitric oxide–donating species.²⁰ Direct comparisons between peroxynitrite and NO· indicate that NO· is 100 to 1000 times more potent than peroxynitrite in its ability to increase cGMP.²¹ Liposome-entrapped SOD treatment likely limited this interaction between ·O₂^- and NO· and thus preserved the vasodilator effect. This conclusion is supported by our findings that liposome-entrapped SOD treatment substantially enhanced endothelium-dependent vascular relaxations and relaxations to exogenous vasodilators. It has been suggested that an important product of the endothelial NO synthase is the nitroxyl anion (NO^-)²² and that nitroxyl can be converted to NO· by SOD. Thus, another mechanism by which liposome-entrapped SOD might lower blood pressure is increased formation of NO· from nitroxyl, although this might have been expected to occur in all groups of animals studied.²³ Finally, it has also been suggested that ·O₂^- itself can serve as a vasoconstrictor agent.¹ It is therefore possible that the blood pressure–lowering effect of the liposome-encapsulated SOD might be simply due to reduction of ·O₂^- levels independent of an effect on NO·.

Liposome-entrapped SOD was used in the present study to augment vascular SOD levels. In previous studies, native SOD has been shown to penetrate poorly into endothelial cells, and when injected intravenously, it does not augment tissue levels of SOD.^{24 25} Furthermore, we¹¹ have found that native SOD, when added directly to a vessel in an organ chamber, has a minimal effect on vascular relaxations to endothelium-dependent vasodilators or to exogenous nitrovasodilators. Likewise, in preliminary studies, we found that intravenous administration of native SOD had little effect on in vivo responses to nitrovasodilators or acetylcholine. Native SOD is negatively charged and is therefore electrostatically repelled from the surface of vascular cells. The delivery of SOD via liposomes overcomes hindrances to vascular delivery. Recent studies²⁶ have shown that intravenous injections of liposome-encapsulated SOD improve endothelium-dependent vascular relaxation in vessels from rabbits with diet-induced atherosclerosis.

The increase in vascular levels of SOD achieved by liposome-encapsulated SOD treatment averaged 30%. Although this may seem modest, when ambient levels of ·O₂^- are low, even small increases in SOD can markedly shorten the half-life of ·O₂^-.²⁷ Therefore, the degree of increase in SOD achieved in the present studies may importantly modulate total vascular ·O₂^- levels. This was confirmed by our estimates of vascular ·O₂^- production using lucigenin-enhanced chemiluminescence. Furthermore, the localization of the liposome-delivered SOD may be important. Previous studies using electron micrographic localization have revealed endothelial cell, smooth muscle cell, and interstitial delivery of SOD. In particular, interstitial delivery may be quite important in preventing degradation of NO· because it traverses interstitial spaces.

To examine the consequences of ·O₂^- scavenging in vivo, we examined responses to an intravenous bolus of acetylcholine. Because these studies were performed in vivo while the animals were conscious, it was difficult to perform a detailed dose-response curve to acetylcholine in all animals. In preliminary studies, however, a dose-response curve was performed in control animals, and the dose selected (40 µg/kg) was found to produce an intermediate hypotensive response. This was selected to avoid excessive hypotension in controls and to allow detection of differences between the controls and the hypertensive animals. The augmented hypotensive response to acetylcholine in the liposome-encapsulated SOD–treated animals suggests that the levels of SOD were augmented in the microcirculation as well as in the larger conduit vessels.

Treatment with liposome-entrapped SOD enhanced the blood pressure response to acetylcholine not only in rats with angiotensin II–induced hypertension but also in controls. This finding is again compatible with the concept that even in the normal situation, a balance exists between NO· and ·O₂^- that importantly influences responsiveness of vessels in the microcirculation to endothelium-dependent vasodilators.

Treatment with liposome-entrapped SOD did not affect the blood pressure response to acetylcholine in the norepinephrine-treated rats. Although this observation is at first difficult to understand, there are complex interactions between norepinephrine and acetylcholine that may make interpretation of these findings difficult. For example, -adrenergic stimulation has been shown to enhance acetylcholine release in brain slices²⁸ and perfused rat hearts.²⁹ If this occurred in vivo, downregulation of muscarinic receptors might result, leading to a relative loss of responsiveness to acetylcholine in these animals.

Beyond simply enhancing the effects of nitric oxide, there are other potential mechanisms whereby liposome-entrapped SOD could prevent the hypertension caused by angiotensin II. These include alterations of baroreceptor responsiveness, interactions with other neurohumoral influences, and changes in blood volume. It has been observed, for example, that nitric oxide can modulate baroreflex activity.³⁰ Likewise, angiotensin II has been shown to stimulate the production of endothelin-1 by vascular smooth muscle.³¹ Whether or not these interactions could be modified by ·O₂^-, other reactive oxygen species, or SOD requires further investigation. Additionally, liposome-entrapped SOD, owing to its lipophilic character, could cross the blood-brain barrier and exert central effects.

These findings may provide insight into the clinical observations that hypertensive populations with elevated renin profiles (and presumably, elevated angiotensin II) have an increase in cardiovascular event rates compared with groups of hypertensive subjects with low renin profiles.³² Superoxide anion and its oxygen radical metabolites can not only alter endothelium-dependent vascular relaxation but may also participate in the oxidation of LDLs,³³ the activation of proto-oncogenes and the promotion of cellular growth,^{34 35} and the activation of proinflammatory molecules, such as the vascular cell-adhesion molecule.³⁶ Many of these properties are shared by angiotensin II,^{37 38 39 40} and at least one mechanism responsible for vascular disease in angiotensin II–induced hypertension is increased production of ·O₂^-.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL-39006 and DK-45215, a merit review grant from the Veterans Administration, and Vascular Biology Program Project grants HL-48667 and HL-48676. Dr Tarpey is a recipient of an NIH Clinician Investigator Award. Dr Bech Laursen is supported by grants from the Danish Heart Foundation, the Jørgen Møller Foundation, and Bristol-Meyers Squibb A/S, Denmark.

Received February 26, 1996; revision received September 11, 1996; accepted September 30, 1996.

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