(Hypertension. 1997;30:314.)
© 1997 American Heart Association, Inc.
Articles |
Enhanced Endothelium-Dependent Relaxations After Gene Transfer of Recombinant Endothelial Nitric Oxide Synthase to Rabbit Carotid Arteries
From the Divisions of Cardiovascular Disease (I.J.K., R.S.S.), Vascular Surgery (G.M., P.G.), Anesthesiology and Pharmacology (M.T., Z.S.K.), and Endocrinology and Metabolism (T.O.), Mayo Clinic and Foundation, Rochester, Minn.
Correspondence to Timothy O’Brien, MD, Assistant Professor, Department of Endocrinology and Metabolism, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail OBrien.Timothy{at}mayo.edu
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Abstract |
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Abstract We tested the effects of overexpression of the endothelial nitric oxide synthase (eNOS) gene in the normal arterial wall by adenoviral-mediated gene transfer. Rabbit carotid arteries were surgically isolated and exposed to adenoviral vectors encoding eNOS (AdeNOS) or ß-galactosidase (AdßGal) on the contralateral side. Vector solutions at a concentration of 1x1010 plaque forming units/mL were instilled for 20 minutes before restoration of flow. Arteries were harvested 4 days later for immunostaining, measurement of cGMP, and vasomotor studies. Endothelium-specific gene transfer was confirmed by staining for ß-galactosidase in the AdßGal arteries. Immunostaining of en face endothelial cell imprints from AdeNOS-transduced arteries with a monoclonal antibody to eNOS showed increased immunoreactivity. Basal cGMP levels were significantly greater in the AdeNOS-transduced arteries (18.4±4.6 versus 4.2±0.5 pmol/mg protein; P<.05). Contractions to phenylephrine were significantly reduced in the AdeNOS-transduced arteries (area under curve, 106±5 versus 119±7; P<.05), but in the presence of the eNOS inhibitor, NG-monomethyl-L-arginine (L-NMMA, 3x10-4 mol/L), there was no difference between the two (area under curve, 148±5 versus 153±6; P=NS). Relaxations to acetylcholine obtained during submaximal contractions to phenylephrine were significantly enhanced in the AdeNOS-transduced arteries (EC50, 7.45±0.05 versus 7.23±0.03; P<.05). We conclude that overexpression of eNOS in the endothelium results in diminished contractile responses, as well as enhanced endothelium-dependent relaxations. These findings imply a possible role for vascular eNOS gene transfer in the treatment of vasospasm and endothelial dysfunction.
Key Words: nitric oxide • gene transfer • endothelium • nitric oxide synthase • adenovirus
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Introduction |
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Nitric oxide plays a key regulatory role in vascular wall homeostasis by its actions on vascular tone,1 2 platelet adhesion-aggregation,3 leukocyte adhesion,4 and smooth muscle growth.5 NO is produced by the enzyme NOS, of which at least three distinct isoforms are known to exist. These isoforms differ in transcriptional regulation, dependence on calcium, and Vmax.6 7 The endothelial NOS (eNOS), also called type III NOS, is a calcium-dependent, constitutively active enzyme that produces small amounts of NO continuously. NO has been shown to account for the biological activity of the endothelium-derived relaxing factor in the cardiovascular system8 and plays a key role in the regulation of blood pressure.9 10 Increased production of eNOS may occur in response to humoral or mechanical stimuli.11 The vascular protective effect of exercise and estrogen may be due to an increase in the messenger RNA for eNOS.12 13 Decreased bioavailability of NO due to decreased production or excessive degradation may be responsible for the endothelial dysfunction and accelerated arterial pathology seen in hypertension, atherosclerosis, transplant vasculopathy, coronary spasm, and diabetes.14 Increasing NO production in the vessel wall by use of gene transfer techniques may have a therapeutic role in these disease states.
Gene transfer to the vascular wall holds promise as a means of controlling local vascular function as well as serving as a tool in the study of vascular biology.15 16 Several attributes of a replication-defective adenovirus make it useful in the study of vascular wall gene transfer, including the ability to achieve relatively efficient transfer and expression of recombinant genes by the vascular wall cells.17 18 Adenoviral-mediated gene transfer of eNOS into endothelial cells may be a unique mode of increasing NO production in the vessel wall.
With use of the hemagglutinating virus of Japan (HVJ) in a DNA-nuclear protein-liposome complex, eNOS gene transfer to the injured rat carotid artery has been reported and shown to result in a reduction of neointimal formation.19 More recently, adenoviral-mediated expression of human recombinant eNOS in rat lungs has been shown to attenuate hypoxic vasoconstriction.20 Recombinant adenovirus was delivered by aerosolization and localized partly to the adventitia of the pulmonary arteries. However, eNOS gene transfer to the intact endothelium has not been reported. The present study was therefore undertaken to determine the effects of overexpression of eNOS in the endothelium on vascular tone and endothelium-dependent relaxations.
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Methods |
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Construction, Propagation, and Purification of Adenoviral Vectors
A recombinant adenovirus encoding the eNOS gene driven by a cytomegalovirus promoter was generated based on previously described methods.21 Briefly, bovine eNOS cDNA (provided by Dr David Harrison, Emory University, Atlanta, Ga) was cloned into the pACCMVpLpA vector (provided by Dr Robert Gerard, University of Texas Southwestern Medical Center, Dallas, Tex). The resulting plasmid was linearized and cotransfected with dl309 into 293 cells by calcium phosphate/DNA coprecipitation. dl309 is a biologically selected, restriction enzyme-site-loss variant of wild-type adenovirus type 5.22 293 Cells are human embryonic kidney carcinoma cells that have been transformed with the left end of human adenovirus type 5 DNA.23 Recombinant adenoviral vectors were generated by homologous recombination.21 Viral plaques were picked and propagated in 293 cells. Viral DNA was enriched by Hirt extraction24 and screened by restriction mapping and polymerase chain reaction for the presence of eNOS cDNA. Positive plaques underwent two further rounds of plaque purification in 293 cells. Stocks were prepared from positive plaques, and these were used to generate high titer preparations. Viral preparations were performed by infecting confluent monolayers of 293 cells in 175-cm2 flasks with viral stock at a multiplicity of infection of 1 to 10. Virus was purified by double cesium chloride gradient ultracentrifugation and was dialyzed against 10 mmol/L Tris, 1.0 mmol/L MgCl2, 1.0 mmol/L HEPES, and 10% glycerol for 4 hours at 4°C. Viral titer was determined by plaque assay.21 eNOS activity was confirmed by positive NADPH diaphorase staining25 in confluent 293 cells transduced with AdeNOS. The defectiveness of AdeNOS for replication was tested by infection of human embryonic lung cell diploid cell cultures. Replication competent viruses at a multiplicity of infection of 10 or more produced cytopathic effects and destroyed the monolayer in less than 3 days. Infection with AdeNOS at a comparable multiplicity of infection produced no observable cytopathic effects after 5 days. A recombinant replication defective adenoviral vector encoding the E. coli ß-galactosidase gene (AdßGal)26 driven by the cytomegalovirus promoter was obtained from Dr James Wilson (University of Pennsylvania, Philadelphia) and used as a control. It was propagated, isolated, and quantitated as described above. Viral stocks were stored at -70°C. Before use, viral preparations were tested in vitro by histochemical detection of NADPH diaphorase and ß-galactosidase activity in transduced porcine coronary artery smooth muscle cells.
Gene Delivery
Twenty-two male New Zealand White rabbits weighing 3.0 to 3.5 kg
were used in these experiments. The animals were housed individually in
stainless steel, wire-bottomed cages in a room with a 12-hour
light/dark cycle. All experimental protocols were approved by the
institutional animal care and use committee and were performed in
accordance with the recommendations of the American Association for the
Accreditation of Laboratory Animal Care. Sedation and induction of
anesthesia were obtained with an intramuscular injection of
ketamine (65 mg/kg), xylazine (13 mg/kg), and
acepromazine (22 mg/kg). Paramedian cervical incisions were made
in the anterior neck, and the common carotid arteries were exposed
bilaterally by blunt dissection. Branches of the carotid artery were
cauterized or tied off using 5-0 ethilon sutures. After the
administration of heparin (100 U/kg), proximal and distal vascular
clamps (Edward Weck and Co) were applied to the carotid artery, and a
24-gauge angiocatheter was inserted into the proximal part of the
isolated segment. The needle was withdrawn and blood removed from the
segment of the artery using a gauze wick at the open end of the
angiocatheter. The solution containing adenoviral vector (100 µL of a
1x1010 pfu/mL concentration) was then instilled
intraluminally via the catheter, the catheter was removed, and the
defect in the arterial wall was closed with a 10-0 ethilon
suture. After 20 minutes, vascular clamps were removed, and flow was
restored. The contralateral vessel was transduced similarly with an
identical concentration of AdßGal. In 4 animals, one of the arteries
was exposed to the vehicle alone (PBS with 0.5% albumin,
PBS-A) and the other to AdßGal as described above. The cervical
incisions were closed with subcuticular sutures, and the animal was
allowed to recover. Four days later, carotid arteries were isolated as
described above and harvested, and the animal was then euthanized. Four
animals were used for confirmation of gene transfer by histochemistry
for ß-galactosidase and immunohistochemistry for eNOS. In the
remaining 18 animals, each artery was divided into 3 to 4 rings, 1 of
which was used for measurement of cGMP and the rest for vascular
reactivity studies.
Detection of ß-Galactosidase Expression
Segments of rabbit carotid arteries transduced with AdßGal or
AdeNOS were washed in PBS and fixed for 30 minutes at 4°C in 2%
formaldehyde and 0.2% glutaraldehyde in PBS, pH 7.4.
One milliliter of a solution of X-Gal was added to the rings, and these
were incubated at 37°C for 2 hours. After a rinsing with PBS, vessel
segments were cut into 3-mm rings and embedded in paraffin.
Five-µm-thick cross sections spaced at least 100 µm apart were
cut from each segment and counterstained with nuclear fast red. The
sections were examined under a light microscope. Efficiency of gene
transfer to the endothelium was determined by counting
stained cells and expressing the number of stained cells as a
percentage of total cells counted.
Immunohistochemistry of Endothelial Cells En Face
(Häutchen Preparation)
En face preparations of the endothelium were
prepared based on methods described previously.27 28 29 After
harvest, carotid arteries were flushed with PBS, cleaned of
periadventitial fat, and cut into 5-mm rings that were then opened
longitudinally. With the endothelial layer upward, each
vessel segment was placed between two glass slides (Superfrost,
Fisher). The glass slides were pressed together on a cold plate
(-70°C to -80°C) for 1 minute and then forced apart, leaving an
endothelial layer attached to the top slide and the
remainder of the vessel wall on the other slide. The identity of the
endothelial monolayer was confirmed by typical
endothelial cell morphology on phase-contrast
microscopy and positive immunostaining for factor VIII.
To examine eNOS immunoreactivity, the endothelial
monolayer was fixed in 10% acetone for 10 minutes and air dried for 30
minutes. Nonspecific antibody binding was blocked with 5% BSA, and
slides were incubated with a monoclonal mouse anti-eNOS antibody, 1:50
dilution (Transduction Laboratories) for 60 minutes. TRITC-conjugated
anti-mouse IgG antibody (1:64 dilution, Sigma Chemical Co) was applied
for 20 minutes, and slides were mounted with PBS-glycerol (1:1) and
viewed under a fluorescence microscope.
Measurement of cGMP
From each harvested artery, a ring was immersed immediately in a
solution of 3-isobutyl-L-methylxanthine (IBMX, 1 mmol/L)
and incubated at 37°C for 30 minutes before being snap-frozen in
liquid nitrogen and stored at -70°C until the time of assay. cGMP
levels were determined with a radioimmunoassay kit (Amersham) as
previously described.30 The stimulatory effect of
acetylcholine on cGMP levels in arteries transduced with AdeNOS or
AdßGal was determined by adding the drug
(3x10-8 mol/L) for the last 90 seconds
of the 30-minute incubation in the IBMX solution. The
arterial rings were then snap-frozen in liquid nitrogen and
stored at -70°C for cGMP assay. The duration of exposure to
acetylcholine and the concentration were chosen based on vascular
reactivity studies which showed that at this concentration the greatest
difference between the two groups was present.
Vascular Reactivity
Rings (4 mm long) from each carotid artery were used for
assessing vascular reactivity. Rings were connected to isometric force
displacement transducers (Grass Instruments) and suspended in organ
chambers filled with 25 mL of gassed (95% O2 and 5%
CO2) modified Krebs-Ringer bicarbonate solution (pH 7.4,
temperature 37°C; composition in mmol/L: 118.3 NaCl, 4.7
KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2
KH2PO4, 25.0 NaHCO3, 0.026 calcium
sodium EDTA, and 11.1 glucose). The rings were allowed to equilibrate
for 1 hour and then stretched to the optimal point on the
length-tension curve (3 g for most rings) as determined by repeated
exposure to 20 mmol/L KCl. The rings were left at this
resting tension throughout the remainder of the study. The maximal
contraction of each ring was determined by KCl 60 mmol/L. A
concentration-response curve to phenylephrine
(3x10-8 to 10-5
mol/L) was obtained in the absence and presence of L-NMMA
(3x10-4 mol/L). All concentration
responses were determined in the presence of
indomethacin (10-5
mol/L) to block any effects mediated by the activation of
cyclooxygenase. In a separate group of animals,
acetylcholine (10-9 to
10-6 mol/L) was added cumulatively
during a submaximal contraction to phenylephrine.
Submaximal contractions were obtained using a
10-6 to 3x10-6
mol/L concentration of phenylephrine, with care
taken to match the contractions in the two groups. Concentration
responses to sodium nitroprusside (10-9 to
10-5 mol/L) were similarly obtained in
arteries transduced with AdßGal or AdeNOS. To rule out a direct
effect of the control vector on contractility and
endothelium-dependent relaxations, concentration
responses to phenylephrine and acetylcholine were obtained
as described above in rings from arteries exposed to vehicle or the
AdßGal vector.
The following drugs were used: acetylcholine chloride, L-NMMA, sodium nitroprusside, indomethacin, and phenylephrine bitartrate (all from Sigma Chemical Co). Drugs were made up immediately before study, and the concentrations are reported as the final molar concentration in the organ chamber. Relaxations are expressed as percent reduction of the phenylephrine-induced contractions.
Statistics
Data are presented as mean±SEM. Statistical
analysis was performed by ANOVA followed by Fisher’s post hoc
test to detect significant differences in multiple comparisons. An
unpaired Student’s t test was used to detect significant
differences when two groups were compared. A value of P<.05
was considered to be statistically significant.
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Results |
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Confirmation of Gene Transfer
Arteries transduced with AdßGal at a concentration of 1x1010 pfu/mL and harvested 4 days later showed endothelium-specific gene transfer as confirmed by X-Gal staining (Fig 1A and 1B). There was no staining in the AdeNOS arteries (Fig 1C and 1D). The efficiency of gene transfer was quantified at 28±12% of endothelial cells. Phase-contrast microscopy of en face preparations (Häutchen) of endothelial cells showed typical endothelial cell morphology (Fig 2A) and positive factor VIII immunostaining (not shown). To demonstrate eNOS immunoreactivity, immunofluorescent staining with a monoclonal eNOS antibody was performed. Positive staining was seen in the imprints from the endothelium of AdßGal arteries due to the presence of endogenous eNOS (Fig 2B). However, in imprints from eNOS-transduced arteries, a marked increase in the intensity of immunofluorescence was observed (Fig 2C), indicating the presence of increased amounts of eNOS in AdeNOS-transduced cells.
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Basal and Stimulated cGMP Levels
Basal cGMP levels were significantly elevated in the
AdeNOS-transduced arteries compared with AdßGal-transduced arteries
or arteries instilled with vehicle alone (Fig 3, P<.05). Exposure of
arterial rings to acetylcholine
(3x10-8 mol/L) for 90 seconds resulted
in a significant increase in cGMP levels in both groups of arteries.
However, in the AdeNOS arterial rings, stimulated cGMP
levels were significantly higher than in the AdßGal rings (Fig 3, P<.05).
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P<.05 stimulated vs basal cGMP levels.
Contractile Responses to Phenylephrine and KCl
Transduction with AdßGal did not affect the contractile
responses to phenylephrine (area under curve, 107±11
[vehicle] versus 109±7 [AdßGal]; P=NS) or
endothelium-dependent relaxations to acetylcholine
(maximal relaxations, 100% in both groups; EC50, 7.36±0.1
[vehicle] versus 7.43±0.9 [AdßGal]; P=NS). Maximal
contractions to KCl were significantly reduced in the AdeNOS-transduced
arteries (4.2±0.2 versus 3.31±0.2 g, P<.05). Contractions
to phenylephrine were significantly reduced in the
AdeNOS-transduced arteries (Table 1, Fig 4). Area under the curve,
EC50, and maximal contractions were significantly different
in the two groups. When the concentration responses to
phenylephrine were repeated in the presence of L-NMMA
(3x10-4 mol/L), no significant
difference was observed in the two groups (Table 1, Fig 4). Responses
to phenylephrine in the presence of L-NMMA were
significantly greater in both groups when compared with responses
obtained in the absence of L-NMMA (Table 1, Fig 4).
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). Data are
mean±SEM, n=8 rabbits.
Endothelium-Dependent Relaxations
Relaxations to acetylcholine obtained during submaximal
contractions to phenylephrine
(10-6 to 3x10-6
mol/L) were significantly enhanced in the AdeNOS-transduced
arteries as measured by a shift in EC50 and by area under
the curve (Fig 5 and Table 2). However, maximal relaxations were
unchanged, being nearly 100% of the phenylephrine
precontraction in both groups. Submaximal contractions to
phenylephrine (2.9±0.2 g in the AdeNOS group and 3.2±0.15
g in the AdßGal group) were not significantly different
(P=.21). Relaxations to sodium nitroprusside were not
different in the two groups (Fig 6),
indicating similar responses to a direct smooth muscle vasodilator.
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) or AdeNOS
(
). Submaximal contractions were obtained with
phenylephrine (10-6 to
3x10-6 mol/L) after a concentration-response
curve was done to determine EC50. Precontractions (2.9±0.2
g in the AdeNOS group and 3.2±0.15 g in the AdßGal group) were not
significantly different (P=.21). Data are mean±SEM, n=6
rabbits.
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Discussion |
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This study demonstrates for the first time that adenoviral-mediated transfer of the eNOS gene to the endothelium alters vascular reactivity. Expression of recombinant eNOS in transduced arteries results in elevated basal cGMP levels, diminished sensitivity to phenylephrine and KCl, and enhanced endothelium-dependent relaxations to acetylcholine. L-NMMA, an inhibitor of eNOS, restores the sensitivity to phenylephrine in arteries expressing the recombinant eNOS gene. These results indicate that adenoviral-mediated transfer of the eNOS gene to the endothelium yields a functionally active recombinant enzyme and increased NO production.
Intraluminal delivery of adenoviral vectors to uninjured arteries results in endothelium-specific gene transfer. In the rat carotid artery, a concentration of 1x1010 pfu/mL of the adenoviral vector yielded relatively efficient gene transfer with minimal effects on the arterial phenotype.31 Up to 35% of endothelial cells are transduced using this method. Using ß-galactosidase staining, we confirmed similar, relatively efficient, endothelium-specific gene transfer in the rabbit carotid artery. To confirm gene transfer with the AdeNOS vector, we used an en face (Häutchen) preparation of endothelial cells from the transduced arteries. The Häutchen preparation is a well-characterized method of studying endothelial monolayers and is useful for immunostaining of endothelial cell imprints obtained from arteries. An increased intensity of fluorescence in imprints obtained from AdeNOS-transduced arteries indicates an increased amount of eNOS protein in the endothelial cells.
Several effects of NO result from the activation of a soluble cytoplasmic guanylate cyclase, the enzyme that catalyzes formation of cGMP from GTP.32 The rise in basal levels of cGMP in arteries transduced with the AdeNOS vector reflects the increased production of NO by the endothelium of these vessels and resulting increase in the cGMP levels of the underlying smooth muscle cells. Although enzymatic activity of recombinant eNOS was not measured, the presence and functionality of the protein is demonstrated by immunohistochemical, biochemical, and pharmacological data. Adenoviral-mediated vascular gene transfer may lead to increased production of cytokines33 and in theory may result in the expression of the inducible NOS isoform (iNOS). In porcine coronary artery smooth muscle cells transduced with the vectors used in this study, calcium-independent NOS enzymatic activity was not detected, confirming the lack of expression of iNOS (I.J. Kullo, R.S. Schwartz, V.J. Pompili, M. Tsutsui, S. Milstein, L.A. Fitzpatrick, V.S. Katusic, T. O’Brien, unpublished data, 1996). In the present study, when compared with arteries instilled with vehicle alone, AdßGal-transduced arteries did not show significant elevations of cGMP or hyporeactivity to contractile agonists. The use of the AdßGal vector as control in the present study therefore helps to exclude the possibility of iNOS expression secondary to adenoviral-mediated gene transfer to the arterial wall.
Both receptor-mediated (to phenylephrine) and receptor-independent (to KCl) contractions were significantly reduced in the AdeNOS-transduced arteries. These observations are best explained by increased NO generation due to expression of recombinant eNOS. The specificity of these findings was confirmed by using the NOS antagonist L-NMMA. In the presence of L-NMMA, no significant difference was found between the contractions to phenylephrine in the two groups. The diminished sensitivity to contractile agonists is typical of states in which there is increased NO production in the vascular wall. Busse and Mulsch34 found decreased contractions to norepinephrine in rat aortas that had been treated with endotoxin. The effect was reversed by the NOS inhibitor NG-nitro-L-arginine. The effects observed in their study were due to the activity of iNOS. In another study, expression of a recombinant eNOS gene in injured rat carotid arteries resulted in reduced contractions to KCl (50 mmol/L).19
The enhancement of endothelium-dependent relaxations to acetylcholine as a result of adenoviral-mediated endothelial overexpression of eNOS is a novel finding. Enhanced endothelium-dependent relaxations in normal arteries may occur when endogenous eNOS is upregulated by an increase in shear stress due to chronic exercise35 or by an estrogen effect.36 Our results offer proof of principle that such effects may also be obtained by vascular gene transfer of recombinant eNOS. The recombinant enzyme is responsive to signals initiated by receptor occupation by an agonist (in this case, acetylcholine) and may respond similarly to other agonists such as serotonin, bradykinin, and thrombin, which are known to cause the release of NO from the endothelium. This effect is not due to an enhanced sensitivity to NO because relaxations to sodium nitroprusside were similar in the AdeNOS and AdßGal arteries.
Transfer and expression of genetic material in endothelial cells is a potentially powerful tool to study endothelial cell biology. Expression of recombinant NOS in endothelial cells may be useful in elucidating the pathogenetic mechanisms of diseases such as hypertension and atherosclerosis, in which the exact defect in the NO-cGMP pathway is not clear. Such an intervention may eventually prove useful in the site-specific therapy of disorders characterized by endothelial dysfunction and attenuated endothelium-dependent relaxations. Local delivery of NO itself is difficult owing to its short half-life, high reactivity, and limited solubility in aqueous media.37 Most in vivo studies of NO rely on specific pharmacological tools such as NO donor compounds rather than authentic NO. The use of NO donors in the clinical setting is complicated by the need for metabolic activation in some instances,38 tolerance after repeated treatment,39 and hypotension at higher doses. Our results show that adenoviral-mediated gene transfer of eNOS to the endothelium results in a functionally active recombinant enzyme, which may provide a continuous supply of NO for the duration of transgene expression. This technique is therefore a feasible method of local delivery of NO that may overcome some of the disadvantages of NO donors for use in site-specific vascular wall therapy.
In summary, the present study demonstrates that in vivo gene transfer of eNOS to the endothelium yields functional expression of the recombinant protein and a resulting biological effect on vasomotor function. Expression of recombinant eNOS in the endothelium results in increased NO production, causing elevated basal levels of cGMP in the arterial wall and diminished sensitivity to contractile agonists. Furthermore, there is enhancement of endothelium-dependent relaxations. These findings imply that expression of recombinant eNOS in the endothelium may be useful in increasing the production of NO in the arterial wall. Such an increase may be beneficial in vascular diseases characterized by a decreased bioavailability of NO.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by Mayo Clinic intramural research grants (Dr O’Brien, Dr Schwartz), the J. Holden DeHaan Foundation (Dr Schwartz), and in part by National Institutes of Health grants HL-44116 and HL-53542 (Dr Katusic). The authors would like to thank Sharon Guy and Leslie Smith for technical assistance.
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Footnotes |
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1 The first two authors contributed equally to this article.
Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10-13, 1996; previously published in abstract form (Circulation. 1996;94:I-44).
Received October 14, 1996; first decision November 5, 1996; accepted February 24, 1997.
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