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(Circulation. 1996;94:2909-2914.)
© 1996 American Heart Association, Inc.

Articles

Endogenous Cell Seeding

Remnant Endothelium After Stenting Enhances Vascular Repair

Campbell Rogers, MD; Sahil Parikh, AB; Philip Seifert, MS; Elazer R. Edelman, MD, PhD

the Department of Medicine, Cardiovascular Division (Cardiac Catheterization Laboratory and Coronary Care Unit), Brigham and Women's Hospital, Harvard Medical School, Boston, Mass, and the Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Mass.

Correspondence to Campbell Rogers, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. E-mail cdrogers@bics.bwh.harvard.edu.

	Abstract

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Background Endothelial integrity is essential for maintaining vascular homeostasis, and endothelial denudation results in neointimal thickening. Balloon-expandable endovascular stents provide a luminal scaffolding within atherosclerotic arteries with minimal direct contact between balloon and endothelium. We wondered whether stents cause diminished endothelial ablation, and if so, whether the degree of endothelial damage might determine later proliferative sequelae.

Methods and Results Stainless steel stents were expanded in normal or previously denuded iliac arteries of New Zealand White rabbits. Stented arteries were harvested 15 minutes, 1 hour, 3 days, or 14 days later. En face staining of the luminal surfaces of stented arteries demonstrated that endothelial cell loss began immediately after stent expansion and was restricted to interstices between stent struts. Remnant endothelium adjacent to struts provided the foundation for complete endothelial regeneration of the stented segment within 3 days. Both early monocyte adhesion and later intimal macrophage accumulation were reduced >80% in nonballooned but stented arteries, in concert with a twofold reduction in intimal thickening after 14 days, compared with arteries completely denuded with a balloon before stent expansion.

Conclusions It is accepted that deep injury caused by balloon-expanded endovascular stents is a critical contributor to experimental stent-induced neointimal hyperplasia. Our data indicate that the degree of endothelial injury may also be an important component of vascular repair after stenting and an important consideration in stent and balloon design and use. The use of stents for primary endovascular intervention may allow partial retention of endothelium within treated arteries, thereby modulating vascular repair with less need for adjunctive pharmacological therapy.

Key Words: stents • endothelium • pathology • restenosis • arteriosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Vascular interventions provoke a cascade of cellular and biochemical events that include an accelerated arteriopathy and restenosis. Experimental models examining this process have traditionally used endothelial denudation as a means of rapidly inducing myointimal thickening.^{1 2 3} In such models, the eventual restoration of endothelial integrity via ingrowth from neighboring normal vessel segments brings an end to the hyperplastic process.^{2 3 4} The importance of endothelial integrity to vascular homeostasis has led to exploration of mechanisms whereby endothelial cells may regulate other vascular cell actions and to a search for practical measures favoring endothelial restoration after injury.^{5 6 7 8}

Clinical vascular interventions such as balloon angioplasty ablate the endothelial lining of diseased vessels,^{9 10 11} and it has been proposed that promoting rapid endothelial regrowth might favorably alter clinical vascular repair. Endovascular stents, although they are expanded on a balloon, separate the balloon from the luminal surface by the thickness of their struts. We wondered whether this might allow for arterial dilation without circumferential balloon-endothelium contact, retention of some native endothelium, and as a result, enhanced vascular healing. En face histological staining techniques revealed endothelial cell loss within minutes of stent expansion in arteries without previous balloon injury but that in contrast to arteries subjected to balloon angioplasty alone, a substantial amount of endothelium remained after stenting and that this remnant endothelium repopulated the stented arterial surface within 3 days. Such rapid reendothelialization resulted in near abolition of early inflammatory cell recruitment and a twofold reduction in later intimal thickening.

Our data demonstrate that endovascular stents may permit arterial dilation without complete endothelial obliteration, and as a result, muted hyperplastic sequelae. Current clinical practice for stent placement often includes denuding interventions such as arterial predilation with a bare balloon before stent expansion. This practice may need to be reconsidered whenever clinically practical. Moreover, in addition to a focus on pharmacological means of modulating the response to vascular injury, further examination of clinically practical methods of reducing endothelial and vessel wall injury may be warranted. Alternative protocols might therefore limit or eliminate dilation of the artery before stent implantation rather than requiring genetically engineered endothelial cell–seeded stents to reendothelialize injured vessels.^{5 6 7} Novel stent designs might facilitate implantation without balloon predilation and focus on means of speeding endogenous endothelial regrowth.

	Methods

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Animals
New Zealand White rabbits (Millbrook Farm Breeding Laboratories, Amherst, Mass) weighing 3 to 4 kg were housed individually in steel mesh cages and fed rabbit chow and water ad libitum. Aspirin (Sigma Chemical Co, 0.07 mg/mL) was added to drinking water beginning 1 day before surgery to achieve a dose of 5 mg·kg^-1·d^-1. Endovascular stents were placed in both external iliac arteries as previously described.^{12 13} Under anesthesia with ketamine (Aveco Co, 35 mg/kg IM) and sodium pentobarbital (Nembutal, Abbott Laboratories, 4 mg/kg IV), both femoral arteries were exposed and ligated. A stainless steel stent (Multi-Link, Advanced Cardiovascular Systems) mounted coaxially on a 3-mm angioplasty balloon was passed retrogradely via arteriotomy into each iliac artery and expanded with a 15-second inflation at a pressure of 8 atm. Iliac arteries for rabbits of this size range from 2.5 to 2.75 mm in diameter, yielding a balloon/stent-to-artery ratio of 1.1:1 to 1.2:1.^{12 13}

To study the importance of endothelial denudation that accompanies stent expansion, some stents were placed in normal arteries and others were placed after the iliac arterial endothelium had been removed with a 3F balloon embolectomy catheter (Baxter Healthcare Corp, Edwards Division). The embolectomy catheter was passed via arteriotomy retrogradely into the abdominal aorta and withdrawn through the iliac artery in the inflated state three times, a procedure known to completely remove the endothelial lining.^{1 2 4 8} Arteries subjected only to balloon angioplasty with the same 3-mm balloons as used for stent deployment were also examined. All animals received a single intravenous bolus of standard anticoagulant heparin (100 U/kg, Elkin-Sinn Inc) at the time of surgery to limit stent thrombosis.

Endothelial Injury and Regeneration
To examine stent-induced endothelial denudation and subsequent regrowth, arteries were harvested 15 minutes (n=3), 60 minutes (n=3), or 3 days (n=4) after stent expansion or 60 minutes after balloon angioplasty (n=3). En face staining was performed with a modified Hautchen preparation.¹⁴ This technique is superior to other means of examining endothelial integrity, including scanning electron microscopy or the injection of tracer dyes such as Evans Blue, because it maintains luminal surface integrity with high fidelity and with the broadest view of the arterial surface. Precise quantitative assessment can be made at all magnifications. After anesthesia with intravenous sodium pentobarbital, inferior vena caval exsanguination, and perfusion with Ringer's lactate solution via left ventricular puncture, the iliac arteries were perfused in situ via the abdominal aorta with modified Ito-Karnovsky fixative (200 mL), AgNO₃ (100 mg in 200 mL for 1 minute), and bromides (COBr₂, 3.0 g, and NH₄Br, 1.0 g, in 200 mL dH₂O for 1 minute). Both iliac arteries were excised, rinsed in modified Ito-Karnovsky fixative on ice for 60 minutes, and placed in 0.1 mol/L NaPO₄ buffer. Arteries were opened longitudinally and the stents lifted gently off the luminal surface so as not to disturb the adjacent structures. Unstained areas underlying stent struts were no wider than the stent struts themselves, attesting to the power of this technique in providing in situ fixation with full preservation of morphological and spatial detail of the intact luminal surface. The arteries were then pinned to dental wax, dehydrated, and transferred to glass slides. The area of endothelial denudation in the stented or angioplastied portion was quantified by computer-assisted digital planimetry.

Cellular Responses
In another series of experiments, the effects of remnant endothelium on cellular responses within the injured vessel wall were assessed in arteries with or without prestent balloon denudation harvested 3 days (n=4 each) or 14 days (n=6 or n=4, respectively) after stent implantation. After euthanatization and perfusion with Ringer's lactate solution as described above, both iliac arteries, as well as a section of spleen (as a positive control tissue for immunohistochemical staining), were excised and fixed in Carnoy's solution (60% methanol, 30% chloroform, 10% glacial acetic acid). Proximal and distal stent ends were identified and marked and the stented arterial segments embedded with a new methyl methacrylate formulation¹⁵ and cut with a tungsten carbide knife (Delaware Diamond Knives) on an LKB Historange microtome. Cross sections 5 µm thick were taken from three sites along each stent-implanted artery, including each end and the middle. To minimize sampling error, the three arterial sites along each stent were analyzed morphometrically and the results averaged.

Tissue and cell structures were examined in histological sections by staining with Verhoeff's elastic tissue stain, hematoxylin-eosin, or modified Russell-Movat pentachrome stain. Neointimal and medial cross-sectional areas and thicknesses were quantified in elastin-stained sections by computer-assisted digital planimetry. The extent of deep arterial injury caused by each stent strut was quantified histologically by the method of Schwartz et al.¹⁶ Each stent strut in an arterial cross section was graded as to the extent of its vessel wall disruption: 0 for no disruption of the internal elastic lamina, 1 for disruption of the internal elastic lamina, 2 for laceration of the tunica media, and 3 for disruption of the external elastic lamina. An overall injury score for each cross section was calculated by averaging the individual scores for all struts within that section. The rigid scaffolding provided to the artery by the stent provided a taut luminal surface on which to examine adherent cells postmortem. Cells adherent to the luminal surface were examined individually under x600 magnification, their nuclear morphology was classified as polymorphonuclear or monocytic, and their numbers were counted.

Specific cell types were identified by standard immunohistochemical techniques based on previously published methods^{15 17} with antibodies to rabbit macrophages (monoclonal mouse RAM 11 IgG, DAKO) or endothelial cells (polyclonal goat anti–human factor VIII, ICN Biomedicals). Methacrylate-embedded sections were deplasticized with xylenes and ethylene glycol monoethyl ether (Sigma) as previously reported.¹⁵ Sections were then rehydrated, digested with 0.1% trypsin (for RAM 11 staining only), quenched, and blocked with 10% serum. Incubation was performed with primary antibody diluted in PBS at 1.2 µg/mL IgG concentration for RAM 11 or 5.8 mg/L protein concentration for anti–factor VIII. For each set of immunohistochemically stained slides, sections of spleen were used as positive controls and nonspecific IgGs were used at the same concentrations as the primary antibodies for negative controls. Sections were then rinsed and incubated with species-appropriate biotinylated secondary antibodies followed by another rinse. Labeling was done with avidin-biotin-peroxidase or avidin-biotin–alkaline phosphatase kits (Vectastain ABC Elite, Vector) with substrates 3,3-diaminobenzidine (Sigma) and alkaline phosphatase substrate kit I (Vector) kits, respectively. Total cell density for a cross section from the midportion of each stented segment was calculated, and the number of cells identified as macrophages by RAM 11 staining was counted and expressed as a percentage of total cells.

Statistics
All data are presented as the mean±SD. Comparisons between groups used ANOVA followed by Scheffe's procedure as a post hoc test. Values of P<.05 were considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Stent Expansion and Endothelial Injury
Endothelial cell preservation after angioplasty or stent implantation was studied by use of en face staining with AgNO₃. Histologically abnormal endothelium was apparent, centered within the areas bordered by stent struts 15 minutes after stent expansion within otherwise uninjured rabbit iliac arteries (Fig 1A, 1B, and 1C). Some areas showed frank endothelial cell loss, revealing underlying medial smooth muscle cells (Fig 1C), whereas other areas showed more subtle signs of endothelial cell injury, with nuclear cell membrane staining (Fig 1B). One hour after stent placement, endothelial cell loss had progressed to a point at which a rim of intact cells remained adjacent to stent wires (Fig 1D and 1E). Of the surface area between struts, 41±22% was devoid of endothelium, presenting denuded medial smooth muscle cells at the luminal surface. In contrast, 1 hour after balloon angioplasty, there was complete endothelial ablation within the angioplastied segment. No remnant endothelium was present, and the internal elastic lamina or underlying medial smooth muscle cells were visible throughout. Between 1 hour and 3 days after stenting, however, endothelial restoration occurred. En face staining 3 days after stenting demonstrated complete reendothelialization of the stented segment (Fig 1F).

View larger version (213K): [in this window] [in a new window]   Figure 1. Rabbit iliac arteries after endovascular stent placement. En face examination shows endothelial cells lining the luminal surface, with underlying orthogonally arranged medial smooth muscle cells evident at sites of endothelial cell loss. White bands are sites at which stent struts have prevented surface staining. A, At 15 minutes after stent placement, a pattern of endothelial cell injury centered in interstices between struts is apparent (magnification x34). At higher magnification (x100) of sites indicated by arrows, areas of early cell injury with nuclear condensation and extrusion (B) or frank cell loss (C) are seen. D, One hour after stent placement, areas of cell loss have widened (magnification x34). E, At higher magnification (x100), preserved endothelial cells adjacent to stent struts are apparent. F, Three days after stent expansion, the luminal surface has become completely relined with regenerated endothelium (magnification x100).

Biological Effects of Remnant Endothelium
The biological effects of remnant endothelium after stent expansion were examined at times corresponding to times of peak monocyte recruitment (3 days) and peak intimal thickening (14 days) in this model.¹⁸ Stents were expanded in arteries with either intact or previously denuded endothelium. Tissue and cellular reactions, including neointimal area and monocyte/macrophage and endothelial cell presence, were evaluated in histological cross sections.

Three days after stent placement in denuded arteries, a monolayer of inflammatory cells was observed adherent to the luminal surface (175±58 cells per section, Figs 2A and 3A¹⁹ ). The luminal accumulation of mural thrombosis and inflammatory cell presence measured 1.41±0.24 mm² at this time.¹⁹ Stent expansion in nondenuded arteries resulted in 83% fewer adherent monocytes after 3 days (30±6 cells per section, P<.003 compared with denuded stented arteries; Figs 2B and 3A) and 81% less intimal area (0.27±0.02 mm², P<.0001 compared with denuded stented arteries). The luminal surface of stented nondenuded arteries was lined after 3 days with a monolayer of cells (Figs 1F and 2B), specifically identified by immunohistochemical staining for factor VIII as endothelial cells (Fig 2C).

View larger version (99K): [in this window] [in a new window]   Figure 2. Rabbit iliac arteries 3 days (A through C, magnification x400) or 14 days (D through F, magnification x200) after endovascular stent placement. Sections are stained with Verhoeff's elastic tissue stain (A and B), with modified Russell-Movat Pentachrome stain (D and E), or immunohistochemically for factor VIII (C and F). A, Three days after stent placement with prestent endothelial denudation, many adherent mononuclear cells are seen lining the internal elastic lamina (arrow). B, Without preceding denudation, adherent monocytes are not seen, and endothelial cells lining the lumen are immunohistochemically identified with brown staining for factor VIII (C). After 14 days, a neointima has formed, separating the lumen from the internal elastic lamina (arrows) and stent struts (black rectangles). Stent placement after endothelial denudation (D) results in twofold more neointimal hyperplasia than does stent placement without preceding denudation (E), and luminal endothelial cells are again immunohistochemically identified in nondenuded arteries by brown staining for factor VIII (F).

View larger version (18K): [in this window] [in a new window]   Figure 3. A, Adherent monocyte number 3 days after stent placement in rabbit iliac arteries showing 83% fewer monocytes adherent to arteries not subjected to prestent balloon denudation (*P<.003). B, Intimal macrophages 14 days after stent placement showing the percentage of intimal cells identified as macrophages with a cell-specific antibody reduced 89% in arteries not denuded before stent placement (**P<.0006).

Fourteen days after stenting, an organized neointima had replaced the initial inflammatory cell–rich mural thrombus (Fig 2D). Arteries denuded of endothelium before stent placement accrued a neointima measuring 0.91±0.19 mm².¹⁹ Monocytes were still observed adherent to the surface (28±14 cells per section¹⁹ ). Within the intima, RAM 11 staining showed intensely labeled cells surrounding the stent struts (Fig 4) as well as in interstrut regions (0.66±0.17% of cells¹⁹ ). Cells staining for factor VIII (endothelial cells) were not apparent. In contrast, when arteries receiving stents without initial balloon denudation were compared with denuded stented arteries, there was less intimal thickening (intimal area, 0.52±0.13 mm², P<.02; Figs 2E and 5), there were fewer luminally adherent monocytes (7±2 cells per section, P<.02), there were fewer RAM 11–positive macrophages within the intima (0.09±0.04% of cells, P<.0006, Fig 3B), and there was an intact layer of factor VIII–positive endothelial cells (Fig 2F). There was no difference in medial area, stent diameter, or deep arterial injury between denuded and nondenuded groups at 3 or 14 days. For all anti–factor VIII and RAM 11 staining protocols, control nonspecific IgG stained negatively, whereas spleen sections stained with primary antibodies stained positively.

View larger version (155K): [in this window] [in a new window]   Figure 4. Tissue macrophages surrounding a stent strut (open rectangle, right) in a rabbit iliac artery 14 days after balloon denudation and stent placement. Lumen is at top of figure. Monocyte/macrophages staining red are labeled with a cell-specific antibody (RAM 11, magnification x600).

View larger version (12K): [in this window] [in a new window]   Figure 5. Neointimal area 14 days after endovascular stent placement in rabbit iliac arteries. Without prestent balloon denudation, neointimal hyperplasia was reduced 43% (*P<.02).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Endothelial Injury and Repair
To date, the most effective experimental models of vascular injury have been those that remove the endothelial monolayer. Physical scraping with repeated passage of an inflated balloon catheter or nylon loop and insufflation of air into the arterial lumen have each induced neointimal hyperplasia in a reproducible fashion.^{1 2 3} In human studies, endothelial dysfunction is an early harbinger of atherosclerosis²⁰ as well as a predictor and epiphenomenon of advanced lesions. The importance of endothelial loss in the pathogenesis of intimal thickening is further demonstrated by the rapidity with which intimal growth ceases once endothelial cells have spread from adjacent normal segments to reline the artery.^{2 3 4}

In contrast to denuding experimental models, clinical interventions induce deep vessel wall damage through stretching as well as endothelial injury through abrasion. Endovascular stents are unique among clinical devices in that they provide arterial dilation without circumferential contact of the device with the vascular endothelium. Our data demonstrate that stent struts limit contact of the expanding balloon with the underlying wall. In contrast to the total denudation that follows balloon angioplasty, balloon-expanded stents left areas of endothelium intact. Endothelial cell loss was apparent within the interstices of the stent 15 minutes after expansion, and the area of denudation encompassed 41% of the luminal surface after 1 hour. Within 3 days, however, the surface of the stented artery had become completely relined with endothelial cells. The difference between balloon- and stent-induced damage allowed study of endothelial injury and regrowth as contributing factors in the pathogenesis of neointimal hyperplasia.

In denuded arteries, endothelial regrowth begins as normal endothelial cells adjacent to the injured segment^{2 21} divide and migrate to reline the arterial surface. When a stent is placed without antecedent balloon denudation, sufficient endothelium remains within the stented segment to allow repopulation with a much reduced requirement for endothelial proliferation and migration. In several animal models, stent placement within denuded arteries produces an intimal response rich in macrophages^{18 19 22} and severalfold greater than that seen after balloon injury alone.^{12 13 23} Our data show that these biological parameters can be dramatically muted by remnant endothelium, presumably because of rapid regeneration of an intact endothelial monolayer. Monocyte recruitment and intimal area after 3 days were reduced by 83% and 81%, respectively; macrophage presence within the intima after 14 days by 89%; and neointimal hyperplasia after 14 days by 43%.

The mechanism of endothelial injury during stent expansion is not clear. Although it was expected that stent struts would themselves scrape the endothelial layer during expansion, the pattern of endothelial loss observed was not consistent with this mechanism. On the contrary, endothelial injury was most severe in stent interstices and was least severe adjacent to struts. Contributing factors to endothelial injury during stent expansion may include balloon-vessel contact between struts; pressure imposed by blood confined to the closed space between balloon, stent struts, and vessel wall during expansion; inhomogeneous circumferential strain applied to the vessel wall during dilation; or acute alterations in flow. Current clinical practice of coronary stent implantation includes high-pressure stent expansion with noncompliant balloons at pressures >14 to 16 atm. Furthermore, adjunctive pharmacological regimens accompanying stent use are evolving and may have unanticipated effects on vascular wall cellular responses. The reproducible localization of endothelial cell loss to stent interstices also implies that factors such as balloon pressure and compliance, strut thickness and configuration, speed of inflation, and vessel oversizing may all be important determinants of endothelial loss. Optimizing these parameters may allow less injurious stent expansion with correspondingly limited vascular responses.

Effects of Endothelial Injury and Repair
Endothelial dysfunction is one of the earliest markers of atherosclerosis,²⁰ and the effectiveness of intact endothelium at inhibiting intimal thickening has been demonstrated in several experimental models.^{2 3 4} Endothelial cell products reduce vessel wall permeability to platelet-derived growth mediators and other circulating factors. They inhibit thrombosis, platelet adhesion, and vascular smooth muscle cell growth.²⁴ They also bind and inactivate mitogens.^{25 26} Aside from classic experiments identifying heparan sulfate proteoglycan as a potent compound in such growth regulation, recent evidence also suggests that nitric oxide may participate in the control of vascular healing after injury.²⁷ If so, production of nitric oxide by regenerating endothelial cells might contribute to the modulation of neointimal hyperplasia observed in this model.

Macrophage presence within the intima in stented rabbit iliac arteries has been shown to be closely related to the appearance of intimal thickening.^{13 18 19} In denuded stented arteries, monocyte recruitment begins early and peaks within the first week.¹⁹ Our data now demonstrate that this endothelium-independent monocyte adhesion was virtually abolished by an intact endothelial monolayer. Although mechanisms underlying the recruitment and endothelium-independent binding of monocytes to stent-injured arteries are not clear, the inhibition of monocyte recruitment and infiltration by early endothelial regrowth may be an important part of the modulation of neointimal hyperplasia in this model.

Study Limitations
Our data support the beneficial effects of endothelial regrowth on vascular repair in nonatherosclerotic vessels and introduce a potential means for endothelial preservation in clinical arterial interventions. Direct extrapolation from biological effects of rapid endothelial restoration in experimental models to clinical settings is limited by uncertainty of endothelial cell presence and function in atherosclerotic vessels. Although there is histological evidence of endothelial cell presence within diseased vessels,^{9 11} further examination of regenerated endothelial cell biological activity will be essential to directing these observations toward clinical utility. Furthermore, direct extrapolation from stent expansion in normal arteries to primary clinical stenting in diseased arteries can only be made with caution. Difficulty in positioning stent-bearing balloon catheters across severe arterial stenoses may necessitate novel stent and catheter designs that will obviate the need for arterial predilation before stent expansion.

Implications
The burgeoning use and possibly reduced restenosis rates of endovascular coronary stents^{28 29} mandate better understanding of the effects of endovascular implants on vascular wall homeostasis. This study has demonstrated the different vascular responses to partial versus complete endothelial denudation caused by balloon-expanded stents. The study also shows that deep vascular injury is not the only mechanism by which stent implantation can provoke intimal thickening.¹⁶

Current clinical stent use often includes denuding interventions such as coronary imaging with an ultrasound catheter or predilation with a balloon before stent deployment. Our data allow the hypothesis that if some endothelium is present in atherosclerotic vessels,^{9 11} stents used without balloon predilation may provide a means for dilating arteries while avoiding complete endothelial ablation. Previous attempts have been made to reintroduce into injured vessels genetically engineered endothelial cells attached to stent struts.^{5 6 7} Alternative approaches may be (1) to use stents in a manner so as to minimize endothelial injury and (2) to design stents and catheters to enable primary stent placement without predilation and to facilitate endogenous endothelial regrowth.

	Acknowledgments

 
This work was supported in part by grants from the American Heart Association (95004400), the National Institutes of Health (GM/HL-49039), the Burroughs Wellcome Fund for Experimental Therapeutics, and the Whitaker Foundation. We are grateful to Drs Morris Karnovsky and Renu Virmani for review of our primary data and to Drs John Bittl and Andrew Selwyn for critical review of the manuscript.

Received March 26, 1996; revision received June 18, 1996; accepted July 2, 1996.

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