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Ann Thorac Surg 1995;59:948-954
© 1995 The Society of Thoracic Surgeons

Effects of a Single Administration of Fibroblast Growth Factor on Vascular Wall Reaction to Injury

Michael A. Parish, MD, Eugene A. Grossi, MD, F. Gregory Baumann, PhD, Tohru Asai, MD, Daniel B. Rifkin, PhD, Stephen B. Colvin, MD, Aubrey C. Galloway, MD

Division of Cardiothoracic Surgery, Departments of Surgery and Cell Biology, New York University Medical Center, New York, New York

Accepted for publication December 28, 1994.


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Expansion of the vascular wall through formation of neointimal fibromuscular lesions is the basic mechanism underlying stenosis of vascular grafts, restenosis of arteries treated by balloon angioplasty, and other major cardiovascular problems. This study examined the effect of a single, systemic, low dose of basic fibroblast growth factor (bFGF) on formation of neointimal fibromuscular lesions in response to injury. New Zealand white rabbits (n = 76) were subjected to balloon injury of the abdominal aorta. Forty-five rabbits were given a single intravenous dose of bFGF (0.5 µg/kg) immediately after injury, and 31 rabbits were given only the vehicle solution as controls. After 2 (n = 15), 5 (n = 21), 14 (n = 29), or 28 (n = 11) days the rabbits were sacrificed. Those rabbits receiving the single administration of bFGF exhibited significantly greater intimal thickening (intima/media ratio) than the control group at 5 days (mean +/- standard error of the mean, 0.091 +/- 0.009 versus 0.058 +/- 0.006; p < 0.002), but not at 14 or 28 days. These results were achieved without any significant differences in mitotic indices, as determined by a mitostatic method, between the two groups at any postinjury interval examined. The findings suggest that a single systemic dose of exogenous bFGF has a relatively long term effect on enhancing the neointimal response to vascular injury. Therefore, local control of endogenous bFGF may be useful in limiting formation of vascular neointimal fibromuscular lesions, thus improving the long-term results of vascular grafts, balloon angioplasty, and other cardiovascular procedures.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Expansion of the vascular wall through formation of a neointimal fibromuscular lesion is the key factor underlying many of the clinical problems faced by cardiovascular surgeons. This phenomenon, seemingly an excessively exuberant response to vascular injury, is an early and basic component of stenosis of vascular grafts, restenosis of arteries that have been treated by balloon angioplasty, microvascular disease associated with rejection of transplanted hearts, and the pathogenesis of spontaneous atherosclerotic lesions. Surgical research directed toward regulating neointimal fibromuscular lesion formation in vascular grafts has been reinforced strongly in recent years by the studies of cardiologists seeking to discover mechanisms for limiting arterial restenosis after balloon angioplasty. A wide variety of pharmacologic agents has been tested for the ability to block the smooth muscle cell (SMC) proliferation and migration that lead to neointimal fibromuscular lesion formation, but none of the agents tested has shown any clinically meaningful success to date [1].

During the past 20 years the role of growth factors in promoting neointimal fibromuscular lesion formation has begun to come under close scrutiny. The growth factor that originally received the greatest attention was platelet-derived growth factor [2], but recent work has demonstrated that this growth factor's role in neointimal fibromuscular lesion formation is probably not a primary one [3–5] and is more closely related to SMC migration than to SMC proliferation [3, 6, 7]. However, another growth factor known as basic fibroblast growth factor (bFGF) has been shown to be a potent mitogen for both smooth muscle [8, 9] and endothelial cells [10–13] and may play an important role in angiogenesis [14]. This heparin-binding polypeptide is present in the nucleus and cytoplasm of smooth muscle and endothelial cells as well as in the intercellular matrix [15–18]. Although the bFGF complementary DNAs are known to lack a classic secretory signal peptide sequence, suggesting that the protein is a nonsecreted cell product, bFGF is released from endothelial cells when they are injured [10, 11, 19] and can also be released from the intercellular matrix [20].

The present study was designed to examine the effects of a single, systemic, low dose of bFGF on neointimal thickening after balloon injury in the rabbit abdominal aorta and to determine whether bFGF preferentially binds to an injured segment of an artery.


   Material and Methods
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Experimental Model
The rabbit was chosen as an experimental model because, among other reasons, in this animal diet can be used to induce development of lipid-laden, atherosclerotic vascular lesions, an attribute that might prove useful in later stages of this line of investigation. The aorta was chosen as the vessel to be studied because in this large, elastic artery the flow hemodynamics, a potential factor in neointimal fibromuscular lesion development [21, 22], are relatively unaffected by vasoconstriction or mural thrombi that might develop after injury. The abdominal aorta was chosen because Goldberg and colleagues [23] have shown that the New Zealand white rabbit abdominal aorta has a greater response to balloon injury in terms of the number of neointimal cells over a 48-day period than the thoracic aorta. In addition, the abdominal aorta of the New Zealand white rabbit normally has a very low rate of DNA replication among the medial cells (0.5%), and the uninjured intima is rarely more than one or two cells thick [24]. The balloon catheter method of arterial injury [25] is used widely, most commonly in the rat carotid artery. Both medial and intimal tissues are injured by dragging the balloon catheter through the lumen of an artery [26].

Eighty-four New Zealand white rabbits (2.2 to 5.1 kg) were anesthetized by intramuscular ketamine (Aveco Co, Inc, Fort Dodge, IA) (35 mg/kg) and xylazine (Rugby Laboratories, Inc, Rockville Center, NY) (5 mg/kg). All animals received humane care in compliance with the ``Guide for the Care and Use of Laboratory Animals'' published by the National Institutes of Health (NIH publication 85-23, revised 1985). Additional injections of a 1:1 mixture of ketamine (100 mg/mL) and xylazine (20 mg/mL) were given as necessary in 0.5-mL increments. One hundred percent oxygen was administered by face mask, and Keflin (cephalothin; Eli Lilly & Co, Indianapolis, IN) (30 mg/kg) was given intravenously. A longitudinal incision was made on the medial aspect of the distal hind limb to expose the superficial femoral artery. The animals were heparinized (Elkins-Sinn, Inc, Cherry Hill, NJ) with 100 U/kg via an ear vein. A 3F Fogarty embolectomy catheter (Baxter Healthcare Corp, Edwards Division, Irvine, CA) with minimal eccentricity in shape when distended was introduced through an arteriotomy in the superficial femoral artery and advanced to the level of the diaphragmatic abdominal aorta. The catheter then was drawn antegrade through the abdominal aorta with the balloon inflated with saline solution so as to maintain a constant resistance as judged by the operator. This maneuver was repeated three times with the balloon rotated approximately 120 degrees between each pass. A consistent ballooning technique is required because the degree of distention during ballooning significantly influences the degree of endothelial denudation [27] and subsequent neointimal proliferation [28, 29]. We performed this ballooning procedure in 6 additional rabbits, immediately perfusion fixed the aorta, and examined the ballooned segment by scanning electron microscopy to ensure that this technique was removing almost all of the endothelial cells from the luminal surface. After ballooning the catheter was removed and the superficial femoral artery was ligated. The wound was irrigated and closed with 4-0 nylon suture.

Neointimal Hyperplasia and Basic Fibroblast Growth Factor
Forty-five of the rabbits were given bFGF (Synergen Inc, Boulder, CO) (0.5 µg/kg) intravenously immediately after completion of the balloon injury procedure. Thirty-one other rabbits similarly were given an equivalent dose of vehicle solution only (0.3 mol/L glycerol in 10 mmol/L NaPO4, pH 7.0) as controls. Animals were sacrificed at 2 (n = 15), 5 (n = 21), 14 (n = 29), or 28 (n = 11) days. Starting at 6 hours before sacrifice most of the rabbits received 1 mg/kg of the mitostatic drug colchicine by intramuscular injection, which was repeated at 2-hour intervals. This regimen prevented any aortic wall or blood cells that entered mitosis during the final 6-hour period from advancing beyond the metaphase stage. A segment of duodenum was removed from each rabbit after sacrifice and examined by light microscopy to verify that the colchicine had been effective in arresting dividing cells in metaphase.

At the time of sacrifice the rabbits were anesthetized with ketamine (35 mg/kg) and xylazine (5 mg/kg) and a median sternotomy was performed. The left ventricle was cannulated using a 14-gauge angiography catheter secured with a 6-0 Prolene (Ethicon, Somerville, NJ) pursestring suture. After the abdominal aorta was exposed through a laparotomy incision, the entire aorta was flushed from the left ventricle with Plasmalyte (Baxter Healthcare Corp, Deerfield, IL) and then perfused with cold 3% glutaraldehyde in Sorensen's phosphate buffer (0.1 mol/L, pH 7.3) at physiologic pressure for 5 minutes. The abdominal aorta was excised, and after further fixation, a portion from the middle of the ballooned segment was embedded in epon.

Longitudinal sections approximately 5 mm long, 1 µm thick, and 50 to 100 µm apart were cut from each specimen. These sections were stained with a polychrome stain [30] and examined under a light microscope with an attached video camera. A video image of each section was digitized, and the entire intimal and medial areas in the first section cut from each specimen were measured using computerized video morphometry software. The degree of neointimal thickening was determined by calculating the ratio of the area of the intima to the area of the media for the entire section. The results were analyzed using Students's t test (SPSS/PC+; SPSS Inc, Chicago, IL) and expressed as the mean +/- the standard error of the mean.

In those rabbits that received colchicine before sacrifice at 2 (n = 15), 5 (n = 9), 14 (n = 15), and 28 (n = 7) days a mitotic index for the 6-hour period immediately before sacrifice was measured. In each of three sections cut from each specimen a minimum of 1,000 cells in the media and all cells in the neointima and adventitia were examined with an oil immersion objective (x 1,000 magnification) and a grid pattern eyepiece. For each section the number of nuclei in a specific region of the aortic wall observed to be in arrested metaphase was expressed as a percentage of the total number of nuclei observed in that region. The mitotic index is presented as the mean +/- the standard error of the mean of all the sections from all colchicine-treated animals sacrificed at each postoperative interval.

To confirm light microscopic findings thin sections were cut from some control and experimental specimens from each postoperative time interval, mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a Siemens transmission electron microscope.

Eight additional rabbits were given 125I-bFGF (1.4 µCi/kg) intravenously immediately after the balloon injury, and were sacrificed 1 hour later. The abdominal aorta was excised using a laparotomy incision, and the uninjured thoracic aorta was excised through a median sternotomy. Both specimens from each rabbit were incised longitudinally for measurements of the area of the luminal surface and then placed in an Auto-Gamma Scintillation Spectrometer (Packard Instrument Company, Downers Grove, IL) to determine the amount of radiolabeled 125I-bFGF bound to each segment. Data were expressed as counts per unit surface area, analyzed using the Wilcoxon matched-pairs test, and expressed as the mean +/- the standard error of the mean.


   Results
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
At 2 days after injury the endothelium was found to be removed uniformly from most of the luminal surface of the aorta. A scattered monolayer of platelets was present on the stripped luminal surface. The media showed little reaction to the injury except for a few scattered, edematous SMCs in the inner half of the media and some mitotic SMCs at various levels within the media (Table 1Go). These mitotic SMCs usually were not clustered but were spread relatively evenly along the length of the section. The medial mitotic SMCs invariably displayed a primarily contractile rather than a synthetic phenotype and retained close contact with adjacent SMCs (Fig 1Go). In addition, mitosis could be observed among some of the outermost medial SMCs and the fibrocytes of the adventitia (Fig 2Go). Little or no interaction between circulating blood elements other than platelets and the denuded luminal surface was observed at 2 days, and no formation of neointima could be detected at this time.


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  		Table 1. . Mitotic Indices of Layers of Aortic Wall at Various Intervals After Balloon Injurya
 


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  		Fig 1. . Electron micrograph of control rabbit aorta at 2 days after balloon injury showing arrested mitosis in three medial smooth muscle cells (SMC) deep in the media near the adventitia. (Uranyl acetate and lead citrate; original magnification, x8,250.)

 


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  		Fig 2. . Light micrograph of basic fibroblast growth factor-treated rabbit aorta at 2 days after balloon injury showing adventitial cells in arrested mitosis (arrows). (Polychrome stain; original magnification, x2,120.)

 
By 5 days, however, there was a small but easily observed neointima being formed with a fairly constant thickness along the length of the injured aorta (Fig 3Go). On electron microscopic examination the neointimal cells appeared to be modified SMCs that had migrated from the media into the intima to colonize a new luminal area and demonstrated a relatively high mitotic index (Table 1Go). The neointima contained few collagen and elastin fibers in its intercellular matrix at this stage of development. Some cells in the media and adventitia also still were undergoing mitosis at 5 days. At this early stage of neointima formation the intima/media ratio was significantly greater in the bFGF-treated group (0.091 +/- 0.009) than in the control group (0.058 +/- 0.006; p = 0.002) (Table 2Go).



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  		Fig 3. . Light micrograph of basic fibroblast growth factor-treated rabbit aorta at 5 days after balloon injury showing a thin, pale-staining neointima (NI). (A = adventitia; M = media.) (Polychrome stain; original magnification, x334.)

 

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  		Table 2. . Intimal/Medial Ratio of bFGF-Treated and Control Animals at Various Time Intervals After Balloon Injurya
 
By 14 days the neointima had continued to expand to a thickness approximately 50% of that of the media (Fig 4Go), but there was no longer any significant difference in intima/media ratio between the bFGF-treated and control groups. Collagen and elastin fiber formation was observed more easily in the neointima at this time, and the luminal surface was lined with modified SMCs. A relatively low level of mitotic activity still could be detected in the neointima, media, and adventitia. At 28 days the neointima had become approximately equal in thickness to the media (Fig 5Go), and mitotic activity could rarely be found in any layer of the vessel wall. A web of elastic fibers was noticeable throughout the outer two-thirds of the neointima, which had begun to assume a more mature appearance overall.



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  		Fig 4. . Light micrograph of basic fibroblast growth factor-treated rabbit aorta at 14 days after balloon injury showing a thicker, darker-staining neointima (NI). (A = adventitia; M = media.) (Polychrome stain; original magnification, x321.)

 


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  		Fig 5. . Light micrograph of basic fibroblast growth factor-treated rabbit aorta at 28 days after balloon injury showing a further thickened neointima (NI) with the same thickness and staining properties as the media (M). (A = adventitia.) (Polychrome stain; original magnification, x334.)

 
In addition, a significantly greater amount of 125I-bFGF per unit area was localized to the denuded abdominal aorta (380.21 +/- 85.11 versus 172.09 +/- 38.48 counts/mm2; p < 0.05). Nonspecific binding using protein A and a control uninjured abdominal aorta was negligible in a subsequent experiment (data not shown).


   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The most striking finding of this study is that a single systemic dose of bFGF (0.5 µg/kg) administered immediately after vascular injury can significantly increase the thickness of the resultant neointimal expansion at 5 days after injury. In a previous study Lindner and co-workers [31] demonstrated that prolonged daily systemic administration of a larger dose of bFGF (12 µg/day) for 2 weeks after ballooning of the rat carotid artery produced a neointima with a cross-sectional area double that of controls. They did not report on neointimal thickening at postinjury periods shorter or longer than 14 days. The present findings show that even a single, relatively small dose of bFGF can have a significantly enhancing effect on neointimal thickening over an interval as long as 5 days after injury.

A possible explanation for the long duration of this bFGF effect is that bFGF can bind to basement membrane [16, 18] and heparan sulfate proteoglycan complexes in the intercellular matrix, which would be exposed by arterial injury, and such bound bFGF could act as a reservoir for the slow release of bFGF [32]. Another possible explanation is that systemic administration of bFGF after arterial injury initiates a cytokine-growth factor cascade mechanism [33], which through a self-stimulatory positive feedback loop could amplify and sustain neointimal thickening over a relatively long period.

The finding that a single application of bFGF can increase neointimal thickening 5 days later implies a possible major role for endogenous bFGF in neointimal fibromuscular lesion formation and, conversely, also suggests that blocking or altering the pathways through which bFGF acts might inhibit production of such lesions. Of course, it is acknowledged that producing an increase in the size of neointimal fibromuscular lesions through introduction of a pharmacologic dose of a specific growth factor does not necessarily prove that endogenously produced amounts of that growth factor play a similar major role in lesion formation.

The principal mechanisms through which bFGF might act to stimulate neointimal lesion formation are SMC proliferation and SMC migration. It is unlikely that by only 5 days after injury the increased thickness of the newly formed neointimal lesions observed in bFGF-treated animals could be accounted for by an increase in the production of intercellular connective tissue within the neointima [31]. The presence of bFGF has been shown to be mitogenic for SMCs in vitro [8, 9] and in vivo [31]. No significant increase in SMC proliferation, however, in either the media or neointima of the bFGF group could be detected by a mitostatic method at any of the postinjury intervals examined (see Table 1Go). The mitostatic method showed that the mitotic indices measured varied considerably among animals from the same group sacrificed on the same day, but were quite consistent in various sections taken from the same aorta.

We chose to use the mitostatic method for measuring SMC proliferation rather than the more commonly applied 3H-thymidine or bromodeoxyuridine immunohistochemistry methods to measure directly actual proliferation rather than DNA replication. Although rates of SMC DNA replication usually are presumed to be equivalent to rates of SMC cell division, there is ample justification for caution regarding assumptions about the relative brevity of the G2 phase in vascular SMCs. In the adult human aorta, carotid artery, and iliac artery 7% of the SMCs have been found to be polyploid, mostly tetraploid but occasionally higher forms, and this subset of SMCs may have important functional roles [34]. Ten percent of normotensive rat aortic SMCs have tetraploid nuclei, but in hypertensive rat aortas a twofold to threefold increase in the frequency of tetraploid and octaploid SMCs develops [35, 36]. Such polyploid SMCs could represent cells arrested in the G2 phase of the cell cycle or true tetraploids in the G0 resting phase of the cycle. Furthermore, in vitro studies of addition of epidermal growth factor to subconfluent quiescent porcine aortic vascular SMCs have shown minimal cell division despite a high rate of DNA synthesis measured by 3H-thymidine [37].

Comparison of vascular SMC mitotic rates between this study and various other studies is extremely difficult due to differences in the animal species used, the vessel chosen for study, the method of producing vascular injury, the length of the interval after injury when the mitotic rate is measured, the method used to measure the mitotic rate, and the length of the interval over which the mitotic rate is measured. In the present study, among the time intervals used the mitotic index in the media was the highest at 2 days, when the means for the two groups ranged from 0.6% to 1% (see Table 1Go). Assuming that the mitotic index for the 6-hour period measured remained constant over 24 hours and does not show diurnal variation, by no means a certain assumption, this medial mitotic index would extrapolate to 2.4% to 4% over a 24-hour period.

In a previous study involving bFGF, Lindner and co-workers [31], using a 3H-thymidine labeling technique, found that intraarterial infusion of 120 µg of bFGF into the rat carotid artery immediately after balloon catheter or filament loop injury caused a highly significant increase in medial SMC labeling or ``proliferation'' (54.8% versus 11.5% after balloon injury, 43.3% versus 1.3% after filament loop injury). They also observed that a similar administration of bFGF 6 weeks after injury caused a significant increase in neointimal SMC labeling (6.9% versus 0.9%). Another study using the balloon-injured rat carotid artery and the 3H-thymidine method found the medial SMC mitotic index over a 1-hour period at 2 days after injury to be 6% [38]. Other studies using similar methods found a medial SMC mitotic index of 29% in the rabbit carotid over the first 24 hours after balloon injury [39] and a medial SMC mitotic index of 13% in the rat carotid artery over 24 hours at 2 days after injury [3].

Furthermore, the data presented here show that at 5 days after injury the SMC mitotic index in the neointima was at its highest rate for the time intervals measured, with the means for the two groups ranging from 6.9% to 9.4% for a 6-hour period or 28% to 38% extrapolated to 24 hours. In the media the mean SMC mitotic index for the groups ranged from 0.075% to 0.082% for 6 hours or 0.30% to 0.33% extrapolated to 24 hours. Other studies using the 3H-thymidine method over a 24-hour period found that in the ballooned nude rat carotid artery the medial SMC mitotic index was 25% at 4 days after injury and 11% at 6 days [5] and that in the ballooned rat carotid artery at 7 days the neointimal SMC mitotic index for a 24-hour period ranged from 33% to 36% and the medial SMC mitotic index ranged from 2.5% to 3.3% [22]. Additional very similar studies showed the medial SMC mitotic index for a 24-hour period to be approximately 12.5% at 4 days after injury and approximately 5% at 7 days [3] and showed the neointimal SMC mitotic index to be 73% at 4 days and 58% at 7 days and the mitotic index of the SMCs of the inner media to be 41% at 4 days and 15% at 7 days [26].

Although direct comparison of vascular SMC mitotic indices between different experimental studies is extremely difficult for the reasons mentioned, the general trend of the comparisons presented above strongly suggest the possibility that methods of gauging SMC ``proliferation'' by measuring the rate of DNA replication rather than the rate of actual cell division may be overestimating the role that SMC proliferation plays in neointimal lesion formation.

Although replication of neointimal and medial SMCs has been investigated heavily with respect to the pathogenesis of neointimal lesions, mitosis in the adventitia of the same vessels rarely has been reported or discussed [39, 40]. In this study we noted a moderate level of outer wall SMC and adventitial fibrocyte mitosis in both the bFGF-treated and control groups, especially at 2 and 5 days after injury. What direct or indirect role, if any, proliferation among adventitial cells plays in neointimal lesion formation is unknown, but certainly merits further study.

Another unexpected result of this study was the finding that the medial SMCs in arrested mitosis displayed a contractile phenotype, with a heterochromatic nucleus and a cytoplasm dominated by microfilaments, rather than a synthetic phenotype, with a euchromatic nucleus and a cytoplasm dominated by an extensive rough endoplasmic reticulum and a large Golgi apparatus [41, 42]. A number of previous studies have suggested that in vascular SMCs loss of the contractile phenotype is a prerequisite for entry into mitosis [41, 43–45]. It is quite possible that administration of the mitostatic drug colchicine, which has antitubulin properties that disrupt microtubules, 6 hours before sacrifice may have slowed or prevented the SMCs' modulation of phenotype from contractile to secretory before the SMCs entered mitosis [46]. Nevertheless, that contractile phenotype SMCs were able to reach metaphase shows that SMC modulation to a contractile phenotype and entry into mitosis usually may be coincident but are not inextricably linked.

The relative importance of the roles of SMC migration and SMC proliferation in neointimal lesion formation are understood poorly. Other studies have suggested that only a small number of SMCs in an artery proliferate in response to an injury stimulus and that nondividing as well as proliferating SMCs can migrate into the neointima [47, 48]. A study of the size of neointimal lesions induced by low flow showed that the size is more affected by SMC migration than proliferation [22]. In studies on the effects of platelet-derived growth factor on the injured rat carotid artery, two groups of investigators concluded that increased migration of SMCs into the neointima probably accounted for most of the growth factor's effect on neointimal thickening [3, 5]. The present findings showing a significant increase in the intima/media ratio of the bFGF-treated group compared with the control group at 5 days without a correspondingly significant increase in the mitotic index of the media or neointima of the bFGF group at 2 days or 5 days also suggest that SMC migration might play a dominant role. The exact mechanisms by which normally immobile medial SMCs suddenly become migratory and rapidly advance into the neointima are understood very poorly. One way, however, by which growth factors might stimulate neointimal fibromuscular lesion formation would be by increasing the SMCs' ability to modify the surrounding intercellular matrix so as to facilitate movement through it [49].

In conclusion, the results presented here demonstrate the potential role of local bFGF with respect to SMC migration and proliferation resulting in neointima formation as a response to moderate vascular injury. The findings show that exogenous bFGF binds to the site of vascular injury and promotes early neointimal lesion formation for up to 5 days. The observation of this link between bFGF and the early stage of neointimal lesion formation suggests that restricting the bioavailability of endogenous bFGF at the site of arterial injury might inhibit neointimal lesion formation. Studies involving the use of anti-bFGF immunoglobulin G antibody to inhibit formation of neointimal lesions in injured vessels are currently in progress.


   Acknowledgments
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
Supported in part by grant CA34282 from the National Institutes of Health to Dr Rifkin.

We gratefully acknowledge the assistance of John Favale.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Address reprint requests to Dr Baumann, Division of Cardiothoracic Surgery, New York University Medical Center, 520 First Ave, Rm 532, New York, NY 10016.


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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