© 2000 American Heart Association, Inc.
Vascular Biology |
Early Effects of Arterial Hemodynamic Conditions on Human Saphenous Veins Perfused Ex Vivo
From the Emory University School of Medicine (K.M., T.F., D.N.K., Z.S.G.) and Georgia Institute of Technology (M.T., N.C., D.N.K., Z.S.G.), Atlanta.
Correspondence to Zorina S. Galis, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, WMB 319, Atlanta, GA 30322. E-mail zgalis{at}emory.edu
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Abstract |
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Abstract—Exposure to the arterial hemodynamic environment is thought to be a potential trigger for the pathological remodeling of saphenous vein grafts. Using matched pairs of freshly isolated human saphenous vein, we analyzed the early effects of ex vivo hemodynamic conditions mimicking the venous (native) compared with arterial (graft) environment on the key components of vascular remodeling, ie, matrix metalloproteinase (MMP)-9 and MMP-2 and cell proliferation. Interestingly, we found that arterial conditions halved latent MMP-9 (50±11%, P=0.01) and MMP-2 (44±6%, P=0.005) levels relative to matched vein pairs maintained ex vivo under venous perfusion for up to 3 days. Immunostaining supported decreased MMP levels in the innermost area of arterially perfused veins. Either decreased synthesis or increased posttranslational processing may decrease MMP zymogen levels. Biosynthetic radiolabeling showed that arterial perfusion actually increased MMP-9 and MMP-2 production. When we then examined potential pathways for MMP zymogen processing, we found that arterial conditions did not affect the expression of MT-MMP-1, a cell-associated MMP activator, but that they significantly increased the levels of superoxide, another MMP activator, suggesting redox-dependent MMP processing. Additional experiments indicated that increased superoxide under arterial conditions was due to diminished scavenging by decreased extracellular superoxide dismutase. Arterial perfusion also stimulated cell proliferation (by 220% to 750%) in the majority of vein segments investigated. Our observations support the hypothesis that arterial hemodynamic conditions stimulate early vein graft remodeling. Furthermore, physiological arterial flow may work to prevent pathological remodeling, particularly the formation of intimal hyperplasia, through rapid inactivation of secreted MMPs and, possibly, through preferential stimulation of cell proliferation in the outer layers of the vein wall.
Key Words: matrix metalloproteinase • vein graft remodeling • hemodynamics • redox • extracellular superoxide dismutase
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Introduction |
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Saphenous vein bypass grafting is an important treatment modality for atherosclerotic disease, but it frequently progresses to graft failure, a cause of ischemic morbidity and mortality.1 2 Development of intimal hyperplasia, through smooth muscle cell (SMC) proliferation and migration,3 4 diminishes lumen diameter and, in association with thrombosis and atherosclerosis, can lead to total vein graft occlusion.1 2 Pathological vein graft remodeling also includes medial and adventitial hypertrophy and sclerosis, whose precise consequences remain to be defined.5 Previous studies6 7 8 9 10 have shown that SMC proliferation and migration and general tissue reshaping depend on the activity of matrix-degrading enzymes called matrix metalloproteinase (MMP)-2 and MMP-9, known also as gelatinase A and B. These enzymes degrade basement membrane collagens and elastin,11 presumably freeing cells to move through the extracellular matrix and enabling the remodeling of tissues.
Two main factors traditionally believed to promote vein graft remodeling are surgical preparation injury and exposure to the arterial hemodynamic environment. We and others have shown that vein graft preparative injury increases MMP-9 and MMP-2 levels and vascular cell proliferation12 13 14 as well as intimal hyperplasia.15 The role of MMPs in pathological graft remodeling is also supported by findings that MMP inhibitors reduce vein graft intimal hyperplasia.16
Vascular remodeling, which occurs in response to changes in blood flow and pressure, normalizes shear and wall stress.17 Cyclic mechanical stretch could stimulate the proliferation of venous SMCs, as suggested by in vitro studies.18 Although pulsatility and increased pressure and flow, experienced by veins in the arterial hemodynamic environment, are thought to contribute to triggering the graft remodeling, their specific effects are somewhat conflicting. Arterial conditions are characterized by increased wall tension, associated with the formation of intimal hyperplasia,19 as well as by increased flow, thought to decrease intimal hyperplasia.20 21 In the present study, we pursued the early effects (up to 3 days) of ex vivo hemodynamic conditions mimicking either the bypass graft (arterial) or native (venous) environment on gelatinase expression/activation and cell proliferation in matching pairs of human saphenous veins.
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Methods |
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Tissue Preparation
Saphenous vein leftover after coronary artery bypass grafting was obtained after the last proximal anastomosis. To minimize variance, in each perfusion experiment, we compared arterial versus venous perfusion by the use of 2 matching vein segments derived from a single vein specimen (single donor and same preparative surgeon). We selected only undamaged vein specimens, >10 cm in length, 3 to 5 mm in diameter, and with a uniform gross morphology. In some cases, a vein specimen ring was immediately embedded as the nonperfused (fresh) control.
Ex Vivo Saphenous Vein Perfusion
The ex vivo perfusion system has been described in detail
elsewhere.22 Matching vein segments were placed in 2
identical systems, mounted in physiological
orientation between 2 cannulas acting as the inflow and outflow
perfusion conduits. Total time from operating room to perfusion was <1
hour. The perfusion medium was DMEM containing 100 U/mL penicillin, 100
µg/mL streptomycin, and 0.25 µg/mL amphotericin B (Fisher). Dextran
(5%) was added to obtain physiological viscosity
(4 cP). Bromodeoxyuridine (BrdU, 10 µmol/L) was added to the
culture medium to study cell proliferation. Venous perfusion conditions
were as follows: steady flow, flow rate 15 mL/min, and pressure 10
mm Hg. Arterial conditions were as follows: pulsatile flow
90 cycles/min, flow rate 90 mL/min, and pressure 120/80 mm Hg.
Static conditions were no flow/no pressure. The perfusion systems were
maintained at 37°C in an incubator containing 95% air/5%
CO2. After perfusion, vein ends were discarded,
and each segment was divided into equal (5-mm) rings, which were either
immediately processed for biochemical or histological
assays or cultured.
SDS-PAGE Zymography
Tissues were extracted in ice-cold 10 mmol/L sodium
phosphate, pH 7.2, containing 150 mmol/L NaCl (PBS), 1% Triton
X-100, 0.1% SDS, 0.5% sodium deoxycholate, and 0.2% sodium azide.
Samples normalized by total protein content (DC Protein Assay, Bio-Rad)
were separated by 10% SDS-PAGE containing gelatin (1 mg/mL) in
parallel with prestained molecular weight markers (Novex), as
previously described.23 Gelatinases, identified as lytic
bands after staining gels with colloidal brilliant blue G, were
quantified by use of the Molecular Analyst software program
(Bio-Rad).
Western Blotting
Tissue extracts normalized by protein were separated on 10%
SDS-PAGE minigels and then transferred onto nitrocellulose (Bio-Rad).
MMP-2 and MMP-9 were detected by using commercially available
antibodies (Oncogene Science). Extracellular (ec) superoxide dismutase
(SOD) was detected by using a polyclonal chicken antiserum (1:1000)
generated against the oligopeptide corresponding to amino acids 211 to
228 (GVCGPGLWERQAREHSER), which was generously provided by Dr
David Harrison (Emory University), followed by rabbit anti-chicken
horseradish peroxidase labeled secondary antibody (1:15000, Jackson
Immunoresearch). Cu/Zn SOD was detected with sheep anti-human Cu/Zn SOD
antibody (1:1000, Biodesign International), followed by anti-sheep
horseradish peroxidase labeled antibody (1:1000, Jackson
Immunoresearch). Reaction was developed with the ECL chemiluminescence
kit (Amersham), as described previously.23 Films were
quantified by use of the Molecular Analyst software.
MMP-2 and MMP-9 De Novo Synthesis
Pulse metabolic radiolabeling was performed by
culturing vein rings in methionine and cysteine-deficient DMEM
containing Expre35S35S (50
µCi/10 mL, DuPont-NEN) for 6 hours. Tissue extracts, normalized by
total protein, or culture media aliquots, normalized by tissue weight,
were incubated with anti-MMP antibodies (1 µg/50 µg) for 2 hours at
room temperature. Immune complexes precipitated with protein
A–Sepharose (10 mg per sample, Sigma Chemical Co) were separated by
SDS-PAGE. Gels were fixed, impregnated with
EN3HANCE (DuPont-NEN), dried, and exposed to
x-ray film at -70°C. Immunoprecipitated proteins were quantified by
use of the Molecular Analyst software program.
Immunohistochemistry
OCT-embedded frozen tissue sections (7 µm) were fixed in
acetone. MMP-2 and MMP-9 were detected by 1-hour incubation with 1:200
primary antibodies, followed by a peroxidase-labeled
streptavidin-biotin kit (LSAB 2, DAKO) and diaminobenzidine as
substrate. Nuclei were counterstained with Gill’s hematoxylin
(Sigma Chemical Co). Omission of primary antibody served as a negative
control. Sections were imaged by use of a Zeiss Axioscope microscope
equipped with a computer-based imaging system with ImagePro Plus
software (Media Cybernetics).
Proliferating cells were detected with 1:20 anti-BrdU antibody (DAKO), followed by Texas red–conjugated secondary antibody (Jackson Immunoresearch). All nuclei were counterstained with Hoechst. For each vein specimen, 3 sections spaced 200 µm apart were analyzed. In each section, the BrdU-positive (red) nuclei and all nuclei counterstained with Hoechst (blue) were counted in 3 microscopic fields, each encompassing the full thickness of the vessel wall. This method resulted in counting of at least 5000 cells per vein by 2 independent observers. The percentage of BrdU-positive nuclei was calculated and averaged per vein.
In Situ Zymography
Gelatinolytic activity was detected in
frozen tissue sections by use of Kodak NTB2
autoradiography emulsion (1:1 in incubation buffer), as
described previously.24
Superoxide Production Assay
Superoxide production was evaluated in vein rings by
lucigenin (5 µmol/L) chemiluminescence,25 reported
to have high specificity, and validated by use of electron spin
resonance.26 Immediately after ex vivo perfusion, rings
from matching vein segments were incubated in Krebs-HEPES buffer
(mmol/L: NaCl 99.01, KCl 4.69, CaCl2 1.87,
MgSO4 1.20,
K2HPO4 1.03,
NaHCO3 25.0, sodium HEPES 20.0, and glucose 11.1)
for 30 minutes at 37°C and then transferred to scintillation vials
containing lucigenin in Krebs-HEPES buffer. Counts were obtained at
1-minute intervals by use of a scintillation counter (LS 7000, Beckman
Instruments, Inc) in out-of-coincidence mode with a single active
photomultiplier tube for 20 minutes. Background counts (lucigenin
solution alone) were subtracted from the total counts. Some rings were
preincubated with Cu/Zn SOD, diphenyleneiodonium, or
diethyldithiocarbamate for 30 minutes. For each condition, 2 different
rings from each vein segment were measured and averaged.
Localization of Superoxide
Dihydroethidium (Molecular Probes), which in the presence of
superoxide is converted to ethidium and intercalates with nuclear DNA,
was used to localize and semiquantitatively assess intracellular
superoxide in vein tissue sections, essentially as described by Miller
et al.27 Frozen tissue sections (30 µm) were
incubated with 2 µmol/L dihydroethidium in Krebs-HEPES buffer at
37° for 30 minutes in the dark. Sections of arterially
and venously perfused matching vein segments were incubated in
parallel, with images collected with the use of a 585-nm long-pass
filter and identical acquisition parameters on a Bio-Rad
MRC 1024 argon confocal microscope.
Statistical Analysis
In each experiment, to minimize individual patient variations,
parameters measured for veins maintained under
arterial conditions were normalized to values obtained from
matched segments perfused under venous conditions. The letter n always
represents the number of independent experiments in which pairs
of matched vein segments were investigated. Means of normalized levels
were compared by 2-tailed Student t test, with a value of
P
0.05 accepted as significant.
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Results |
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Vein Graft Morphology and Viability
Perfused and fresh vein segments showed similar typical saphenous vein morphology, including areas of intimal thickening, a tunica media with longitudinally and circumferentially organized SMCs, and a loosely organized adventitia with multiple vasa vasora. Regardless of hemodynamic conditions, or general morphology, endothelial immunostaining (not shown) demonstrated variable (25% to 90%) luminal coverage, similar to that previously reported for vein grafts in situ.28 29
Gelatinolytic bands secreted by veins corresponded
to latent (pro) MMP-9, pro-MMP-2, and active MMP-2, as previously found
for human saphenous vein SMCs23 and as confirmed by
Western blotting. Phorbol myristate acetate (25 nmol/L) or 10
ng/mL tumor necrosis factor-
(R&D Systems) stimulated pro-MMP-9
tissue levels in human saphenous vein maintained in perfusion systems
for 3 days and left the MMP-2 level unchanged (see Figure
I, which can
be accessed online at http://atvb.ahajournals.org), as previously found
in cultured human saphenous vein SMCs,23 indicating that
veins remained viable with intact gelatinase metabolic
pathways for up to 5 days in the ex vivo system.
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Arterial Hemodynamics Affect MMP-9 and
MMP-2 Levels and Distribution
Comparison of MMP-9 and MMP-2 tissue levels in veins maintained in
different hemodynamic conditions showed that although
there was no statistically significant difference between static and
venous conditions (pro-MMP-9, P=0.95; pro-MMP-2,
P=0.60; and active MMP-2, P=0.20; n=4 independent
experiments), levels were significantly affected in
arterial conditions. After 3 days of perfusion,
arterially perfused vein segments contained 50±8% less
pro-MMP-9 (P=0.01), 44±6% less pro-MMP-2
(P=0.005), and 35±14% less active MMP-2
(P=0.08) than their venously perfused counterparts (n=4), by
gelatin zymography (Figure 1
). Lower
levels of MMP-9 and MMP-2 were also detected in the culture media (not
shown). To examine whether the decreased levels of zymogens are due to
decreased synthesis, we investigated the effects of
hemodynamic conditions on de novo MMP
production by metabolic labeling of matching
perfused vein segments. Interestingly, immunoprecipitation of MMP-9 and
MMP-2 showed that arterial perfusion actually increased the
level of newly synthesized MMP-9 and MMP-2 (Figure 2), suggesting that the apparent decrease
in zymogen levels may be due to an increased posttranslational
processing, eventually leading to MMP protein
degradation.30 Immunoprecipitation and
immunostaining support a different distribution of
MMP-9 and MMP-2. Newly synthesized MMP-9 was exclusively detected in
the culture media, suggesting rapid secretion and low tissue retention,
also supported by low levels of tissue specimen
immunostaining (Figure 3). In contrast, radiolabeled MMP-2 was
detected primarily in tissue extracts, supporting the higher retention
in the extracellular space, reflected by the intensely positive tissue
immunostaining. MMP-2 was detected extracellularly and
intracellularly, whereas MMP-9 staining was mostly intracellular.
Although no overall quantitative differences in MMP
immunostaining were detected in frozen tissue sections
of arterially or venously perfused vein segments, in the
arterially perfused vein sections, staining for both MMPs
appeared less intense in the inner layers and more intense in the outer
layers of the wall (Figure 3). Interestingly, increased
gelatinolytic activity and increased cell
proliferation were detected in the outer layers, suggesting the
preferential early remodeling of outer vein wall layers of the graft
and supporting the contribution of MMP activity.
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Cellular Proliferation
Cellular proliferation, identified by BrdU nuclear incorporation,
occurred predominantly in the adventitia and outer media after 3 days
of perfusion, with only occasional inner medial or intimal
proliferating cells. The proliferation index of arterially
perfused vein segments was higher than that of venously perfused vein
segments, with values between 200% and 750%, with a mean of
288±127% (P=0.07, n=9 microscopic fields per vein segment)
in 4 of 6 matched pairs, whereas in 2 other experiments, there was no
difference (not shown). Staining of serial sections with an
endothelial cell marker identified some of these
proliferating cells as endothelial cells of the vasa
vasorum (not shown), whereas others were presumed to be SMCs or
myofibroblasts.
Potential Mechanisms of Gelatinase Activation
On the basis of our results showing that arterial
conditions actually stimulate MMP production, we further
investigated potential activation pathways that could explain the
decreased zymogen levels. First, we analyzed the expression of
MT-MMP-1, an activator of MMP-2 expressed by human vascular
cells,31 and found that perfusion conditions did not
affect its levels (see Figure II, which can be accessed online at
http://atvb.ahajournals.org), as measured by Western blotting.
We then investigated the possible regulation of MMP processing due to
the redox environment. We showed previously that activation of
gelatinase zymogens produced by human SMCs can be triggered by
interaction with reactive oxygen species.32 Shear stress
and pulsatile stretch were reported to increase the release of such
species by isolated vascular cells.33 34 Indeed, when we
compared intact vein segments, we found that compared with venous
conditions, arterial perfusion increased the superoxide
levels by 240% to 580% (average increase 350±100%,
P<0.05, n=4), as measured by chemiluminescence (Figure 4). The addition of Cu/Zn SOD eliminated
the counts, indicating that chemiluminescence was solely due to
superoxide. Furthermore, because the exogenous Cu/Zn SOD does not cross
the cell plasma membrane, the result suggests that all superoxide
detected was present in the extracellular space. Superoxide
detected in the extracellular space may have been generated
extracellularly or may have come from intracellular sources. When we
examined semiquantitatively the intracellular levels of superoxide with
the use of dihydroethidium staining (not shown), we found no
differences between arterial and venous conditions.
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Mechanisms of Increased Superoxide Levels in
Arterially Perfused Vein Grafts
To sort out potential pathways of increased superoxide levels
under arterial conditions, we tested the effects of several
inhibitors (Figure 4). We
found that the addition of diphenyleneiodonium, a flavoprotein
inhibitor, completely inhibited chemiluminescence, ruling
out nonflavoproteins, such as xanthine/xanthine oxidase. Although we
cannot rule out the contribution of other flavoprotein-dependent
enzymes, such as NO synthase, which under certain circumstances may
produce superoxide, the vascular NAD(P)H oxidase is the most likely
major source of superoxide production35 in the
vein wall. Treatment of veins with diethyldithiocarbamate, a copper
chelator inhibitor of SOD, eliminated the difference
between superoxide levels in venously and arterially
perfused veins (782±190% versus 786±20%, relative to matched
nontreated venously perfused controls; n=4; P=0.98; Figure
4), suggesting that increased superoxide production in
arterially perfused veins is due to decreased scavenging by
SOD.
When we further investigated this possibility, we found that vein
tissue content of ecSOD was greatly reduced in arterially
perfused compared with venously perfused tissue (Figure 5). In
contrast, hemodynamic conditions did not affect Cu/Zn
SOD levels, suggesting that arterial
hemodynamic conditions specifically affect superoxide
scavenging in the extracellular compartment. Dihydroethidium staining,
which primarily reflects intracellular superoxide, supported this
hypothesis. We detected similar levels and patterns of staining in
venously and arterially perfused veins (not shown), further
supporting the concept that differences in total superoxide levels are
due to different levels of extracellular superoxide.
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Discussion |
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In the present investigation, we specifically questioned the effect of arterial hemodynamic conditions on human vein graft MMPs. Because all the veins used in the present study had been prepared for grafting, they were predisposed to intimal hyperplasia.15 Strict inclusion criteria on the specimens obtained from the bypass surgery limited the number of experiments that we were able to carry out, but at the same time, they minimized variances. Porter et al21 demonstrated previously that arterial shear stress inhibits the development of intimal hyperplasia in cultured vein pieces, but the additional impact of increased pressure and pulsatility has not been assessed. Furthermore, in their system, vein pieces were cut open and encased in silicon, disrupting the natural vein structure and consequently altering the forces experienced by vascular cells. Our perfusion system allowed us to create aggregate conditions similar to the venous or arterial hemodynamic environments and to study their effects on freshly harvested intact vein segments surgically prepared for grafting.
We found that MMP-9 and MMP-2 levels under venous perfusion conditions were similar to those in static conditions, previously used for study of vein intimal hyperplasia.36 Contrary to what we expected, our findings showed that MMP zymogen levels were decreased under arterial perfusion conditions. We also examined effects of different perfusion conditions on the levels of major endogenous inhibitors of gelatinases (tissue inhibitor of metalloproteinase-1 or -2, data not shown), but we found no significant differences. Thus, hemodynamic conditions seem to influence the vein-degradative capacity through direct effects on MMPs. MMP-9 and MMP-2 distribution appeared decreased in the intima and inner media of the arterially perfused vein grafts, areas affected by changes during the formation of intimal hyperplasia. We suggest that this mechanism could work to limit the development of intimal hyperplasia triggered by surgical preparative injury in grafts experiencing normal arterial hemodynamics. This scenario is supported by the clinical observation that vein grafts with normal arterial flow rates develop less intimal hyperplasia20 and have higher patency rates37 than either technically compromised grafts or grafts with poor distal runoff, both subjected to lower flow rates.
We found that although arterial conditions stimulate MMP de novo synthesis, tissue levels under the same conditions are decreased. Among the potential explanations that we considered was an increased mass transfer, or a "washout" of tissue MMPs at high arterial perfusion pressure and flow rates. However, we found no differences in MMP-9 or MMP-2 levels in the perfusates of venously or arterially perfused tissues (data not shown). We then considered potential pathways for increased posttranslational proteolytic processing of MMP zymogens, which, once initiated, results in subsequent MMP degradation. We found that arterial conditions increased the superoxide levels 3- to 4-fold, supporting the contribution of increased redox stress under arterial conditions to rapid processing and inactivation of gelatinase zymogens. Taken together, the results from the biochemical assays and dihydroethidium staining indicate that hemodynamic conditions specifically affected the levels of extracellular superoxide. Furthermore, in our experiments with whole vein segments, the effect seemed to be due to modulation of the superoxide-scavenging capacity of veins rather than to increased superoxide production, as previously demonstrated in isolated vascular cells with cyclic stretch33 and oscillatory shear.34 Differences may be due to the fact that previous studies investigated expression in isolated cells. Although the source of extracellular superoxide cannot be definitively demonstrated, positive dihydroethidium staining indicated that superoxide was present in cells throughout the vein wall. The cell type was not specifically investigated.
We found that arterially perfused veins had substantially decreased tissue levels of ecSOD, but not of cytosolic Cu/Zn SOD, which was consistent with previous observations in endothelial cells. To our knowledge, regulation of ecSOD expression directly by mechanical forces has not been described previously. Although the ecSOD activity was not directly measured, inasmuch as activity levels characteristically parallel protein levels,38 we suggest that its scavenging activity was decreased in veins under arterial hemodynamic conditions. Besides triggering the processing of MMP zymogens secreted by vascular cells in the extracellular space, decreased superoxide scavenging may increase the oxidative modification of other extracellular proteins, such as inspissated plasma lipoproteins, predisposing grafts to the formation of atherosclerotic lesions.
In the ex vivo conditions used in the present experiments, early cell proliferation occurred primarily in the outer layers of the vein wall. Differences found in the majority of specimens further support the contribution of hemodynamics, because in our experiments, comparisons were made in matched segments derived from the same vein, which minimized the contribution of surgical preparative injury, a major stimulus.14 Preferential localization of cell proliferation in the outer layers of vein specimens suggests that in conditions of adequate perfusion of grafts, the compensatory vein wall thickening, necessary to normalize wall stress, may take place through adventitial and medial wall hyperplasia instead of intimal hyperplasia. It is also possible, as suggested previously, that proliferating myofibroblasts migrate into the intima of vein grafts, contributing to intimal hyperplasia39 at later time points. In the present study, we did not perform morphometric analysis of vein grafts, because noticeable changes are likely to occur at times >3 days and because specimen fixation is incompatible with the biochemical functional analysis we performed. Thus, long-term effects of arterial hemodynamic conditions remain to be demonstrated.
Therefore, we identified likely components of early remodeling that are influenced by arterial hemodynamics: upregulation of MMP synthesis, increased MMP zymogen processing, and cellular proliferation. All these processes may be mediated via effects on the redox state of the vessel wall, as suggested by our present observations and by previous studies showing the many regulatory functions of reactive oxygen species. Detection of increased superoxide levels in vein grafts, which are likely due to diminished scavenging by ecSOD, may provide insight into future therapeutic strategies for vein graft disease. We previously proposed the use of antioxidants to limit the degrading activity of vascular MMPs.40 Antioxidant treatment has already been associated with reduced restenosis and remodeling in patients who undergo angioplasty.41 Similar treatment, or direct vein graft gene therapy, may result in reduced intimal hyperplasia and atherosclerosis, lowering the incidence of recurrent ischemia in those undergoing bypass grafting.
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Acknowledgments |
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These studies were supported through a research grant awarded by the Whitaker Foundation (to Dr Galis) and through funds from an Emory-Georgia Tech Biomedical Alliance grant. Dr Kreton Mavromatis was the recipient of a National Research Service Award Fellowship. We gratefully thank Drs David Harrison and W. Robert Taylor for many helpful discussions. We also thank the Emory Cardiac Surgery Operating Room staff for their assistance in obtaining remnant bypass grafting material.
Received July 2, 1999; accepted April 16, 2000.
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