© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Effects of Hypercholesterolemia on Myocardial Ischemia-Reperfusion Injury in LDL Receptor–Deficient Mice
From the Department of Molecular and Cellular Physiology (S.P.J., N.S., T.Y.A., D.J.L.) and the Department of Surgery (W.G.G.), LSU Medical Center, Shreveport, La.
Correspondence and reprint requests to David J. Lefer, PhD, Department of Molecular and Cellular Physiology, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130. E-mail dlefer{at}lsumc.edu
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Abstract |
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Abstract—Hypercholesterolemia is a primary risk factor for atherosclerosis, coronary artery disease, and myocardial infarction. We subjected low density lipoprotein receptor–deficient (LDLr –/–) and control (wild-type) mice to 30 minutes of myocardial ischemia and 120 minutes of reperfusion. Myocardial infarction per area at risk (AAR) was noted under baseline conditions to be significantly (P<0.05) smaller in the LDLr –/– mice compared with wild-type mice (24.7±3.2% and 38.8±4.3% of AAR, respectively). Subsequently, mice were fed a high-cholesterol diet (HCD) for 2 or 12 weeks, which resulted in significant increases in serum cholesterol levels in both LDLr –/– and wild-type groups. After 2 weeks of the HCD, the LDLr –/– mice demonstrated a significant elevation (P<0.01) in myocardial necrosis per AAR (50.2±5.36% of AAR) compared with the normal-diet LDLr –/– group, whereas the short-term HCD-fed wild-type mice demonstrated no significant difference from baseline. In contrast, wild-type mice fed the HCD for 12 weeks revealed a significant (P<0.05) decrease in necrosis per AAR, which was 22.5±3.2% of the AAR in comparison with that in the normal-diet wild-type mice (38.8±4.3% of AAR). LDLr –/– mice on the same long-term HCD showed a similar significantly (P<0.05) decreased infarct size, which was 13.2±4.0% of the AAR. In additional experiments, we determined that myocardial tissue total glutathione (GSH) levels were reduced after 2 weeks of the HCD and were significantly increased after 12 weeks of the HCD in the LDLr –/– mouse heart. These data suggest that short-term cholesterol feeding renders the myocardium of LDLr –/– mice more susceptible to ischemia-reperfusion injury, whereas more long-term hypercholesterolemia confers cardioprotection in the LDLr –/– mouse heart.
Key Words: infarct • cholesterol • neutrophils • mutant mice
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Introduction |
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There is a large body of evidence indicating that the best-known risk factor for the development of coronary artery disease is elevated plasma lipid levels.1 2 3 Furthermore, a number of prospective studies have established that the risk of cardiac morbidity and mortality is directly related to the concentration of plasma cholesterol.2 3 Despite the development of a number of agents that effectively reduce serum cholesterol levels in patients, coronary arteriosclerosis and subsequent myocardial infarction (MI) still represent a major health concern in our society. The majority of previous studies concerning the pathobiology of myocardial ischemia-reperfusion (MI-R) injury have been derived from animals that are otherwise normal. Although the data derived from these studies provide meaningful insights into the mechanisms of cellular injury after MI-R, the relevance of these findings to those human populations that are at greatest risk for developing myocardial reperfusion injury remains unclear.
Previous studies investigating the effects of MI-R in the setting of hypercholesterolemia have focused primarily on rabbit models of ischemia alone4 5 or on I-R.6 7 8 Osborne et al4 reported that creatine kinase release after 5 hours of regional ischemia was significantly greater in rabbits fed a high-cholesterol diet (HCD) for 10 to 12 weeks. Furthermore, a subsequent study5 of MI in hypercholesterolemic rabbits reported an increased severity of myocardial tissue injury that was significantly reversed by treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor lovastatin. More recently, hypercholesterolemic rabbits have been subjected to MI-R injury.6 7 8 Golino and colleagues6 demonstrated that MI size was dramatically increased in rabbits subjected to I-R after only 3 days of cholesterol feeding. It was also reported7 that platelet depletion markedly reduced MI size and the extent of no-reflow in rabbits fed a 2.0% cholesterol diet for 3 days. Enhanced MI-R injury in the setting of hypercholesterolemia was also demonstrated in isolated rabbit hearts after global I-R.8
In recent years, a number of mutant mice have been developed in which genes that regulate lipoprotein metabolism and circulating cholesterol levels have been manipulated. The LDL receptor knockout (LDLr –/–) mouse is one of the earliest gene-targeted strains developed for atherosclerosis research. The LDLr –/– mouse closely resembles the condition of familial hypercholesterolemia in humans and has been widely utilized for experimental studies of hypercholesterolemia and how this genetic alteration leads to atherosclerosis.9 10 11 The LDLr –/– mouse develops profound hypercholesterolemia and arterial lesions when placed on a high-fat diet.9 10 Thus, the LDLr –/– mouse appears to be an ideal animal model for the investigation of the effects of hypercholesterolemia on MI. In the present study, we investigated the effects of acute and prolonged hypercholesterolemia on MI-R injury in both wild-type and LDLr –/– mice.
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Methods |
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Transgenic Mice
This study focused on the LDL receptor–deficient (ie, LDLr –/–) mouse as an animal model of hypercholesterolemia. Male LDLr –/– mice, as previously described,9 and male wild-type (C57BL/6) mice (age and weight matched) were purchased from Jackson Laboratories (Bar Harbor, Me). All experimental procedures complied with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services publication No. (NIH) 86-23, revised 1985, Animal Resources Program, DRR/NIH, Bethesda, Md), approved by the Council of the American Physiology Society, and with federal and state regulations. All experimental procedures were approved by the Louisiana State University Medical Center Animal Care and Use Committee.
LDLr –/– (n=9) and wild-type (n=10) mice were maintained on standard laboratory rodent chow No. 8640 (normal diet; ND) purchased from Harlan Teklad. A second group of LDLr –/– (n=6) and wild-type (n=6) mice were placed on an HCD (Teklad 90221, Harlan Teklad) containing 1.25% cholesterol and 15.8% fat for 2 weeks, and a third group of LDLr –/– (n=6) and wild-type (n=6) mice were fed the same HCD but for a longer time (12 weeks).
Surgical Procedures
Animals were initially anesthetized with sodium
pentobarbital (100 mg/kg IP) before any surgical procedure.
Anesthesia was maintained via supplemental doses of sodium
pentobarbital (30 mg/kg IP) as needed. Mice were secured to the
operating table by taping the extremities. A 4-0 silk ligature was
placed behind the upper incisors and pulled tautly to extend the neck.
A midline incision was made from the xiphoid process to the submentum.
The salivary glands were separated from the midline to allow access to
the trachea. A tracheotomy was then performed and a section of
polyethylene-90 tubing was inserted into the animal’s trachea and
connected via a loose junction to a Harvard respirator (model 683
rodent respirator, Harvard Apparatus). The respirator’s
tidal volume was set at 1.4 mL/min and the rate at 120 strokes/min, and
it was supplemented with 100% O2. The right
carotid artery was then cannulated with polyethylene-10 tubing to
monitor mean arterial pressure. The arterial
cannula was connected to a blood pressure transducer and a BP-1 (World
Precision Instruments) blood pressure monitor.
After an equilibration period of 10 minutes, a thoracotomy was performed. With the use of electrocautery (model 100, Geiger Instrument Co, Inc), an incision was made to the left of the sternum. The pericardial sac was then removed. Ligation of the left anterior descending (LAD) coronary artery was performed using a 7-0 silk suture attached to a BV-1 needle (Ethicon, Inc). A small piece of polyethylene tubing was used to secure the ligature without damaging the artery. The chest wall was approximated and covered with Parafilm wax paper to prevent desiccation.
MI-R Protocols
The protocol for the MI-R experiments is depicted in Figure 1
. One group each of wild-type mice on
the ND (n=10) and of LDLr –/– mice also on the ND (n=9) was subjected
to 30 minutes of LAD coronary artery occlusion and 120 minutes
of reperfusion. In subsequent studies, a second group of wild-type
(n=6) and LDLr –/– (n=6) mice was subjected to 2 weeks of the HCD
before undergoing 30 minutes of LAD occlusion and 120 minutes of
reperfusion. A third group of wild-type (n=6) and LDLr –/– (n=6) mice
was subjected to 12 weeks of the HCD before being placed through the
same MI-R model as the previous groups.
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Determination of Area at Risk and Infarct Size
At the conclusion of the 2-hour period of reperfusion, the LAD
was religated with 7-0 silk sutures. Evans blue dye (1.3 mL of a 1.0%
solution, Sigma Chemical Co) was retrogradely injected into the carotid
artery catheter to delineate the in vivo area at risk (AAR). The heart
was excised and fixed in a 1.5% solution of agarose gel (SeaPlaque,
FMC BioProducts). After the gel had solidified, the heart was
sectioned perpendicular to the long axis in 1-mm slices by using a
McIlwain tissue chopper (Brinkman Instruments Inc). The 1-mm slices
were placed in individual wells of a 6-well cell-culture plate (Cell
Wells, Corning Glass Works) with the basal side exposed. Each slice was
then counterstained with 3.0 mL of 1.0%
2,3,5-triphenyltetrazolium chloride (Sigma)
solution for 5 minutes at 37°C. The right ventricle was excised and
each slice was weighed and visualized under an Olympus SZ4045 (Olympus
America Inc) dissecting microscope equipped with a Sony CCD Iris color
video camera (Sony Electronics Inc). The left ventricular
area, AAR, and area of infarction for each slice were then determined
by computer planimetry by using National Institutes of Health Image
(version 1.57) software. The size of the MI was determined by the
following previously described equation: weight of
infarction=(A1xWT1)+
(A2xWT2)+(A3xWT3)+(A4xWT4)+(A5xWT5),
where AX is the percent of infarcted area as
determined by planimetry from 5 sections numbered 1 to
5,12 and WTX is the weight of the
corresponding numbered section.
Hematology of Peripheral Blood
Total white blood cell, neutrophil, and platelet counts were
performed by LabCorp, Inc (BioVet Division). Whole-blood samples from
LDLr –/– ND (n=6), 2-week HCD (n=6), and 12-week HCD (n=7) and from
wild-type ND (n=12), 2-week HCD (n=7), and 12-week HCD (n=5) mice were
obtained from the carotid artery and collected into pediatric (
1 mL)
lavender-topped, evacuated, EDTA-containing tubes (Microtainers, Becton
Dickinson and Co).
Cholesterol Measurement
Blood was obtained from wild-type mice on the ND (n=12), the
2-week HCD (n=7), and the 12-week HCD (n=5) as well as from LDLr –/–
mice fed an ND (n=6), a 2-week HCD (n=6), and a 12-week HCD (n=7)
through a polyethylene-10 tubing cannula placed in the right common
carotid artery. Once collected, blood was centrifuged in an
Eppendorf microfuge at 14 000 rpm for 10 minutes to isolate the
plasma. Total, LDL, and HDL cholesterol fractions were
assayed using enzymatic determination kits from Sigma
Diagnostics. The total cholesterol assay
was performed on 10-µL aliquots of plasma. LDL
cholesterol and HDL cholesterol were separated
by using latex beads coated with affinity-purified goat polyclonal
antisera to specific human apolipoprotein and phosphotungstic
acid/MgCl2 precipitation of apoB-containing
lipoproteins, respectively. Next, LDL and HDL cholesterol
levels were measured on 50-µL aliquots of separated fractions.
Neutrophil Accumulation
Midventricular tissue slices (1 mm in
thickness) were prepared from all of the hearts subjected to the MI-R
protocol after the completion of all experimental procedures. The
tissue sections were immediately fixed and stored in a 10% neutral
buffered formalin solution (Sigma Diagnostics). The tissue
slices were paraffin embedded, cut into 10-µm sections, and placed on
slides. The tissue specimens were then stained with Gill’s No. 3
hematoxylin and eosin. The slides were then viewed microscopically, and
the number of neutrophils (polymorphonuclear cells) per high-power
field was determined. For each of the hearts examined, the number of
neutrophils was counted in 6 fields of 3 separate tissue sections.
Myocardial Tissue Glutathione Measurement
In additional experiments, wild-type (C57BL/6) and LDLr –/–
mice were fed an ND (n=5 each for wild type and LDLr –/–) or the HCD
for 2 weeks (n=9 for wild-type and n=6 for LDLr –/– mice) or 12 weeks
(n=8 for wild-type and n=7 for LDLr –/– mice). Mice were
anesthetized with sodium pentobarbital (100 mg/kg IP), and the
hearts were excised and rapidly snap-frozen in liquid nitrogen. The
heart tissue was derivatized with iodoacetic acid and Sanger’s
reagent, and total glutathione (GSH) levels were quantified by
high-performance liquid chromatography as
described previously.13
Statistical Analyses
The infarct size, AAR, left ventricle size, leukocyte counts,
cholesterol levels, and hemodynamic data
were analyzed with an ANOVA coupled with post hoc
analysis with Scheffe’s test for significance. All statistics
were calculated with StatView 4.5 (Abacus Concepts). All values are
reported as mean±SEM. Statistical significance was set at
P<0.05.
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Results |
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Plasma Lipid Levels
Plasma total cholesterol and triglyceride levels for wild-type and LDLr –/– mice are presented in Table 1
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Circulating Blood Cells
Data for circulating white blood cells and neutrophils are
presented in Table 2.
High-cholesterol feeding for 12 weeks’ duration resulted
in a significant (P<0.05) increase in the number of
circulating white blood cells, neutrophils, and platelets in
wild-type mice compared with baseline conditions. Similarly,
hypercholesterolemia significantly
(P<0.05) increased the number of white blood cells and
neutrophils at 12 weeks after initiation of the HCD when compared with
baseline. In contrast, circulating platelet levels were
significantly (P<0.05) reduced in the LDLr –/– mice after
12 weeks of high-cholesterol feeding.
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Hemodynamic Data
Mean arterial blood pressure and heart rate were
recorded throughout the infarct size determination protocol, and
the rate-pressure product was calculated. Summary
hemodynamic data for wild-type mice are
presented in Table 3, and LDLr
–/– hemodynamic data are also presented in
Table 3. There were no significant differences in heart
rate, blood pressure, or rate-pressure product at any time during
the experimental protocol in wild-type mice. In LDLr –/– mice, heart
rate was significantly (P<0.05) increased in the 2-week HCD
group at baseline compared with the ND group. Heart rate was also
significantly higher at 120 minutes of reperfusion in the ND and the
2-week HCD groups compared with the 12-week HCD mice. In addition, the
rate-pressure product was significantly less in the 12-week HCD
group compared with the ND group at 30 minutes of ischemia.
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Myocardial Neutrophil Accumulation
Neutrophil infiltration into the I-R myocardium was
determined in wild-type (Figure 2A) and
LDLr –/– (Figure 2B) animals. Neutrophil accumulation induced
by MI-R was unchanged in wild-type mice after 2 weeks of
high-cholesterol feeding and was significantly
(P<0.05) reduced after 12 weeks of an HCD. After 2 weeks of
an HCD, the extent of neutrophil infiltration into the I-R
myocardium was significantly (P<0.01) increased
in LDLr –/– mice. In contrast, postischemic neutrophil
infiltration into the ischemic zone returned to levels
significantly (P<0.05) lower than baseline in the LDLr
–/– animals after 12 weeks of high-cholesterol
feeding.
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Myocardial AAR and Infarct Size
Figure 3A represents AAR
data for the left ventricle and infarct size in wild-type mice fed an
ND, a 2-week HCD, and a 12-week HCD. We observed no significant
differences in the size of the myocardial AAR placed at risk by
coronary artery occlusion. There was no significant difference
in infarct size between ND wild-type hearts (38.8±4.3%) and
2-week-HCD wild-type hearts (42.6±3.9%). However, 12-week-HCD
wild-type hearts (22.5±3.2%) presented significantly
(P<0.05) smaller areas of necrosis per AAR compared with ND
wild-type hearts (38.8±4.3%).
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AAR and infarct data for LDLr –/– mice are presented in
Figure 3B
. Although all 3 groups of LDLr –/– hearts
experienced similarly sized areas of ischemia (AAR per left
ventricle), 2 weeks of high-cholesterol feeding resulted in
significantly (P<0.01) larger infarcts (50.2±5.4% of the
AAR) compared with the ND LDLr –/– hearts (24.7±3.2% of the AAR).
Continued HCD feeding of LDLr –/– mice for 12 weeks attenuated the
infarct size to 13.2±4.0% of the AAR (P<0.05 versus ND
LDLr –/–).
Myocardial Tissue GSH Levels
In additional studies, myocardial GSH levels were measured as an
index of the oxidant status of the hearts. These data are depicted in
Figure 4. In wild-type animals (Figure 4A) subjected to either 2 or 12 weeks of an HCD, we did not
observe any significant changes in GSH levels. In contrast, we observed
a significant (P<0.05) reduction in total GSH levels 2
weeks after initiation of an HCD in the LDLr –/– hearts (Figure 4B). Furthermore, with prolonged
hypercholesterolemia (to 12 weeks in duration),
we observed a significant (P<0.05) increase in total GSH
levels in the LDLr –/– mouse heart.
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Discussion |
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Elevated serum cholesterol has long been associated with an increased risk for coronary artery disease and the development of MI. In the present study, we examined the extent of myocardial tissue injury in hypercholesterolemic murine hearts subjected to acute MI-R. We investigated MI-R injury in both wild-type and LDL -/- mice that were fed an HCD for both 2 and 12 weeks of duration. These studies revealed a number of novel and interesting findings related to the response of the hypercholesterolemic heart to I-R.
One very interesting finding of the present study is the response
of the LDL -/- mouse heart to myocardial reperfusion injury under
conditions of an ND. Myocardial infarct size was 
50% less in the
LDLr –/– mice compared with wild-type mice when fed an ND and then
subjected to I-R. This result is somewhat unexpected, in light of the
fact that serum cholesterol levels are markedly elevated in
LDLr –/– mice compared with the wild-type mice when both animals are
fed an ND.
An additional finding of interest in the present study is the response of both wild-type and LDLr –/– mice to MI-R at 2 and 12 weeks after initiation of cholesterol feeding. In wild-type mice, we observed no change in infarct size after 2 weeks of an HCD and a significant reduction in myocardial necrosis after 12 weeks. This result is in sharp contrast to the majority of previous studies regarding hypercholesterolemia. In the LDLr –/– mouse, the extent of MI was significantly increased after 2 weeks and then was significantly reduced to below baseline levels at 12 weeks after high-fat-diet administration. Thus, it appears that short-term (ie, 2-week) exposure to high cholesterol levels does not alter the development of MI in wild-type mice but markedly enhances MI in mice deficient in the LDL receptor. Furthermore, prolonged (ie, 12-week) exposure to elevated circulating cholesterol levels appears to protect the myocardium from I-R injury in both wild-type and LDLr –/– mice.
The effects of hypercholesterolemia on atherogenesis and vascular function have been extensively studied by a number of investigators. One of the earliest and most easily detected events associated with elevated serum cholesterol levels is a diminution in NO release by the vascular endothelium.14 Impaired endothelium-dependent vascular reactivity has been reported in a number of blood vessel types and a variety of animal models of hypercholesterolemia.14 15 16 17 18 In addition, endothelium-dependent vascular reactivity is impaired in the forearm19 and coronary microcirculation20 of humans. The endothelial cell dysfunction resultant from an HCD has been shown to promote interactions between circulating leukocytes and the endothelium.21 22 This response to hypercholesterolemia is thought to be a pivotal step in the development of atherosclerotic lesions and has been shown to be effectively reversed by treatment with NO therapy21 or with the NO precursor L-arginine.20 Enhanced vascular immune responses to hypercholesterolemia are considered a hallmark of this condition, and it is now believed that hypercholesterolemia induces an inflammatory condition.23 24
Along these lines, it has been reported that
hypercholesterolemic rats exhibit enhanced leukocyte
adhesion and microvascular protein leakage in the setting of mesenteric
I-R.25 In addition, a recent study11 of LDLr
–/– mice reported enhanced leukocyte adhesion and emigration in
response to inflammatory stimuli. In that study, mice were
subjected to 4 or 8 weeks of an HCD and then exposed to various
stimuli, including leukotriene B4,
platelet-activating factor, or tumor necrosis factor-
. The
authors reported that the magnitude of
leukocyte–endothelial cell interactions increased with
the duration of high-cholesterol feeding. Taken together,
these data suggest that the extent of I-R injury would be enhanced in
hypercholesterolemic animals.
Previous experimental investigations have been reported in which hypercholesterolemic animals have been subjected to various forms of I-R injury.4 5 6 7 8 11 Previous reports have uniformly indicated that hypercholesterolemia results in larger infarct development in rabbit hearts exposed to ischemia4 5 or ischemia in combination with reperfusion.6 7 8 These results are somewhat contradictory to our observations, in that we observed significantly less reperfusion injury in the LDLr –/– mice compared with wild-type mice under baseline conditions. In addition, our studies of LDLr –/– mice indicate that more prolonged exposure to hypercholesterolemia actually protects the heart from reperfusion injury. It is conceivable that species differences between the mouse and rabbit might help to reconcile some of the differences that were observed.
More specifically, we observed that
hypercholesterolemia in LDLr –/– mice induces
a biphasic response to MI-R injury, in which the extent of infarction
is elevated at 2 weeks and then significantly reduced at 12 weeks after
initiation of the high-fat diet. This response is unique and does not
parallel what has been reported previously in other studies. This may
be due in part to the extremely high levels (ie, >2000 mg/dL) of
cholesterol that are obtained in these mice after induction
of a high-fat diet. In contrast, in previous rabbit
studies,4 5 6 7 8 circulating cholesterol levels in
hypercholesterolemic animals were increased to 300
mg/dL, which is significantly lower than the cholesterol
levels attained in the present study. Exposure to extremely high
levels of circulating lipids may render the heart more tolerant to
ischemia and reperfusion after more prolonged periods of
time.
Additional experiments were performed in which the levels of myocardial tissue GSH were measured in normocholesterolemic and hypercholesterolemic animals. These data may provide insight into the oxidative stress of the myocardium in response to the various diet regimens. Our data indicate that exposure to acute hypercholesterolemia (ie, 2 weeks) significantly reduces total GSH levels in the LDLr –/– mice. This suggests that the defensive antioxidant enzyme levels of the heart have been reduced and that the cardiac myocytes may be more susceptible to myocardial reperfusion injury, which we observed as an increase in infarct size. In sharp contrast, more prolonged hypercholesterolemia (ie, 12 weeks) resulted in a significant increase in myocardial GSH levels in the LDLr –/– mouse hearts, and this may be partially responsible for the reduction in myocardial infarct size that was observed. Thus, it may be possible that whereas acute hypercholesterolemia results in enhanced injury in the setting of MI-R, prolonged exposure to high circulating levels of cholesterol may serve to "precondition" the murine myocardium and reduce tissue injury resulting from subsequent I-R. Clearly, additional studies are required to fully elucidate the mechanisms responsible for this bimodal response in the LDLr –/– mouse. One might also speculate that understanding how hypercholesterolemia can actually protect the murine myocardium may lead to the development of novel therapeutic strategies for the treatment of MI in humans.
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Acknowledgments |
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We gratefully acknowledge the technical support of Micah B. Strange. We thank DeRoyal Surgical (Powell, TN) and Ethicon Surgical (Somerville, NJ) for the generous donation of surgical supplies. This research was supported by National Institutes of Diabetes and Digestive and Kidney Diseases grant PO1 DK44510 (T.A.) and DK43785 (T.A.). This research was also supported by National Institutes of Health Heart, Lung, and Blood Institute Grant HL60849 (D.J.L.).
Received July 28, 1998; accepted April 12, 1999.
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