© 1997 American Heart Association, Inc.
Articles |
Effect of Streptozotocin-Induced Hyperglycemia on Lipid Profiles, Formation of Advanced Glycation Endproducts in Lesions, and Extent of Atherosclerosis in LDL Receptor-Deficient Mice
From the Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, California.
Correspondence to Peter Reaven, MD, Division of Endocrinology and Metabolism, Department of Medicine, 0682, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0682.
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Abstract |
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Abstract Investigations into the mechanisms by which diabetes accelerates atherosclerosis have been hampered by the lack of suitable animal models. We hypothesized that streptozotocin-treated LDL receptor-deficient mice would be a good model of diabetic atherosclerosis because streptozotocin causes diabetes in the parent C57BL/6 strain and because in these mice diet-induced hypercholesterolemia leads to the formation of advanced atherosclerotic lesions throughout the aorta. Diabetes was induced in 18 mice by intraperitoneal injection of streptozotocin. Low-dose insulin was given subcutaneously to prevent excessive mortality and extreme elevations in triglyceride levels. The control group was subjected to sham injections. Both groups were fed a diet containing .075% cholesterol for six months. Average blood glucose was higher in the diabetic group than in the control group (257±67 mg/dL versus 111±7 mg/dL, P<0.05). Although plasma cholesterol was similar (966±399 versus 1002±180 mg/dL) in both groups, VLDL cholesterol was higher whereas LDL cholesterol was lower in the diabetic group. Immunocytochemical analysis demonstrated significantly more advanced glycation endproduct (AGE) epitopes in the artery wall of the diabetic group, whereas staining for oxidation-specific epitopes was similar in both groups. Sera of diabetic mice also contained significantly more IgG autoantibodies that bound to several AGE epitopes than did sera from control mice. Despite the presence of hyperglycemia, diabetic dyslipidemia, and enhanced AGE formation in the diabetic mice, both groups had a similar extent of atherosclerosis (diabetic, 17.3±5.2; control, 16.5±6.6% of the aortic surface). These data suggest that, at least under conditions of marked hypercholesterolemia, hyperglycemia and enhanced AGE formation do not contribute significantly to atherogenesis in LDL-/- mice.
Key Words: arteriosclerosis • advanced glycation endproducts • diabetes • autoantibodies • lipid peroxidation • LDL oxidation
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Introduction |
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Individuals with diabetes have an increased prevalence of atherosclerosis, with increased rates of coronary artery disease, cerebrovascular disease, and peripheral artery disease.1 2 More than 50% of deaths in noninsulin-dependent diabetes mellitus are related to atherosclerosis, with coronary artery disease being the most frequent cause. Although diabetes is frequently associated with coronary artery disease risk factors such as increased levels of total cholesterol, triglycerides, and blood pressure, much of the increased risk for coronary artery disease is not explained by these and other standard cardiovascular risk factors.3 4 This raises the possibility that other less recognized risk factors present in individuals with diabetes such as increased lipoprotein peroxidation and formation of advanced glycation endproducts (AGE) may enhance the atherogenic process. The mechanisms by which lipoprotein oxidation may accelerate atherosclerosis have been extensively reviewed.5 6 7 Moreover, there is increasing evidence that lipoproteins from individuals with diabetes are more susceptible to oxidation8 9 and that plasma from these individuals contains higher levels of lipid peroxides.10 11 12 13 AGE have been demonstrated to induce expression of adhesion molecules by vascular cells, to enhance cellular oxidative stress, and when injected into the blood stream, to accelerate atherosclerosis in rabbits.14 15 16 AGE-modified proteins also accelerate lipid oxidation, and conversely, lipid oxidation enhances AGE formation, at least in vitro.17 18 It has, therefore, been suggested that these two processes, lipid peroxidation and AGE formation, may be mutually reinforcing19 and that this interaction may further enhance development of atherosclerosis. However, it has been difficult to evaluate the impact of these processes on diabetic atherosclerosis because of a lack of appropriate animal models.
The recently developed LDL receptor-deficient (LDLR-/-) mouse appears to be a good model of macrovascular atherosclerosis. As in humans with this condition, there is a marked increase in plasma cholesterol levels, particularly when ingesting a high-fat, cholesterol-enriched diet. When mice are fed such a diet, not only does atherosclerosis develop in the vicinity of the aortic valve, as seen in other atherosclerosis-susceptible strains of mice, but also extensive lesion formation occurs in the aortic arch, the thoracic aorta, and in the abdominal aorta.20 21 22 These aortic plaques show many morphologic similarities to human atherosclerotic lesions. Histopathology reveals a mixture of fatty streaks as well as more advanced and complex lesions. Lesions contain macrophages, foam cells, T-cells, smooth muscle cells, and immunoglobulins, and in more advanced lesions, cholesterol crystals are present.22 Medial involvement and aneurysms have also been observed in murine aortas, and death from cardiovascular causes is not uncommon in cholesterol-fed LDLR-/- mice. Atherosclerotic lesions in this animal model also contain "oxidation-specific" epitopes.22 Finally, a strong humeral immune response to oxidative neoepitopes accompanies atherogenesis in LDLR-/- mice,22 similar to that observed in apo E-deficient mice.
The current paper describes the effects of streptozotocin (STZ)-induced diabetes on lipoprotein profiles, AGE formation, and atherosclerosis in LDLR-/- mice.
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Methods |
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Animals and Diets
Male LDLR-/- mice, 6 to 10 weeks of age from our own colonies, were used in this 6-month intervention study. These mice were C57BL/6x129Sv hybrids, bred from founders obtained from Jackson Laboratories. Mice were housed in a temperature-controlled facility, which maintained a 12-hour light/dark cycle, and were given free access to food and water. Mice were fed standard mouse chow (Harlan Teklad 8604, Madison, Wisc) for 4 to 6 weeks before being divided into two litter-matched groups with similar initial cholesterol levels. At the beginning of the intervention period, one group received intraperitoneal STZ (160 mg/kg) dissolved in saline (injected within 3 minutes of preparation), and the control group received intraperitoneal saline only. This dose of STZ induces extensive pancreatic destruction, leading to insulinopenic diabetes.24 Tail vein blood glucose samples were measured (One Touch II Glucometer, Lifescan, Inc., Milpitas, Calif) three times per week to insure induction of diabetes. Several mice required more than one STZ injection during the first several weeks to induce diabetes. To keep animal handling comparable between groups, the same number of control mice also received additional intraperitoneal saline injections. All mice were then provided an atherogenic diet (Harlan Teklad 94248), which contained .075% cholesterol and 21% anhydrous milkfat, but no cholic acid, for 6 months. Blood was collected in heparinized capillary tubes from the retro-orbital sinus of anesthetized mice every 4 weeks for determination of plasma cholesterol levels. Before each blood withdrawal, all mice were fasted for 4 hours. Plasma aliquots for all other analyses were stored at -80°C. Tail vein punctures were performed on restrained mice approximately every two weeks to measure blood glucose levels.
Pilot studies demonstrated that severely hyperglycemic mice became ill and had reduced long-term survival. Additionally, marked hyperglycemia was frequently associated with severe hypertriglyceridemia. To avoid these complications, hyperglycemic mice received low-dose, slow-release insulin by implantation of insulin pellets (Linshin Canada, Inc., Ontario, Canada). These pellets release approximately .2 U of bovine insulin over 24 hours for up to 4 weeks. Pellets were placed subcutaneously in the area of the upper back through a 20-gauge trocar. Placement of pellets was initiated shortly after the induction of diabetes in each mouse and repeated every 3 to 4 weeks during the course of the study. This low dose of insulin was insufficient to normalize glucose levels but prevented weight loss and greatly improved long-term survival. In a few mice, only moderate hyperglycemia developed after injection of STZ and those mice did not require supplementation of insulin.
Lipid Levels and Lipoprotein Profiles
Plasma cholesterol and triglyceride
levels were measured by enzymatic methods on plasma samples using an
automated bichromatic analyzer (Abbott
Diagnostics). Plasma lipoprotein cholesterol
analysis was also performed using fast performance
liquid chromatography gel filtration. One hundred
microliters of each mouse plasma sample was added to a Superose
6B-filled column (.7x50 cm), and 250-µL sample fractions were
collected for cholesterol analysis.20
Mouse lipoproteins isolated by ultracentrifugation were
run as standards to facilitate lipoprotein peak identification.
Determination of Autoantibody Titers and Specificity
The binding of autoantibodies to several AGE-modified
lipoproteins and proteins was determined by ELISA using a
chemiluminescence detection system developed in the immunology core
laboratory of the SCOR on Arteriosclerosis under
the direction of Dr. Joseph Witztum.25 AGE-modified
proteins were generated by prolonged incubations of glucose or
glucose-6-phosphate with BSA (Gly BSA and G-6-P BSA) or
4-furanyl-2-furoyl-[1H]-imidazole with LDL (FFI-LDL) as
described previously.26 In addition,
carboxymethyllysine-BSA; (a generous gift from Dr. John Baynes) was
also used as an antigen. In this assay, 50 µL of antigen (5
µg/mL) in Tris-buffered saline (TBS) containing 0.27 mM EDTA,
.02% NaN3, .001% aprotinin, and 20 µM butylated
hydroxytoluene was incubated overnight at 4°C in 96-well
polyvinylchloride microtitration plates (Dynatech, Chantilly, VA). The
wells were then washed 4 times with TBS containing 0.27 mM EDTA using a
microtiter plate washer. A 50-µL aliquot of a 1:200 dilution of each
murine serum in TBS containing 3% BSA (dilution buffer) was added and
incubated 1 hour at room temperature. Plates were thoroughly washed
with TBS, and the murine IgG and IgM autoantibodies binding to the
plated antigen were detected by incubating the wells with 50 µL of
alkaline-phosphatase-labeled goat anti-mouse IgG or IgM (Sigma Chemical
Co.). After washing with TBS, 25 µL of a 30% solution of Lumi-Phos
530 (Lumigen Inc., Southfield, Mich) was added to each well, and the
plates were incubated for 2 hours at room temperature in the dark.
Luminescence was determined using a Lucy 1 luminometer supported by
WINLCOM software (Anthos Labtec Instruments, Salzburg, Austria). Data
were expressed as number of flashes of light in 100 milliseconds. Each
determination was performed in triplicate.
Evaluation of Atherosclerosis
At the end of the intervention period, mice were killed by an
overdose of anesthesia. A canula was inserted into the left
ventricle. The heart and aortic tree were perfused with PBS containing
20 µM butylated hydroxytoluene and 2 µM EDTA, followed by perfusion
fixation with formal-sucrose (4% paraformaldehyde, 5%
sucrose). The aorta was then dissected for morphometry as
described.27 The extent of atherosclerosis
was determined in the Sudan IV-stained aortas.21 In brief,
three digital images (arch and proximal half of the thoracic aorta,
lower thoracic and upper abdominal aorta, and lower abdominal aorta)
were captured with a Sony DXC-960MD color video camera (Sony Corp of
America, San Jose, Calif). Image analysis was performed on
24-bit color images, using Optimas 4.1 (Bioscan, Seattle, Wash) image
analysis software, an Oculus TCX true color frame grabber with
4 Mbytes of frame buffer memory (Coreco, St-Laurent, Quebec, Canada),
and a separate VGA image monitor (for a detailed description of the
analysis method, see Reference 2121 ). Results were expressed as
percent of aortic surface area covered by atherosclerotic lesions.
Tissue Preparation and Immunocytochemistry
After the determination of the extent of
atherosclerosis, 7-mm-wide segments of the opened aorta
containing prominent lesions were paraffin-embedded, and 8-µm-thick
serial sections were prepared for immunocytochemical evaluation of AGE-
and oxidation-specific epitopes. Sections were rehydrated and
immunostained using an avidin-biotin-alkaline phosphatase
system (Vector Labs, Burlingame, Calif), as previously
described.22 27 The following antibodies were used: FLI-1
(an antiserum prepared by immunization of guinea pigs with FFI
conjugated to homologous LDL, which recognizes FFI lysine epitopes on
FFI-LDL and other FFI protein adducts,26 GPA-1 (a
guinea-pig antiserum generated with AGE-modified guinea-pig
albumin,26 and MAL-2 (a guinea-pig antiserum to
malondialdehyde-lysine epitopes.28 29 Control slides were
incubated without primary antibody and were devoid of any staining.
Immunocytochemistry using an amplification step yields only semiquantitative results. Therefore, to detect differences in the presence of AGE and oxidation epitopes in lesions by comparative immunocytochemistry, staining of all sections with the same antibody was performed in a single assay, using rigidly controlled conditions (i.e., reagent volumes, incubation times), and alternating slides from diabetic and control animals. Furthermore, only large lesions of similar stage and composition were used for comparison. These lesions were obtained from three diabetic mice with high blood glucose levels (ranging from 320 to 440 mg/dL) and three control mice, which had the same overall extent of aortic atherosclerosis but were euglycemic. Results were quantitated by the same investigator, using a scoring system for the different staining intensities. The scores were defined as 0=sections devoid of staining, 2=mild staining, 4=strong staining, 6=very strong staining (as well as 1, 3, and 5 for intermediate staining intensities). All sections were read twice, alternating between control and diabetic animals. A total of 64 sections from diabetic mice and 64 sections from control mice was evaluated for each of the three antibodies.
Statistics
Mean values between groups were compared by unpaired
t tests and correlations between variables by Pearson's
coefficient of correlation. A P<.05 was defined as
significant. Data analysis was performed with the statistical
package SYSTAT (Evanston, Ill).
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Results |
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During the 6-month intervention phase, 6 of the 24 diabetic mice and 3 of 17 control mice died. In the diabetic group, this was a result of peritoneal infection in one case and excessive anesthesia during insulin pellet placement in two cases. Uncontrolled hyperglycemia and subsequent demise occurred in three other mice despite insulin treatment. The three mice in the control group all died during procedures (peritoneal injections or blood withdrawals). All other animals were healthy, with normal coat condition and stable or increasing weights. Initial body weights for the control and diabetic groups were not significantly different (Table 1
). During the 6 months of the high-fat,
cholesterol-enriched diet, the weight of the control group
increased significantly whereas that of the STZ group remained stable.
However, the difference in final weights between the two groups was not
statistically significant (Table 1).
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Hyperglycemia developed in the STZ-treated group as shown in Table 1
and Fig 1. The average glucose level per
day over the course of the study was 111±7 mg/dL in the control
group and 257±67 mg/dL in the STZ group (P<.05).
Hyperglycemia was present throughout a typical day in the
STZ-treated group as shown in Table 2.
Values on this particular day were 2- to 2.4-fold higher in diabetic
mice than in controls and are consistent with the average daily
glucose levels over the entire course of the study.
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Total plasma cholesterol levels increased rapidly and to a
similar extent in both groups; the average total
cholesterol level per day over the course of the study was
1002±180 mg/dL in the control group and 966±399 mg/dL
in the STZ group (Table 1). Triglyceride levels were higher
in the STZ-treated group, although the difference was not statistically
significant. To determine whether there were differences in
distribution of plasma cholesterol among lipoproteins, we
measured the cholesterol content of VLDL, IDL/LDL, and HDL
lipoproteins in a subset of mice (Table 3). The percent of total plasma
cholesterol carried in VLDL from STZ-treated animals was
nearly 2-fold higher than in VLDL from control animals
(P<.05), whereas the percent carried in LDL was higher in
control animals (P<.05). The percent of total plasma
cholesterol carried in HDL was also higher in control
animals, although this did not reach statistical significance
(P<.15). The average cholesterol content within
each lipoprotein fraction is graphically represented in Fig 2.
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After 6 months of cholesterol feeding, the extent of
atherosclerosis was similar in both control and
STZ-treated groups (Fig 3). The
distribution of lesions in the aortic arch or thoracic or abdominal
aorta was also similar in the two groups.
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To determine the relationship of lipid and glucose variables to the extent of atherosclerosis, we determined correlation coefficients. For both groups combined, there was no significant correlation between extent of atherosclerosis and average plasma cholesterol (r=0.13) or triglyceride level (r=0.14). The correlation of the above parameters with atherosclerosis was also not significant when analyzed separately within the diabetic and the control groups. Given the narrow range of cholesterol values in both groups, this finding is not surprising. No significant correlation between average plasma glucose levels and atherosclerosis was obtained within the diabetic group or when data from both groups were combined. Average glucose levels did correlate moderately with levels of IgG autoantibody binding to model AGE epitopes (G-6-P BSA: r=.51, P=.005; carboxymethyllysine-BSA: r=.43, P=.022; GlyBSA: r=.30, P=.115; FFI-LDL: r=.15, P=.43).
To provide a direct measure of the extent of AGE formation in the
artery wall, we quantified the intensity of
immunostaining with antibodies to AGE- and
oxidation-specific epitopes in advanced atherosclerotic lesions from
diabetic and control mouse aortas. Great care was taken to ensure that
identical assay conditions were used for all tissue sections stained
with the same antibody (see "Methods"), such that quantitative
comparisons between groups could be made for each antibody. Staining of
arterial sections with the two anti-AGE antibodies was
markedly more intense in the diabetic group than the staining in
comparable control lesions (Table 4).
Examples of the comparative immunostaining with FLI-1,
the antibody generated with FFI-LDL, are shown in Fig 4. In contrast, the extent of intimal
staining with MAL-2, an antiserum that recognizes malondialdehyde
lysine (a model epitope of oxidized LDL), was similar in both groups
(Table 4 and Fig 5).
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We hypothesized that increased AGE formation would be reflected
by an increased humoral immune response to these highly immunogenic
neoepitopes, similar to the increased titers of antibodies to epitopes
of oxidized LDL found in subjects with increased
atherosclerosis.30 31 32 We therefore
determined the binding of circulating autoantibodies to model epitopes
of AGE-modified lipoproteins and proteins in both control and diabetic
mice. The level of binding of IgG (but not IgM) autoantibodies to
several AGE-proteins was elevated in plasma from diabetic animals,
although statistically significant differences were not obtained for
all of the model compounds tested (Table 5). Representative data
are shown for IgG autoantibodies to Gly-BSA (Fig 6).
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Discussion |
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Much of the difficulty in investigating the mechanism of enhanced atherosclerosis in diabetes has resulted from the lack of appropriate animal models. Although atherosclerosis has been studied in diabetic rabbits, this model is of limited value because it is particular difficult to generate and maintain diabetic rabbits. Additionally, the development of marked hypertriglyceridemia and large VLDL particles in this diabetic model has made it more, not less, resistant to the development of atherosclerosis.33 Thus, the diabetic rabbit does not constitute a good model of human diabetes, in which increased atherosclerosis is well documented. Although the rat is frequently used as a model of streptozotocin-induced diabetes, it is generally resistant to the development of atherosclerosis, even when given high-fat diets. After the identification of a murine strain susceptible to atherosclerosis and the generation of several gene knockout models that develop extensive aortic lesions, there has been interest in using mice as possible models of diabetic atherosclerosis. As demonstrated in this study, inducing diabetes in LDL receptor-deficient mice appears to offer many advantages. Extensive hyperglycemia was initially achieved, but the plasma glucose could be moderated by providing small amounts of slow-release insulin. This also prevented the extreme hypertriglyceridemia and excess mortality frequently seen in other models. Over a six-month period, average plasma glucose levels of 257 mg/dL in diabetic mice led to increased AGE formation in their aortic tissue, as has been demonstrated in tissues of human diabetic patients.34 35 The lipid profiles of diabetic mice were also similar to those of poorly controlled diabetic patients with increased VLDL levels and slightly reduced HDL levels.36 As is commonly seen in moderately hypertriglyceridemic diabetic patients, LDL cholesterol levels in diabetic mice were slightly lower than in nondiabetic controls. Most importantly, these mice developed advanced aortic lesions that could be easily and reliably quantitated. Overall, this model would thus appear useful for investigations of the impact of diabetes on atherosclerosis.
Given these considerations and, in particular, the fact that the aortic
AGE content was increased, it is surprising that we did not find more
extensive atherosclerosis in the diabetic mice than in
the controls. Several possible explanations for this may exist. In both
groups, plasma cholesterol levels of approximately 1000
mg/dL were achieved. It is conceivable that at these high
levels, plasma cholesterol becomes the predominant force in
driving the development of atherosclerosis and that it
overshadows the atherogenic effect of other risk factors, such as
hyperglycemia and increased AGE formation. Alternatively, the apparent
lack of increased atherosclerosis in the diabetic mice
may be explained by conflicting protective and proatherogenic effects.
It is well known that the distribution of cholesterol
between lipoprotein fractions may modulate lesion formation. In
particular, it has been demonstrated that diabetes associated
hypertriglyceridemia reduces
atherosclerosis in rabbits.33 This would
be consistent with the hypothesis that larger
triglyceride-rich VLDL are less atherogenic than IDL/LDL
particles. As demonstrated in Fig 2, our diabetic mice showed nearly a
2-fold increase in VLDL cholesterol and a reduction in LDL
cholesterol. Thus, in the diabetic mice, a lipid profile of
reduced atherogenic potential could have counterbalanced the
proatherogenic effects of diabetes. Finally, it is possible that
hyperglycemia and increased AGE formation do not significantly enhance
atherogenesis in LDLR-/- mice.
Kunjathoor et al37 recently studied the effect of diabetes on atherosclerosis in wild-type C57BL/6 mice (i.e., the parent strain of the LDLR-/- mice) fed an atherogenic diet. In their study, plasma cholesterol levels in both the diabetic and control animals were only modestly elevated (average plasma cholesterol, 290 mg/dL). Although the extent of atherosclerosis was much smaller and lesions were limited to the aortic origin in their model, atherosclerosis was similar between diabetic and control groups. These findings are consistent with our results and suggest that even at low cholesterol levels diabetes does not necessarily accelerate development of atherosclerosis. However, the same authors also studied BALB/c mice and found that in this strain diabetes did increase lesion formation, although again the overall extent of lesion formation was quite small. This raises the possibility that there may be differences in the susceptibility to proatherogenic effects of diabetes between murine strains.
Additional studies are needed to test whether under conditions of moderate hypercholesterolemia, diabetes enhances the formation of atherosclerosis. The LDLR-/- model is very well suited for such studies, because various levels of hypercholesterolemia can be induced by varying the cholesterol content of the diet and because advanced atheroma occur in this model, in contrast to C57BL/6 mice or other murine strains.
It is generally accepted that increased glucose levels lead to increased nonenzymatic glycation and AGE formation. Our results provide direct experimental evidence for increased presence of AGE epitopes in advanced atherosclerotic lesions of diabetic mice. In contrast, there was no evidence that oxidation-specific epitopes were correspondingly increased, at least within the sensitivity of immunocytochemistry. It has been proposed by many laboratories, including ours, that AGEs would accelerate lipoprotein oxidation via a variety of mechanisms, including enhancing lipoprotein trapping in the artery wall, decomposing into free radicals, and stimulating cellular oxidative stress.15 16 17 18 38 This hypothesis is supported by a number of reports of accelerated lipoprotein oxidation and enhanced in vivo lipid peroxidation in diabetics.8 9 10 11 12 13 26 However, in the current study, oxidation-specific epitopes were similar in distribution and quantity in lesions of both diabetic and control mice, despite markedly greater AGE staining in aorta of diabetic mice. These findings speak against a proatherogenic effect of artery wall AGE through an enhancement of lipoprotein oxidation.
In addition to the direct demonstration of increased AGE immunostaining, we observed increased levels of circulating autoantibodies to a number of AGE epitopes in diabetic mice. This provides indirect evidence for increased formation of AGE in vivo. The AGE found in the aortic wall are likely to be part of the antigen load that induced these autoantibodies. However, it can be assumed that increased AGE formation occurs in other organs as well. The correlation of the autoantibody levels with the degree of hyperglycemia supports the assumption that hyperglycemia enhances AGE formation and that this is reflected by increased autoantibodies. The above findings also constitute the first demonstration of increased plasma autoantibodies to AGE epitopes in diabetes. An analogous observation, i.e., the fact that the titers of plasma autoantibodies to epitopes of oxidized LDL are increased in subjects with increased atherosclerosis or conditions promoting atherogenesis, is well documented in the literature.22 30 31 32 39 40 However, when considering the significance of autoantibody titers, one has to bear in mind that the relationship between these titers and the severity of disease activity is not a direct one. Autoantibody titers may be influenced by many factors, including antigen load, antibody production, and antibody clearance. Nevertheless, our finding of increased levels of autoantibody binding to AGE in diabetic mice supports the hypothesis that plasma autoantibody levels may reflect the degree of the hyperglycemia, AGE formation, and/or the duration of diabetes.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank Dr. Joseph Witztum for valuable advice and comments and Elizabeth Miller and Rich Elam for excellent technical assistance in the conduction of these studies. This work was supported by Grant HL-14197 (SCOR) from the National Heart, Lung, and Blood Institute, National Institutes of Health.
Received December 18, 1996; accepted February 10, 1997.
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