American Journal of Pathology, Vol. 140, No. 2, February 1992 Copright © American Association of Pathologists
Porcine von Willebrand Disease and Atherosclerosis Influence of Polymorphism in Apolipoprotein B100 Genotype
Timothy C. Nichols,*tt Dwight A. Bellinger,tts Keir E. Davis,11 Gary G. Koch,til Robert L. Reddick,tt Marjorie S. Read,tt Jan Rapacz,# Judith Hasler-Rapacz,# Kenneth M. Brinkhous,tt and Thomas R. Griggstt From the Departments of Medicine' and Pathologyt and the Center for Thrombosis and Hemostasis* School of Medicine, the Department of Biostatistics,ll School of Public Health, and the Division of Laboratory Animal Medicine,$ University of
North Carolina, Chapel Hill, North Carolina; and the Departments of Genetics and Meat and Animal Sciences, # University of Wisconsin, Madison, Wisconsin
The relationship of apolipoprotein-B genotype (Lpb) to diet-induced hypercholesterolemia and atherosclerosis was studied in von Willebrand disease (vWD) and normal pigs. Von Willebrand and normal pigs developed comparable levels ofhypercholesterolemia (respectively, 757.9 ± 49.4 versus 772.8 ± 47.9 mg/ dl, P = 0.95). Pigs with Lpbl/5 and Lpb5I8 genotypes, however, developed significantly higher serum cholesterol levels than those with other Lpb genotypes (866.1 ± 64.0 mg/dl, P = 0.0343). Coronary and aortic atherosclerosis, measured by computerassisted automated image analyzer, were not significantly different between vWD and normalpigs. Pigs with an Lpb5 allele developed significantly more atherosclerosis than those with the Lpb3/8 or Lpb8/8 genotypes or the rare Lpbl allele (r 0.434, P S 0.05). Polymorphism in apolipoprotein B100 genotype, then, significantly influenced the severity of diet-induced hypercholesterolemia and atherosclerotic plaque formation in vWD and normal swine without regard to the vWD genotype. (Am J Pathol 1992, 140:403-415) -
A role of von Willebrand factor (vWF) in atherogenesis has been suggested by studies in normal and von Willebrand disease (vWD) pigs.-1 1 In some of these studies, atherosclerosis was induced by high-cholesterol and high-fat diets.2-611 These diets have yielded pigs with a variable degree of hypercholesterolemia, and, in some studies, there was a trend toward higher levels of serum cholesterol in one or the other of the two groups of pigs. Also, within these two groups of atherosclerotic pigs, variation in the extent of coronary and aortic atherosclerosis has been noted.1-11 This variability in diet-induced hypercholesterolemia and atherosclerosis between normal and vWD pigs suggests that vWF may influence cholesterol metabolism in such a manner as to influence atherogenesis. We have conducted a series of studies on the effect of vWF on coronary atherogenesis and thrombosis.4i In the first study, the amount of coronary atherosclerosis was related to the degree of hypercholesterolemia that an individual pig developed and not to the presence of vWF. In addition, balloon injury of one of the three coronary arteries at the start of the experiment did not augment the amount of coronary atherosclerosis an individual pig developed.4 In the subsequent two studies, atherosclerosis was produced by diet alone. In the second study, balloon injury of coronary atherosclerotic plaques in vWD and normal swine at death induced a monolayer of attached platelets similar to what was seen in exposed, nonatherosclerotic subendothelium.5 The presence of atherosclerosis, however, did not promote platelet-fibrin thrombus formation in either phenotype. The presence of atherosclerosis, however, did support platelet activation Supported in part by National Institutes of Health grants HL26309 and HL01 648. Accepted for publication August 26, 1991. Address reprint requests to Dr. Timothy C. Nichols, Department of Medicine, University of North Carolina School of Medicine, CB #7075, 349 Burnett-Womack Building, Chapel Hill, NC 27514.
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on balloon-injured coronary plaques independent of vWF as judged by platelet ultrastructure in the vWD pigs. In the third study, it was shown that vWF was required to support the development of occlusive thrombosis even in the presence of coronary atherosclerosis and hypercholesterolemia.6 In each of these studies, variations in the degree of hypercholesterolemia and coronary atherosclerosis were noted within the two phenotypic groups of
pigs. Employing immunogenetic and molecular genetic techniques, Rapacz et al12 have documented considerable variability in porcine apolipoprotein B100 structure.12-14 Apolipoprotein Bi 00 is the major protein component of plasma low-density lipoprotein (LDL). Lowdensity lipoprotein is the predominant cholesterol transport protein in several species, including pigs.12-14 Eight Lpb alleles have been identified at the apolipoprotein B locus in swine.12'13 The codominant expression of 16 Lpb allotypes (Lpb 1 to 8 and Lpb 1 1 to 18) allows for the determination of any of the 36 Lpb genotypes directly from the immunological phenotype.12 13 Feeding high-fat diets has shown that some Lpb genotypes enhance atherogenesis.14 In the present study, the fourth in a continuation of this series, we described the Lpb polymorphism in the normal and vWD swine from the three earlier studies.4' The purpose of this study was to determine if the variability in susceptibility of these vWD and normal swine to dietinduced hypercholesterolemia and atherosclerosis was related to the polymorphism in porcine apolipoprotein Bi 00. The results showed that Lpb polymorphism but not von Willebrand disease status significantly influenced the development of diet-induced hypercholesterolemia and coronary and aortic atherosclerosis. These data have been reported in part previously.15
Methods
Experimental Animals All pigs were obtained from the Francis Owen Blood Research Laboratory at the University of North Carolina, Chapel Hill, where the vWD pigs have been maintained as a closed colony since it was established in 1971. All animals were treated according to the standards set in 'Guide for the Care and Use of Laboratory Animals' (National Institutes of Health publication no. 85-23). Porcine vWD is characterized by a prolonged bleeding time, decreased levels of plasma factor VIII coagulant activity, and less than 1% of normal plasma vWF antigen and
activity.1 --19 The following criteria were used to select pigs for this analysis: male sex, complete data on vWF levels and genotype, sufficient stored plasma to allow Lpb genotyp-
ing, ingestion of the atherogenic diet for 16 or 22 to 26 weeks for response of serum cholesterol and for quantification of coronary and aortic atherosclerosis by standard methods. Thirty-six pigs (17 vWD and 19 normal) met the criteria for analysis of the effect of the vWD genotype on the response of serum cholesterol to the atherogenic diet, 33 (16 vWD and 17 normal) for the effect of Lpb genotype on serum cholesterol, 27 (14 vWD and 13 normal) for the effect of vWD, serum cholesterol, and Lpb genotypes on coronary atherosclerosis, and 21 (12 vWD and 9 normal) for aortic atherosclerosis. The data on Lpb genotype and aortic atherosclerosis for these pigs have not previously been reported.
Administration of Atherogenic Diet At initiation of the atherogenic diet, pigs ranged in age from 10 to 15 weeks and weighed between 13 and 32 kg. The atherogenic diet was fed to all pigs for 16 or 22 to 26 weeks (Tables 1 and 2). At killing, there was no significant difference in the weights of animals in the normal and vWD groups (normal, 51.79 ± 1.85 kg; and vWD, 52.81 ± 2.94, P = 0.900, Wilcoxon rank sum test). The atherogenic diet was composed of pig chow supplemented with 1% or 2% cholesterol, 20% beef tallow, and 0.75% cholate4- (Tables 1 and 2).
Hemostatic Profiles Plasma was assayed for vWF activity by the tap-tube macroscopic agglutination test using paraformaldehydefixed human platelets and porcine plasma.17 19 Platelet counts, hematocrits, and leukocyte counts were also determined.4i Bleeding times were done by the method of Mertz.17 Except for the tests for vWF, no significant differences were found between vWD and normal pigs for these values.'4
Serum Cholesterol Concentration Serum cholesterol concentration was determined by Bio Vet Laboratories, Burlington, North Carolina, by the method of Allain et al,20 as modified by American Monitor Corporation, Indianapolis, Indiana, for automated determination. Values were obtained before feeding the atherogenic diet and every month until killing. The mean level of hypercholesterolemia for each pig was defined as the average of monthly values while receiving diet.
Determination of Lpb Genotype The Lpb phenotypes were determined in the Immunogenetics Laboratory, University of Wisconsin, Madison, Wis-
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Table 1. Lpb Genotype and Increase in Serum Cholesterol Levels in 19 Normal Swine Fed an Atherogenic Diet Serum cholesterol (mg/dl) Pig no. Weeks on diet Lpb genotype Dietary cholesterol (%) Initial Mean* 174M 1/1 2 16 86 921 43N 1/1 16 2 110 642 32R 1/8 25 115 1080 34R 1 1/8 26 101 985 1 59R 1/8 24 110 745 03N 1/8 16 107 732 56-0 1/8 16 657 70-0 1/8 1 16 73 856 1 43M 1/5 2 16 130 638 04N 1/5 2 16 125 762 1/5 05N 16 97 739 1/5 85N 16 92 880 118Q 1 5/8 25 94 884 35R 2 5/8 22 105 1319 141-0 5/8 16 104 620 2 1510 3/8 25 69 528 145N 8/8 2 22 511t 205NI 2 24 86 479 106MI 16 123 706 101.6 ± 4.2 772.8 ± 47.9t * Mean = the average of monthly values while receiving the atherogenic diet. t The cholesterol value was only present at month 5 for this pig. t Mean + SE of the average monthly values. 1 Pigs included in the analysis of response to diet-induced hypercholesterolemia and coronary atherosclerosis only (Lpb genotype not available).
consin, by the double immunodiffusion test using 16 Lpb epitope specific alloimmune sera as described by Rapacz et al.12,13 Those testing the samples in the immunogenetics laboratory had no knowledge of the von Willebrand disease genotype nor of the degree of hypercholesterolemia and atherosclerosis documented in individual animals.
Coronary Atherosclerosis All animals were killed with an overdose of pentobarbital while under halothane anesthesia. The heart and coronary arteries were perfusion fixed.' Cross sections of each coronary artery were taken perpendicular to the direction of blood flow at 1-cm intervals from their origin. In
Table 2. Lpb Genotype and Increase in Serum Cholesterol Levels in 17 von Willebrand Disease Swine Fed an Atherogenic Diet Serum cholesterol (mg/dl) Pig no. Lpb genotype Dietary cholesterol (%) Weeks on diet Initial Mean*
111R 112R 151-0 21S 75N 60-0
79-0 22R 16S 22N 15N 25N 154M 16N 86R 199M 88Mt
1/1 1/1 1/1 1/3 1/3 1/3 1/3 1/8 1/8 1/8 1/8 1/5 5/8 5/8 3/8 3/8
1 1 1 1 2 1 1 1 1 2 2 2 2 2 1 2 2
24 25 16 26 16 16 16 23 25 16 16 16 16 16 25 16 16
145 140 83 122 112 62 108 94 101 126 94 102 121 96 102 78 105.4 + 5.5
627 597 685 497 808 1049 764 1044 475 612 1005 1005 921 893 432 699 774 757.9 + 49.4t
Mean = the average of monthly values while receiving the atherogenic diet.
t Mean ± SE of the average monthly values.
t Pig included in the analysis of response to diet-induced hypercholesterolemia and coronary atherosclerosis only (Lpb genotype not
available).
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this report, the term 'coronary artery cross section' (CACS) refers to these specimens. The amount of coronary atherosclerosis was calculated three ways for each pig: A) the mean percentage luminal narrowing by intima obtained by averaging results for all CACS; B) the number of CACS per pig with at least 15% luminal narrowing by intima; and C) the percentage of the total number of CACS per pig with at least 15% luminal narrowing by intima.4'5
Aortic Atherosclerosis The aorta was either fixed under systemic pressure in 10% buffered formalin or immersion fixed in 1% gluteraldehyde and 4% formaldehyde when the animal was killed.4'6 The fixed aorta was divided into 10 to 12 equalsized segments taken perpendicular to the direction of blood flow from just below the celiac artery to the origin of the iliac arteries. Aortas that were immersion fixed were opened longitudinally along the ventral aspect and then divided into segments. Aortas that were fixed under pressure were cut into cross sections. Light microscopic sections were prepared from the proximal half of each segment. The extent of aortic atherosclerosis was defined as both the intimal area in square millimeters and the intimal area as a percentage of medial area.2122 The summary statistics are reported as the mean of all values for each group of pigs for the entire abdominal aorta (ie, from the celiac axis to the aortoiliac junction) and for the distal half (ie, sections from the distal five or six of the 10 to 12 segments) of the abdominal aorta.
disease, and serum cholesterol in explaining variation in degree of coronary and aortic atherosclerosis. These methods also were applied to relationships between serum cholesterol and different Lpb genotypes and von Willebrand disease. These analyses supported grouping of the seven Lpb genotypes as follows: Group 1 with Lpbl/1, Lpbl/3, and Lpbl/8 genotypes (ie, the Lpbl allele but not the Lpb5 allele) (n = 19); Group 2 with the Lpbl/5 and Lpb5/8 genotypes (ie, genotypes containing the Lpb5 allele) (n = 10); and Group 3 with Lpb3/8 or Lpb8/8 genotypes (ie, neither the Lpbl nor the Lpb5 allele) (n = 4).
Results vWD and Lpb Genotypes The results of the Lpb phenotypic classification of the individual swine are shown in Tables 1 and 2. Table 1 lists the normal pigs that had bleeding times of 2.38 + 0.2 minutes and vWF activity of 94.3 ± 8.1%. Table 2 lists the vWD pigs, which had bleeding times of more than 10 minutes and less than 1% vWF activity. Four Lpb alleles were identified in the normal and vWD atherosclerotic pigs: Lpb 1, 3,5, and 8. The Lpbl and Lpb8 alleles were present in homozygous and heterozygous genotypes whereas the Lpb3 and Lpb5 alleles were present only as heterozygotes. No mutant Lpb5 alleles for spontaneous hypercholesterolemia were found.23
Serum Cholesterol: Effect of vWD and Lpb Genotypes
Statistical Methods Primary Evaluation and Correlations The data for serum cholesterol and degree of coronary and aortic atherosclerosis were described for Lpb genotypes with means, standard errors, and minimum and maximum values. The association of the respective Lpb genotypes with serum cholesterol and degrees of atherosclerosis was primarily evaluated with Spearman rank correlation coefficients. Unless otherwise stated, r and P values are from this correlation analysis. Also, comparisons across groups based on Lpb genotypes were made with the Kruskal-Wallis test.
Groups of Lpb Genotypes Multiple linear regression methods for the analysis of variance (ANOVA) were used to investigate further the relative roles of different Lpb genotypes, von Willebrand
The response of serum cholesterol to the atherogenic diet, percentage dietary cholesterol, and length of feeding diet according to genotypes are tabulated in Tables 1 and 2. No significant difference was found between the average of mean values in cholesterol levels between normal and vWD pigs (respectively in mg/dl, 772.8 ± 47.9 versus 757.9 + 49.4, P = 0.95, Wilcoxon rank sum test). No significant differences were found in the degree of hypercholesterolemia related to Lpb genotype in pigs that were fed either a 1% or 2% diet (mean cholesterol for pigs receiving 1% cholesterol was 769.0 ± 58.5 mg/dl; and for those receiving 2% cholesterol diet, 762.6 ± 36.4 mg/dl, P = 0.776, Kruskal-Wallis). In addition, when the individual cholesterol levels were compared at each month of receiving the diet for 16 weeks or 22 to 26 weeks and the average of monthly values, no significant differences were found in the levels achieved (data not
shown). Mean cholesterol levels for the pigs are listed in Table
von
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uninvolved. The flat lesions in the aorta contained accumulations of foam cells that ranged from a few cells to several cells thick. Sections from the raised plaques in the abdominal aorta contained foam cells, smooth muscle cells, and a fibrous cap (Figures 1-3). Sections were chosen for illustration based on two criteria. First, each is from one of three distal sections taken from the abdominal aorta (ie, section 9, 10, or 1 1 of the 10 to 12 from each aorta as described in Materials and Methods). Second, the amount of atherosclerosis was equivalent to the mean standard error values for the normal or vWD pigs contained within the three groups of Lpb Genotypes (data in figure legends).
3 according to the groups of Lpb genotypes and the respective homozygous and heterozygous Lpb genotypes. The degree of diet-induced hypercholesterolemia was not significantly associated with the presence of any particular allele, although the correlation with the Lpb5 allele (r = 0.263, P = 0.1) was somewhat stronger than for the other alleles. A comparison of mean cholesterol levels for each of the three groups of Lpb genotypes, however, showed that pigs in group 2 (ie, pigs with the Lpb5 in their genotypes) developed a significantly higher mean cholesterol than those in groups 1 or 3 (P = 0.0343, Kruskal-Wallis test) (Table 3). An overall comparison of mean cholesterol for each of six respective Lpb genotypes (ie, with Lpb3/8 and Lpb8/8 pooled because of small sample size) showed the highest mean cholesterol value in Lpb5/8 genotype (927.4 112.0, n = 5, P = 0.128, Kruskal-Wallis test) and the lowest mean cholesterol in pigs with Lpb3/8 and Lpb8/8 genotypes (542.5 56.2, n = 4).
±
Coronary Atherosclerosis: Effect of vWD and Lpb Genotypes and Serum Cholesterol
±
±
There were no significant differences in the three criteria used to describe coronary atherosclerosis between normal and vWD pigs as previously noted when pigs were reported separately (P 0.628, Wilcoxon rank sum test) (Table 4).4 For the three groups of Lpb genotypes, the presence of the Lpb5 allele correlated significantly with the development of the greatest amount of coronary atherosclerosis. This finding was significant for all three criteria (Table 5). Multiple regression methods for ANOVA also were used to analyze relationships between degree of coronary atherosclerosis and combinations of Lpb alleles and genotypes as well as presence or absence of von Willebrand disease. Descriptive statistics for the three groups of Lpb genotypes are given in Table 5. A significantly greater degree of coronary atherosclerosis was present in the coronary arteries of pigs with the Lpb5 allele (ie, group 2) when compared with pigs in group 1 (0.026 P < 0.107, ANOVA) or group 3 (0.002 P < 0.007) (Table 5). The presence of the Lpbl allele in the absence of the Lpb5 allele (ie, group 1) was associated with an intermediate degree of coronary atherosclerosis. These results also suggested that the Lpb3/8 or Lpb8/8 genotypes (ie, group 3) were associated with somewhat lower degrees of coronary atherosclerosis. The data on pigs with these genotypes should be viewed cautiously be-
Morphology of Atherosclerosis
-
Coronary Arteries The morphology of the coronary atherosclerosis found in these normal and vWD pigs has been described in detail.46 In general, examination of the serial cross sections of all three coronary arteries showed that certain areas were uninvolved, others had accumulations of foam cells and smooth muscle cell proliferation with a fibrous cap, and in some sections calcification, necrosis, and hemorrhage were also evident. Aortic Lesions
-
Two types of aortic lesions were evident on gross specimens in both normal and vWD pigs. The aortic arch and thoracic aorta contained flat, translucent lesions as described in previous studies.23 In contrast, the lesions found in the abdominal aorta not only contained flat, translucent lesions but also ones that were raised, pale gray, and fibrous.23 By light microscopy, some regions of the aorta were Table 3.
Group
Lpb Genotype and Diet-induced Hypercholesterolemia Lpb genotypes Genotypes
Lpbl"/'113,1/8 Lpbl15.5/8 Lpb3/8,8/8
1. 2. 3.
-
Serum cholesterol (mg/dl)* n
19 10 4
Mean + SE 777.8 + 43.8 866.1 ± 64.0t 542.5 ± 56.2
Average of mean of monthly values for the groups while receiving the atherogenic diet. t P = 0.0343, Kruskal-Wallis test for cholesterol across the three Groups of Lpb Genotypes. *
Minimum 475 620 432
Maximum 1080 1319 699
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Figure 1. Diet-induced atherosclerosis of abdominal aorta in a von Willebrand disease pig uith Lpb'8genotype (pig number 165, Table 2, dietfedfor 25 weeks). Morphometric anallsis indicated the atheromatous plaque area was 0.938 mmu?' or 6.6% of medial area. The mean values of these measurements for the z'WD pigs in group 1 on Table 7 considered separately was 0.811 + 0.201 mm2 and 8.2 + 2.3% (n 8). For comparison, the mean 'alues of these measurements for normal pigs in group 1 on Table 7 considered separately uwas 1.854 + 0.723 mm' and 20.5 + 8.4% (n = 4). Pigs with the Lpb' allele developed an intermediate amount of coronary and aortic atherosclerosis relative to pigs with other alleles (group 1 on Tables 5 and 7); (A) is a whole mount; (B) is a x4.6 magnification which includes lesion. Aorta opened and immersion fixed Verhoeff-van Gieson. Bar = 1 mm in both (A) and (B). =
cause group 3 only contained four pigs. These differences in coronary atherosclerosis between pigs in groups 1, 2, and 3 were similarly evident in these regression analyses when serum cholesterol was taken into ac-
count. Also with Lpb genotype taken into account, neither serum cholesterol nor presence or absence of vWD was significantly associated with severity of coronary atherosclerosis. When analyzed without adjustment for Lpb ge-
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Figure 2. Diet-induced atherosclerosis of abdominal aorta in a Willebrand disease pig with Lph5 8 genotype (pig number 1 54M, Table 2, dietfedfor 16 weeks). The atheromatous plaque contains lipid-ladet foam cells and afibrous cap encroaching on the lumen ofthe vessel Morphometric analysis indicated intinmal plaque area uwas 4.445 mm-' or 34% of medial area. The mean values of these measurements for the vWD pigs in group 2 on Table 7 considered separately u'as 1.886 ± 0.6738 mm12 and 21.6 ± 9.4% (n = 3). For comparison, the mean values of these measurenmentsfor the normalpigs in group 2 on Table 7 considered separatelly uas 1.478 0.445 mm2 and 15.5 -+- 3. 1% (n = 4); Pigs with the Lpbs allele developed the most severe atherosclerosis (group 2 on Tables 5 and 7); (A) is a whole mount of the cross section; (B) is x3 magnffication uhich includes lesion. Aorta pressure perfused. Methblene blue and basic fuscin, bar = 1 nim in both (A) and (B). ion
notype, serum cholesterol was significantly correlated with the three criteria used to describe coronary atherosclerosis, as has been previously reported4: for A), r = 0.39, P = 0.045; for B), r = 0.31, P = 0.091; for C), r = 0.39, P = 0.035.
Analyses were performed to determine if there was an effect of 1 % versus 2% cholesterol diet and the duration of diet feeding (16 versus 22 to 26 weeks) on the severity of coronary atherosclerosis. Analyses that adjusted for these variables supported the principal conclusions con-
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Figure 3. Diet-induced atherosclerosis of abdominal aorta in a von Willebrand diseasepig uith Lpt' genotype (pig number 86R, Table 2, diet fed for 25 uweeks). Morphometric analysis indicated lesion area uwas 0.0672 mm or 1% of medial area. The mean value of these measurements for the vWD pig in group 2 on Table 7 considered separately was 0.121 mm and 1. 1% (n = 1). For comparison, the mean value of these measurements for the normal pig in group 3 on Table 7 considered separately was 0.1-35 mm2 and 2.1% (n = 1); (A) is a uhole mount; (B) is a x 4 magnification which includes lesion. Pigs in this group ofLpb Genotvpes developed the least severe coronary and aortic atherosclerosis (group .3 on Tables 5 and 7). Aorta opened and immersion fixed. Verhoeff-van Gieson, bar = 1 mm in both (A) and
(B).
cerning Lpb genotypes, as did analyses that did not adjust for these variables (data not shown). To determine the effect of including diet and balloon injury methods of inducing coronary atherosclerosis, analyses were performed that excluded the subset of coronary arteries that were ballooned. There was no change in the association of severity of coronary atherosclerosis with Lpb genotype when the ballooned coronary artery was excluded (data not shown).
Aortic Atherosclerosis: Effect of vWD and Lpb Genotypes and Serum Cholesterol The extent of aortic atherosclerosis, expressed as both the intimal area in square millimeters and the mean intimal area as a percentage of media, tended to be lower in the vWD pigs when compared with normal pigs (Table 6). The differences were not significant considering either
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Table 4. Coronary Atherosclerosis Morphometry in Normal and vWD Swine Normal and vWD swine genotypes
Normal vWD P valuet
Coronary
atherosclerosis
morphometry
n
Percent luminal narrowing (A)
Number CACS 15% (B)
Percent CACS ,15% (C)
14 13
14.44 ± 2.68% 13.97 ± 3.22% 0.628
4.14 ± 1.01 3.92 ± 0.93
34.05 ± 7.91% 33.90 ± 8.95%
0.752
0.950
Summary statistics of the coronary atherosclerosis morphometry are reported for the normal and vWD pigs as mean ± SE. The severity of coronary atherosclerosis is reported by three criteria: A) mean percent luminal narrowing by intima by genotype; B) the mean number of coronary artery cross sections (CACS) per genotype with a15% luminal narrowing by intima; and C) the mean percent of the total number of coronary artery cross sections per genotype with 2 15% luminal narrowing by intima. The individual values were calculated for each pig and were averaged, and then the result was averaged with those for all other pigs with normal or vWD genotypes. Coronary atherosclerosis morphometry data and calculations are from the formulas reported in references 4 and 5. t P values are from Wilcoxon rank sum test. *
the entire abdominal aorta or the distal half (P 0.201, Wilcoxon rank sum test, Table 6). For the Lpb genotypes, the correlation of the Lpb5 allele with severity of atherosclerosis approached significance for the entire abdominal aorta (for mean intimal area in square millimeters, r = 0.35, P = 0.120; for intimal area as a percentage of medial area, r = 0.38, P = 0.086). Because most of the raised fibrous plaques appeared in the distal aorta, as has been noted before,23 the distal half of the abdominal aorta was evaluated separately. The Lpb5 allele was significantly correlated with the development of atherosclerosis in the distal half (for mean intimal area in square millimeters, r = 0.450, P = 0.041; and for intimal area as a percent medial area: r = 0.434, P = 0.0495) (Table 7, Figure 2). The degree of diet-induced hypercholesterolemia was significantly correlated with aortic atherosclerosis expressed as intimal area as a percentage of media for both the entire abdominal aorta and the distal half (respectively, r = 0.42, P = 0.058; and r = 0.44, P = 0.045). The same trend was suggested when aortic atherosclerosis was considered as intimal area in square millimeters (for the entire abdominal aorta, r = 0.31, P = 0.165; for the distal half, r = 0.39, P = 0.08). -
Multiple regression analyses for analysis of variance (ANOVA) were used also for both measures of abdominal aortic atherosclerosis to analyze relationships between severity of atherosclerosis and combinations of Lpb alleles and genotypes as well as presence or absence of von Willebrand disease. In addition, these analyses were made with and without adjustment for cholesterol. The correlation of the Lpb5 allele or serum cholesterol with the amount of aortic atherosclerosis in these analyses tended to be significant when considered separately (P < 0.05, Wilcoxon rank sum test) or at least suggested trends. With adjustment for serum cholesterol, however, differences in the degree of atherosclerosis between Lpb genotypes were no longer significant (P 3 0.15, ANOVA); these differences were also not significant when there was adjustment for 1% versus 2% cholesterol diet and duration of diet feeding (16 versus 22 to 26 weeks) (data not shown). Similarly serum cholesterol was no longer significant with adjustment for Lpb status. These latter findings could be mainly due to the small sample size for analysis of aortic atherosclerosis. Importantly, comparisons of severity of aortic atherosclerosis based on the absence or presence of von Willebrand disease were clearly not significant (P 3 0.15, Wilcoxon rank sum test).
Table 5. Lpb Genotype and Coronary Atherosclerosis Morphometry Lpb genotypes Coronary atherosclerosis morphometry* n Group Genotypes Percent luminal narrowing (A) Number CACS >15% (B) Percent CACS a15% (C) it Lpbl/1"1/3,1/8 14 12.9 + 2.4% 3.5 t 0.8 31.5 ± 8.1%
2t
Lpbl/5t5t./8
9
3t
Lpb3/8a8/8
4
(0.1, 29.4)
(0,8)
21.6 t 3.3% (9.0, 39.7)
6.4 t 1.1 (2, 13)
(0, 4.8)
0.3 ± 0.3 (0, 1)
2.3 ± 1.2%
(0,100) 50.8 t 8.5% (13.3, 92.9) 2.1 t 2.1%
(0, 8.3)
Summary statistics of the severity of coronary atherosclerosis are reported for the three groups of Lpb genotypes as mean ± SE with min and max values listed below in parenthesis. Coronary atherosclerosis is reported for each group with three criteria as defined in Table 4 and
in Materials and Methods. The individual values were calculated for each pig and were averaged, and then the result was averaged with those for all other pigs in a given group. Calculations are from the formulas reported in references 4 and 5. t 0.002 - P < 0.007 for the three criteria for coronary atherosclerosis for group 2 versus group 3 and 0.026 - P - 0.107 for group 2 versus group 1 in analysis of variance. t The Lpb5 allele correlates with all three criteria for coronary atherosclerosis: for A) r = 0.52, P = 0.005; for B) r = 0.48, P = 0.01 1; and for C) r = 0.46, P s 0.015.
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Table 6. Abdominal Aortic Atherosclerosis Morphomety in Normal and vWD Swine Abdominal aortic atherosclerosis* Normal and vWD swine genotypes Normal vWD P valuet
Entire Intimal area
Distal half Intimal area
n
mm2
% of media
mm2
% of media
9 12
1.495 ± 0.391 1.022 ± 0.260 0.320
16.24 ± 4.16% 10.99 ± 3.16% 0.227
2.358 ± 0.569 1.510 ± 0.436 0.201
25.09 ± 6.46 16.14 ± 4.98 0.256
* Aortic atherosclerosis was defined as the mean intimal area expressed as percent of media and the mean intimal area in mm2 for 10-12 sequential sections per pig for both the entire abdominal aorta and the distal half (see Materials and Methods, "Aortic Atherosclerosis"). The values were calculated for each pig and were averaged, and then the result was averaged with those for all other pigs in a given group. The summary statistics are listed as mean ± SE. t P values are from Wilcoxon rank sum test.
Discussion The relationship between vWD genotype, polymorphism in Lpb genotype, and diet-induced atherosclerosis and hypercholesterolemia was studied in normal and vWD pigs. Four different Lpb alleles were identified in the these pigs: Lpbs 1, 3, 5, and 8. The presence of the Lpb5 allele correlated significantly with development of greatest amounts of coronary and aortic atherosclerosis. The Lpb5 allele occurs frequently in populations of pigs of various breeds and has been associated with atherosclerosis.12-14 In contrast, the Lpbl allele is rare and untested in studies of atherosclerosis.14 In the normal and vWD pigs, the presence of the Lpbl allele correlated with an intermediate amount of disease relative to the other alleles. Pigs with Lpb3/8 or Lpb8/8 genotypes developed the least amount of atherosclerosis. Although pigs with the Lpb5 allele tended to have higher cholesterol levels, this relationship was not as strong as that for atherosclerosis. Importantly, vWD genotype did not influence the development of either hypercholesterolemia or coronary or aortic atherosclerosis. These results suggest that apolipoprotein B100 genotype is a major factor in determining the severity of coronary and aortic atherosclerosis that develops in this porcine model.
For simplicity, the atherosclerosis and cholesterol data in the seven different genotypes found in the normal and vWD pigs were presented in three groups. It is important to note, however, that the pigs with the Lpb5 allele had the most amount of coronary and aortic atherosclerosis when analyzed before genotype grouping. In addition, these pigs developed higher cholesterol levels, particularly ones with the Lpb5/8 genotypes, which developed the highest cholesterol levels and the greatest amount of coronary atherosclerosis. This analysis then identified pigs with Lpb3/8 and Lpb8/8 genotypes as having the least amount of atherosclerosis and lowest serum cholesterol levels. All other pigs were in between these two extremes and shared the Lpbl allele. Although other groupings may be possible, the three Lpb groups were identified by having mild, moderate, or severe disease when each pig was considered individually. Previous studies with varying protocols have found that the Lpb5 allele is associated with diet-induced hypercholesterolemia and aortic atherosclerosis.14 Pigs with the Lpb5 allele fed a cholesterol-free, high-fat diet developed both higher mean serum cholesterol levels and aortic intimal lipidosis than pigs with the Lpb8 allele. In a separate study, Thompson miniature swine with the Lpb5 allele developed hypercholesterolemia when fed a
Table 7. Lpb Genotype and Abdominal Aortic Atherosclerosis Morphomety Abdominal aortic atherosclerosis* Entire Distal half Intimal area Lpb genotypes Intimal area n mm2 % media mm2 % media Group genotypes 1. 12 Lpbl /11/3l1/8 1.159 ± .0294 12.3 ± 3.4% 1.589 ± 0.438 17.9 ± 5.4% (0.049, 3.771) (0.8, 41.9) (0.085, 5.616) (1.4, 65.9) 7 2. Lpbl/5t.5t/8 1.65 ± 0.375 18.1 ± 4.1% 2.826 ± 0.574t 28.5 t 6.4%t (0.442, 2.877) (4.8, 37.3) (0.706, 4.768) (6.5, 58.0) 2 3. 0.128 ± 0.007 1.6 + 0.5% 0.195 t 0.47 2.6 t 0.3% Lpb3/8 (0.121, 0.135) (1.1, 2.1) (0.147, 0.242) (2.3, 2.9) Aortic atherosclerosis is reported as defined in the legend to Table 6. The summary statistics are listed as mean + SE with min and max values listed below in parenthesis. t The Lpb5 allele correlates with the degree of aortic atherosclerosis in the distal half of the abdominal aorta (for mean intimal area in mm2, r = 0.450, P = 0.041; and for intimal area as a percent medial area: r = 0.434, P = 0.0495).
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2% cholesterol, 25% fat diet, whereas pigs with the Lpb3/3 genotype were nonresponders.12 No data on the degree of hypercholesterolemia or atherosclerosis found in these animals were reported. The present study confirms and extends these associations of Lpb genotypes and degree of atherosclerosis to normal and vWD male pigs fed a 1% to 2% cholesterol, high-fat diet. Pigs with the Lpb5 allele not only developed the most severe aortic atherosclerosis and highest cholesterol levels but also the most severe coronary atherosclerosis. In addition, the normal and vWD pigs with the Lpb3/8 and Lpb8/8 genotypes developed the smallest rise in serum cholesterol and the least amount of coronary and aortic atherosclerosis. It should be noted that this group contained only four pigs: one received 2% cholesterol in the diet for 16 weeks and three received 1% or 2% cholesterol for 22 to 25 weeks. The influence of Lpb genotype on diet-induced atherosclerosis is further supported by the additional new finding of an intermediate amount of disease in pigs with the rare Lpb1 allele. These findings have identified groups of normal and vWD pigs with specific Lpb genotypes suited for further study of graded susceptibility to diet-induced atherosclerosis. How different Lpb genotypes might influence lipid metabolism and atherogenesis is understood in part. One strain of pigs with a mutant Lpb5 allele phenotypically exhibits spontaneous hypercholesterolemia and accelerated atherosclerosis.2325 These Lpb5 mutant pigs have normal LDL receptor binding activity but have abnormal low density lipoproteins and defective LDL catabolism, resulting in hypercholesterolemia even while receiving a cholesterol-free diet.23 24 Also a direct relationship exists between the mutant Lpb5 genotype and the concentration and composition of LDL subfractions.26 Although there appears to be considerable homology between human and porcine LDL molecules at the proposed LDL receptor binding sites, LDL from pigs with Lpb2 and Lpb4 alleles has a higher affinity than human LDL for both the pig and human receptor.27 Interestingly the LDL receptor binding regions of alleles Lpb2,3,7 and mutant Lpb5 do not have major differences by DNA sequence analysis.28 The differences in the phenotypic expression of these alleles, then, may result from differences outside of the LDL receptor binding region. These findings are also consistent with the observation in this present study that the correlation of Lpb5 allele with amount of atherosclerosis was greater than that found for degree of hypercholesterolemia. These studies indicate, then, that the polymorphism at the apo-B locus exerts a strong influence on lipid metabolism and atherogenesis in pigs. It is possible that the various Lpb genotypes produce important differences in cholesterol distribution among different lipoproteins, lipid particle size, or lipoprotein
clearance, similar to what has been found in the spontaneously hypercholesterolemic mutant Lpb5 pig.242629 Such differences would not have been detected by measurement of total cholesterol as reported in this study. Further study is needed to document whether or not the Lpb 1, 3, 5, and 8 alleles alter specific aspects of lipoprotein metabolism and atherogenesis in this model of dietinduced atherosclerosis. Lpb genotypes as determinants of the severity of coronary and aortic atherosclerosis may provide insight into the wide range of differences in the degree of atherosclerosis noted in the earlier studies.1'- In this study, vWD pigs with severe hypercholesterolemia, male gender, and especially the Lpb5 allele developed the most severe coronary and aortic atherosclerosis, which was not significantly different from normal pigs that also had the Lpb5 allele. With different Lpb genotypes, both normal and vWD pigs still developed comparable degrees of coronary and aortic atherosclerosis but at graded levels of severity. It is clear that polymorphism in Lpb genotypes could have affected the results of the atherogenesis experiments in the vWD stock, confusing the interpretation of the results either to cause apparent enhancement of the perceived effect of vWF or to mask a more important effect of vWF. It seems likely that any effect of vWF on the development of atherosclerosis is part of a complex array of variables affecting atherogenesis, including the important contribution of lipids and lipoproteins. A mechanism linking vWF with atherogenesis would most likely involve support by vWF of platelet adhesion and activation on arterial walls. Von Willebrand factor functions as a ligand binding platelets to exposed subendothelium, particularly under high shear conditions3033 and in the development of occlusive arterial thrombosis.6.3.3 Platelets thus attached to the vessel wall and activated could release platelet-derived growth factor (PDGF) and thereby promote atherogenesis.3536 It is probable that PDGF can be delivered to the vessel wall by a vWF-independent fashion or that other in vivo factors play a significant role in the proliferative response of smooth muscle cells to injury.37-42 Data from our laboratory have shown that both pseudopod formation and spread of platelets adhering to injured arterial walls is impaired in normocholesterolemic, vWD pigs.543 Interestingly the number of adherent platelets in these injured coronary arteries is not significantly influenced by the presence or absence of vWF.543 Reddick et al, however, have recently found that platelets adhering to ulcerated or damaged coronary artery plaques in vWD pigs fed a high-cholesterol diet are spread or activated to 73% of the degree found in pigs with normal levels of vWF.5 This raises the possibility that the Lpb5 allele leads to a greater uptake of lipid by the vessel wall and that the lipid-rich vessel wall enhances platelet activation by a mechanism
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that bypasses the need for vWF as demonstrated by Reddick et al.5 Such a series of interactions would explain to some degree the loss of protection from atherosclerosis in vWD pigs when Lpb5 and hypercholesterolemia are present.
11.
12.
Acknowledgments The authors thank Mr. Jeff Sigman for excellent technical assistance and Ms. Robin Raymer, manager of the Francis Owen Blood Research Laboratory.
References 1. Brinkhous KM, Reddick RL, Griggs TR: Arterial thrombosis, atherosclerosis, and the factor VlIl/vWF complex, The Role of Factor VIII and von Willebrand Factor. Edited by TS Zimmerman, ZN Ruggeri. New York, Marcel Dekker, 1989, pp 283-303 2. Griggs TR, Reddick RL, Sultzer D, Brinkhous KM: Susceptibility to atherosclerosis in aortas and coronary arteries of swine with von Willebrand's disease. Am J Pathol 1981, 102:137-145 3. Sultzer DL, Brinkhous KM, Reddick RL, Griggs TR: Effects of carbon monoxide on atherogenesis in normal pigs and pigs with von Willebrand's disease. Atherosclerosis 1982,
43:303-319 4. Griggs TR, Bauman RW, Reddick RL, Read MS, Koch GG, Lamb MA: Development of coronary atherosclerosis in swine with severe hypercholesterolemia. Lack of influence of von Willebrand factor or acute intimal injury. Arteriosclerosis 1986, 6:155-165 5. Reddick RL, Read MS, Brinkhous KM, Bellinger DA, Nichols TC, Griggs TR: Coronary atherosclerosis in the pig: Induced-plaque injury and platelet response. Arteriosclerosis
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1990,10:541-550 6. Nichols TC, Bellinger DA, Tate DA, Reddick RL, Read MS, Koch GG, Brinkhous KM, Griggs TR: von Willebrand factor and occlusive arterial thrombosis: A study in normal and von Willebrand's disease pigs with diet-induced hypercholesterolemia and atherosclerosis. Arteriosclerosis 1990, 10:449461 7. Bowie EJW, Fuster V, Owen CA Jr, Brown AL: Resistance to the development of spontaneous atherosclerosis in pigs with von Willebrand's disease. Thromb Diathes Haemorrh 1975, 34:599 8. Fuster V, Bowie EJW, Lewis JC, Fass DN, Owen Jr CA, Brown AL: Resistance to arteriosclerosis in pigs with von Willebrand's disease. J Clin Invest 1978, 61:722-730 9. FusterV, Fass DN, Kaye MP, Josa M, Zinsmeister AR, Bowie EJW: Arteriosclerosis in normal and von Willebrand pigs: Long-term prospective study and aortic transplantation study. Circ Res 1982, 51:587-593 10. Badimon L, Steele P, Badimon JJ, Bowie EJW, Fuster V: Aortic atherosclerosis in pigs with heterozygous and von Willebrand disease. Comparison with homozygous von
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Willebrand and normal pigs. Arteriosclerosis 1985, 5:366370 Fuster V, Lie JT, Badimon L, Rosemark JA, Badimon JJ, Bowie EJW: Spontaneous and diet-induced coronary atherosclerosis in normal swine and swine with von Willebrand disease. Arteriosclerosis 1985, 5:67-73 Rapacz J: Lipoprotein immunogenetics and atherosclerosis. Am J Med Genet 1978, 1:377-405 Rapacz J, Hasler-Rapacz J, Kuo WH: Immunogenetic polymorphism of lipoproteins in swine. Immunogenetics 1978, 6:405-424 Rapacz J, Elson CE, Lalich JJ: Correlation of an immunogenetically defined lipoprotein type with aortic intimal lipidosis in swine. Exp Mol Pathol 1977, 27:249-261 Nichols TC, Bellinger DA, Koch GG, Davis K, Reddick RL, Read MS, Rapacz J, Hasler-Rapacz J, Brinkhous KM, Griggs TR: Porcine von Willebrand disease and atherosclerosis: Influence of polymorphism in apolipoprotein Bi 00 genotype in 35 male pigs. Arteriosclerosis 1990, 10(5):858a Hogan AG, Muhrer ME, Bogart R: A hemophilia-like disease in swine. Proc Soc Exp Biol Med 1941, 48:217-219 Mertz ET. The anomaly of a normal Duke's and very prolonged bleeding time in swine suffering from an inherited bleeding disease. Am J Physiol 1942,136:360-362 Griggs TR, Webster WP, Copper HA, Wagner RH, Brinkhous KM: Von Willebrand factor: Gene dosage relationships and transfusion response in bleeder swine-A new bioassay. Proc NatI Acad Sci USA 1974, 71:2087-2090 Allain JP, Cooper HA, Wagner RH, Brinkhous KM: Platelets fixed with paraformaldehyde: A new reagent for assay of von Willebrand factor and platelet aggregating factor. J Lab Clin Med 1975, 85:318-328 Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC: Enzymatic determination of total serum cholesterol. Clin Chem 1974, 20:470-475 Clarkson TB, Alexander NJ, Morgan TM: Atherosclerosis of cynomolgus monkeys hyper- and hyporesponsive to dietary cholesterol: Lack of effect of vasectomy. Arteriosclerosis 1988, 8:488-498 Parks JS, Kaduck-Sawyer J, Bullock BC, Rudel LL: Effect of dietary fish oil on coronary artery and aortic atherosclerosis in African green monkeys. Arteriosclerosis 1990, 10:11021112 Rapacz J, Hasler-Rapacz J, Taylor KM, Checovich WJ, Attie AD: Lipoprotein mutations in pigs are associated with elevated plasma cholesterol and atherosclerosis. Science 1986, 234:1573-1577 Checovich WJ, Fitch WL, Krauss RM, Smith MP, Rapacz J, Smith CL, Attie AD: Defective catabolism and abnormal composition of low density lipoprotein from mutant pigs with hypercholesterolemia. Biochemistry 1988, 27:1934-1941 Prescott MF, McBride CH, Hasler-Rapacz J, Von Linden J, Rapacz J: Development of complex atherosclerotic lesions in pigs with inherited hyper-LDL cholesterolemia (IHLC) bearing mutant alleles for apolipoprotein B. Am J Pathol 1991, 139:139-147 Lee DM, MokT, Hasler-Rapacz J, Rapacz J: Concentrations and compositions of plasma lipoprotein subfractions of
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Lpb5-Lpu1 homozygous and heterozygous swine with hypercholesterolemia. J Lipid Res 1990, 31:839-847 Ebert DL, Maeda N, Lowe SW, Hasler-Rapacz J, Rapacz J, Attie AD: Primary structure comparison of the proposed low density lipoprotein (LDL) receptor binding domain of human and pig apolipoprotein B: Implications for LDL-Receptor Interactions. J Lipid Res 1988, 29:1501-1509 Maeda N, Ebert DL, Doers TM, Newman M, Hasler-Rapacz J, Attie AD, Rapacz J, Smithies 0: Molecular genetics of the apolipoprotein B gene in pigs in relation to atherosclerosis. Gene 1988, 70:213-229 Checovich WJ, Aiello RJ, Attie AD: Overproduction of a buoyant low density lipoprotein subspecies in spontaneously hypercholesterolemic mutant pigs. Arteriosclerosis 1991, 11:351-361 Tschoop T, Weiss HJ, Baumgartner HR: Decreased adhesion of platelets to subendothelium in von Willebrand's disease. J Lab Clin Med 1974, 83:296-300 Sakariassen KS, Bolhuis PA, Sixma JJ: Human blood platelet adhesion to artery subendothelium is mediated by factor VIII/von Willebrand factor bound to the subendothelium. Nature 1979, 279:636-638 Turitto VT, Weiss HJ, Baumgartner HR: Decreased platelet adhesion on vessel segments in von Willebrand's disease: A defect in initial platelet attachment. J Lab Clin Med 1983, 101:551-564 Nichols TC, Bellinger DA, Johnson AJ, Lamb MA, Griggs TR: Von Willebrand's disease prevents occlusive thrombosis in stenosed and injured porcine coronary arteries. Circ Res 1986, 59:15-26 Bellinger DA, Nichols TC, Read MS, Reddick RL, Lamb MA, Brinkhous KM, Evatt BL, Griggs TR: Prevention of occlusive coronary artery thrombosis by a murine monoclonal anti-
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body to porcine von Willebrand factor. Proc Natl Acad Sci USA 1987, 84:8100-8104 Ross R, Glomset J, Kariya B, Harker L: A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc Natl Acad Sci USA 1974, 71:1207-1210 Ross R: The pathogenesis of atherosclerosis-an update. N Engl J Med 1986, 314:488-500 Friedman RJ, Burns ER: Role of platelets in the proliferative response of the injured artery. Prog Hemost Thromb 1978, 4:249-278 Ingerman-Wojenski CM, Silver MJ: Model system to study interaction of platelets with damaged arterial wall. Exp Mol Pathol 1988, 48:116-134 Schwartz SM, Reidy MA: Common mechanisms of proliferation of smooth muscle in atherosclerosis and hypertension. Hum Pathol 1987,18:240-247 Barrett TB, Benditt EP: Platelet-derived growth factor gene expression in human atherosclerotic plaques and normal artery wall. Proc NatI Acad Sci U S A 1988, 85:2810-2814 McCusker RH, Campion DR, Jones WK, Clemmons DR: The insulin-like growth-factor binding proteins of porcine serum: Endocrine and nutritional regulation. Endocrinology 1989,125:501-509 Antoniades HN, Galanopoulos T, Neville-Golden J, Kiritsy CP, Lynch SE: Injury induces in vivo expression of plateletderived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts. Proc Natl Acad Sci USA 1991, 88:565569 Reddick RL, Griggs TR, Lamb MA, Brinkhous KM: Platelet adhesion to damaged coronary arteries: Comparison of normal and von Willebrand diseased swine. Proc Natl Acad Sci U S A 1982, 79:5076-5079
Porcine von Willebrand Disease and Atherosclerosis Influence of Polymorphism in Apolipoprotein B100 Genotype
Timothy C. Nichols,*tt Dwight A. Bellinger,tts Keir E. Davis,11 Gary G. Koch,til Robert L. Reddick,tt Marjorie S. Read,tt Jan Rapacz,# Judith Hasler-Rapacz,# Kenneth M. Brinkhous,tt and Thomas R. Griggstt From the Departments of Medicine' and Pathologyt and the Center for Thrombosis and Hemostasis* School of Medicine, the Department of Biostatistics,ll School of Public Health, and the Division of Laboratory Animal Medicine,$ University of
North Carolina, Chapel Hill, North Carolina; and the Departments of Genetics and Meat and Animal Sciences, # University of Wisconsin, Madison, Wisconsin
The relationship of apolipoprotein-B genotype (Lpb) to diet-induced hypercholesterolemia and atherosclerosis was studied in von Willebrand disease (vWD) and normal pigs. Von Willebrand and normal pigs developed comparable levels ofhypercholesterolemia (respectively, 757.9 ± 49.4 versus 772.8 ± 47.9 mg/ dl, P = 0.95). Pigs with Lpbl/5 and Lpb5I8 genotypes, however, developed significantly higher serum cholesterol levels than those with other Lpb genotypes (866.1 ± 64.0 mg/dl, P = 0.0343). Coronary and aortic atherosclerosis, measured by computerassisted automated image analyzer, were not significantly different between vWD and normalpigs. Pigs with an Lpb5 allele developed significantly more atherosclerosis than those with the Lpb3/8 or Lpb8/8 genotypes or the rare Lpbl allele (r 0.434, P S 0.05). Polymorphism in apolipoprotein B100 genotype, then, significantly influenced the severity of diet-induced hypercholesterolemia and atherosclerotic plaque formation in vWD and normal swine without regard to the vWD genotype. (Am J Pathol 1992, 140:403-415) -
A role of von Willebrand factor (vWF) in atherogenesis has been suggested by studies in normal and von Willebrand disease (vWD) pigs.-1 1 In some of these studies, atherosclerosis was induced by high-cholesterol and high-fat diets.2-611 These diets have yielded pigs with a variable degree of hypercholesterolemia, and, in some studies, there was a trend toward higher levels of serum cholesterol in one or the other of the two groups of pigs. Also, within these two groups of atherosclerotic pigs, variation in the extent of coronary and aortic atherosclerosis has been noted.1-11 This variability in diet-induced hypercholesterolemia and atherosclerosis between normal and vWD pigs suggests that vWF may influence cholesterol metabolism in such a manner as to influence atherogenesis. We have conducted a series of studies on the effect of vWF on coronary atherogenesis and thrombosis.4i In the first study, the amount of coronary atherosclerosis was related to the degree of hypercholesterolemia that an individual pig developed and not to the presence of vWF. In addition, balloon injury of one of the three coronary arteries at the start of the experiment did not augment the amount of coronary atherosclerosis an individual pig developed.4 In the subsequent two studies, atherosclerosis was produced by diet alone. In the second study, balloon injury of coronary atherosclerotic plaques in vWD and normal swine at death induced a monolayer of attached platelets similar to what was seen in exposed, nonatherosclerotic subendothelium.5 The presence of atherosclerosis, however, did not promote platelet-fibrin thrombus formation in either phenotype. The presence of atherosclerosis, however, did support platelet activation Supported in part by National Institutes of Health grants HL26309 and HL01 648. Accepted for publication August 26, 1991. Address reprint requests to Dr. Timothy C. Nichols, Department of Medicine, University of North Carolina School of Medicine, CB #7075, 349 Burnett-Womack Building, Chapel Hill, NC 27514.
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on balloon-injured coronary plaques independent of vWF as judged by platelet ultrastructure in the vWD pigs. In the third study, it was shown that vWF was required to support the development of occlusive thrombosis even in the presence of coronary atherosclerosis and hypercholesterolemia.6 In each of these studies, variations in the degree of hypercholesterolemia and coronary atherosclerosis were noted within the two phenotypic groups of
pigs. Employing immunogenetic and molecular genetic techniques, Rapacz et al12 have documented considerable variability in porcine apolipoprotein B100 structure.12-14 Apolipoprotein Bi 00 is the major protein component of plasma low-density lipoprotein (LDL). Lowdensity lipoprotein is the predominant cholesterol transport protein in several species, including pigs.12-14 Eight Lpb alleles have been identified at the apolipoprotein B locus in swine.12'13 The codominant expression of 16 Lpb allotypes (Lpb 1 to 8 and Lpb 1 1 to 18) allows for the determination of any of the 36 Lpb genotypes directly from the immunological phenotype.12 13 Feeding high-fat diets has shown that some Lpb genotypes enhance atherogenesis.14 In the present study, the fourth in a continuation of this series, we described the Lpb polymorphism in the normal and vWD swine from the three earlier studies.4' The purpose of this study was to determine if the variability in susceptibility of these vWD and normal swine to dietinduced hypercholesterolemia and atherosclerosis was related to the polymorphism in porcine apolipoprotein Bi 00. The results showed that Lpb polymorphism but not von Willebrand disease status significantly influenced the development of diet-induced hypercholesterolemia and coronary and aortic atherosclerosis. These data have been reported in part previously.15
Methods
Experimental Animals All pigs were obtained from the Francis Owen Blood Research Laboratory at the University of North Carolina, Chapel Hill, where the vWD pigs have been maintained as a closed colony since it was established in 1971. All animals were treated according to the standards set in 'Guide for the Care and Use of Laboratory Animals' (National Institutes of Health publication no. 85-23). Porcine vWD is characterized by a prolonged bleeding time, decreased levels of plasma factor VIII coagulant activity, and less than 1% of normal plasma vWF antigen and
activity.1 --19 The following criteria were used to select pigs for this analysis: male sex, complete data on vWF levels and genotype, sufficient stored plasma to allow Lpb genotyp-
ing, ingestion of the atherogenic diet for 16 or 22 to 26 weeks for response of serum cholesterol and for quantification of coronary and aortic atherosclerosis by standard methods. Thirty-six pigs (17 vWD and 19 normal) met the criteria for analysis of the effect of the vWD genotype on the response of serum cholesterol to the atherogenic diet, 33 (16 vWD and 17 normal) for the effect of Lpb genotype on serum cholesterol, 27 (14 vWD and 13 normal) for the effect of vWD, serum cholesterol, and Lpb genotypes on coronary atherosclerosis, and 21 (12 vWD and 9 normal) for aortic atherosclerosis. The data on Lpb genotype and aortic atherosclerosis for these pigs have not previously been reported.
Administration of Atherogenic Diet At initiation of the atherogenic diet, pigs ranged in age from 10 to 15 weeks and weighed between 13 and 32 kg. The atherogenic diet was fed to all pigs for 16 or 22 to 26 weeks (Tables 1 and 2). At killing, there was no significant difference in the weights of animals in the normal and vWD groups (normal, 51.79 ± 1.85 kg; and vWD, 52.81 ± 2.94, P = 0.900, Wilcoxon rank sum test). The atherogenic diet was composed of pig chow supplemented with 1% or 2% cholesterol, 20% beef tallow, and 0.75% cholate4- (Tables 1 and 2).
Hemostatic Profiles Plasma was assayed for vWF activity by the tap-tube macroscopic agglutination test using paraformaldehydefixed human platelets and porcine plasma.17 19 Platelet counts, hematocrits, and leukocyte counts were also determined.4i Bleeding times were done by the method of Mertz.17 Except for the tests for vWF, no significant differences were found between vWD and normal pigs for these values.'4
Serum Cholesterol Concentration Serum cholesterol concentration was determined by Bio Vet Laboratories, Burlington, North Carolina, by the method of Allain et al,20 as modified by American Monitor Corporation, Indianapolis, Indiana, for automated determination. Values were obtained before feeding the atherogenic diet and every month until killing. The mean level of hypercholesterolemia for each pig was defined as the average of monthly values while receiving diet.
Determination of Lpb Genotype The Lpb phenotypes were determined in the Immunogenetics Laboratory, University of Wisconsin, Madison, Wis-
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Table 1. Lpb Genotype and Increase in Serum Cholesterol Levels in 19 Normal Swine Fed an Atherogenic Diet Serum cholesterol (mg/dl) Pig no. Weeks on diet Lpb genotype Dietary cholesterol (%) Initial Mean* 174M 1/1 2 16 86 921 43N 1/1 16 2 110 642 32R 1/8 25 115 1080 34R 1 1/8 26 101 985 1 59R 1/8 24 110 745 03N 1/8 16 107 732 56-0 1/8 16 657 70-0 1/8 1 16 73 856 1 43M 1/5 2 16 130 638 04N 1/5 2 16 125 762 1/5 05N 16 97 739 1/5 85N 16 92 880 118Q 1 5/8 25 94 884 35R 2 5/8 22 105 1319 141-0 5/8 16 104 620 2 1510 3/8 25 69 528 145N 8/8 2 22 511t 205NI 2 24 86 479 106MI 16 123 706 101.6 ± 4.2 772.8 ± 47.9t * Mean = the average of monthly values while receiving the atherogenic diet. t The cholesterol value was only present at month 5 for this pig. t Mean + SE of the average monthly values. 1 Pigs included in the analysis of response to diet-induced hypercholesterolemia and coronary atherosclerosis only (Lpb genotype not available).
consin, by the double immunodiffusion test using 16 Lpb epitope specific alloimmune sera as described by Rapacz et al.12,13 Those testing the samples in the immunogenetics laboratory had no knowledge of the von Willebrand disease genotype nor of the degree of hypercholesterolemia and atherosclerosis documented in individual animals.
Coronary Atherosclerosis All animals were killed with an overdose of pentobarbital while under halothane anesthesia. The heart and coronary arteries were perfusion fixed.' Cross sections of each coronary artery were taken perpendicular to the direction of blood flow at 1-cm intervals from their origin. In
Table 2. Lpb Genotype and Increase in Serum Cholesterol Levels in 17 von Willebrand Disease Swine Fed an Atherogenic Diet Serum cholesterol (mg/dl) Pig no. Lpb genotype Dietary cholesterol (%) Weeks on diet Initial Mean*
111R 112R 151-0 21S 75N 60-0
79-0 22R 16S 22N 15N 25N 154M 16N 86R 199M 88Mt
1/1 1/1 1/1 1/3 1/3 1/3 1/3 1/8 1/8 1/8 1/8 1/5 5/8 5/8 3/8 3/8
1 1 1 1 2 1 1 1 1 2 2 2 2 2 1 2 2
24 25 16 26 16 16 16 23 25 16 16 16 16 16 25 16 16
145 140 83 122 112 62 108 94 101 126 94 102 121 96 102 78 105.4 + 5.5
627 597 685 497 808 1049 764 1044 475 612 1005 1005 921 893 432 699 774 757.9 + 49.4t
Mean = the average of monthly values while receiving the atherogenic diet.
t Mean ± SE of the average monthly values.
t Pig included in the analysis of response to diet-induced hypercholesterolemia and coronary atherosclerosis only (Lpb genotype not
available).
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this report, the term 'coronary artery cross section' (CACS) refers to these specimens. The amount of coronary atherosclerosis was calculated three ways for each pig: A) the mean percentage luminal narrowing by intima obtained by averaging results for all CACS; B) the number of CACS per pig with at least 15% luminal narrowing by intima; and C) the percentage of the total number of CACS per pig with at least 15% luminal narrowing by intima.4'5
Aortic Atherosclerosis The aorta was either fixed under systemic pressure in 10% buffered formalin or immersion fixed in 1% gluteraldehyde and 4% formaldehyde when the animal was killed.4'6 The fixed aorta was divided into 10 to 12 equalsized segments taken perpendicular to the direction of blood flow from just below the celiac artery to the origin of the iliac arteries. Aortas that were immersion fixed were opened longitudinally along the ventral aspect and then divided into segments. Aortas that were fixed under pressure were cut into cross sections. Light microscopic sections were prepared from the proximal half of each segment. The extent of aortic atherosclerosis was defined as both the intimal area in square millimeters and the intimal area as a percentage of medial area.2122 The summary statistics are reported as the mean of all values for each group of pigs for the entire abdominal aorta (ie, from the celiac axis to the aortoiliac junction) and for the distal half (ie, sections from the distal five or six of the 10 to 12 segments) of the abdominal aorta.
disease, and serum cholesterol in explaining variation in degree of coronary and aortic atherosclerosis. These methods also were applied to relationships between serum cholesterol and different Lpb genotypes and von Willebrand disease. These analyses supported grouping of the seven Lpb genotypes as follows: Group 1 with Lpbl/1, Lpbl/3, and Lpbl/8 genotypes (ie, the Lpbl allele but not the Lpb5 allele) (n = 19); Group 2 with the Lpbl/5 and Lpb5/8 genotypes (ie, genotypes containing the Lpb5 allele) (n = 10); and Group 3 with Lpb3/8 or Lpb8/8 genotypes (ie, neither the Lpbl nor the Lpb5 allele) (n = 4).
Results vWD and Lpb Genotypes The results of the Lpb phenotypic classification of the individual swine are shown in Tables 1 and 2. Table 1 lists the normal pigs that had bleeding times of 2.38 + 0.2 minutes and vWF activity of 94.3 ± 8.1%. Table 2 lists the vWD pigs, which had bleeding times of more than 10 minutes and less than 1% vWF activity. Four Lpb alleles were identified in the normal and vWD atherosclerotic pigs: Lpb 1, 3,5, and 8. The Lpbl and Lpb8 alleles were present in homozygous and heterozygous genotypes whereas the Lpb3 and Lpb5 alleles were present only as heterozygotes. No mutant Lpb5 alleles for spontaneous hypercholesterolemia were found.23
Serum Cholesterol: Effect of vWD and Lpb Genotypes
Statistical Methods Primary Evaluation and Correlations The data for serum cholesterol and degree of coronary and aortic atherosclerosis were described for Lpb genotypes with means, standard errors, and minimum and maximum values. The association of the respective Lpb genotypes with serum cholesterol and degrees of atherosclerosis was primarily evaluated with Spearman rank correlation coefficients. Unless otherwise stated, r and P values are from this correlation analysis. Also, comparisons across groups based on Lpb genotypes were made with the Kruskal-Wallis test.
Groups of Lpb Genotypes Multiple linear regression methods for the analysis of variance (ANOVA) were used to investigate further the relative roles of different Lpb genotypes, von Willebrand
The response of serum cholesterol to the atherogenic diet, percentage dietary cholesterol, and length of feeding diet according to genotypes are tabulated in Tables 1 and 2. No significant difference was found between the average of mean values in cholesterol levels between normal and vWD pigs (respectively in mg/dl, 772.8 ± 47.9 versus 757.9 + 49.4, P = 0.95, Wilcoxon rank sum test). No significant differences were found in the degree of hypercholesterolemia related to Lpb genotype in pigs that were fed either a 1% or 2% diet (mean cholesterol for pigs receiving 1% cholesterol was 769.0 ± 58.5 mg/dl; and for those receiving 2% cholesterol diet, 762.6 ± 36.4 mg/dl, P = 0.776, Kruskal-Wallis). In addition, when the individual cholesterol levels were compared at each month of receiving the diet for 16 weeks or 22 to 26 weeks and the average of monthly values, no significant differences were found in the levels achieved (data not
shown). Mean cholesterol levels for the pigs are listed in Table
von
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uninvolved. The flat lesions in the aorta contained accumulations of foam cells that ranged from a few cells to several cells thick. Sections from the raised plaques in the abdominal aorta contained foam cells, smooth muscle cells, and a fibrous cap (Figures 1-3). Sections were chosen for illustration based on two criteria. First, each is from one of three distal sections taken from the abdominal aorta (ie, section 9, 10, or 1 1 of the 10 to 12 from each aorta as described in Materials and Methods). Second, the amount of atherosclerosis was equivalent to the mean standard error values for the normal or vWD pigs contained within the three groups of Lpb Genotypes (data in figure legends).
3 according to the groups of Lpb genotypes and the respective homozygous and heterozygous Lpb genotypes. The degree of diet-induced hypercholesterolemia was not significantly associated with the presence of any particular allele, although the correlation with the Lpb5 allele (r = 0.263, P = 0.1) was somewhat stronger than for the other alleles. A comparison of mean cholesterol levels for each of the three groups of Lpb genotypes, however, showed that pigs in group 2 (ie, pigs with the Lpb5 in their genotypes) developed a significantly higher mean cholesterol than those in groups 1 or 3 (P = 0.0343, Kruskal-Wallis test) (Table 3). An overall comparison of mean cholesterol for each of six respective Lpb genotypes (ie, with Lpb3/8 and Lpb8/8 pooled because of small sample size) showed the highest mean cholesterol value in Lpb5/8 genotype (927.4 112.0, n = 5, P = 0.128, Kruskal-Wallis test) and the lowest mean cholesterol in pigs with Lpb3/8 and Lpb8/8 genotypes (542.5 56.2, n = 4).
±
Coronary Atherosclerosis: Effect of vWD and Lpb Genotypes and Serum Cholesterol
±
±
There were no significant differences in the three criteria used to describe coronary atherosclerosis between normal and vWD pigs as previously noted when pigs were reported separately (P 0.628, Wilcoxon rank sum test) (Table 4).4 For the three groups of Lpb genotypes, the presence of the Lpb5 allele correlated significantly with the development of the greatest amount of coronary atherosclerosis. This finding was significant for all three criteria (Table 5). Multiple regression methods for ANOVA also were used to analyze relationships between degree of coronary atherosclerosis and combinations of Lpb alleles and genotypes as well as presence or absence of von Willebrand disease. Descriptive statistics for the three groups of Lpb genotypes are given in Table 5. A significantly greater degree of coronary atherosclerosis was present in the coronary arteries of pigs with the Lpb5 allele (ie, group 2) when compared with pigs in group 1 (0.026 P < 0.107, ANOVA) or group 3 (0.002 P < 0.007) (Table 5). The presence of the Lpbl allele in the absence of the Lpb5 allele (ie, group 1) was associated with an intermediate degree of coronary atherosclerosis. These results also suggested that the Lpb3/8 or Lpb8/8 genotypes (ie, group 3) were associated with somewhat lower degrees of coronary atherosclerosis. The data on pigs with these genotypes should be viewed cautiously be-
Morphology of Atherosclerosis
-
Coronary Arteries The morphology of the coronary atherosclerosis found in these normal and vWD pigs has been described in detail.46 In general, examination of the serial cross sections of all three coronary arteries showed that certain areas were uninvolved, others had accumulations of foam cells and smooth muscle cell proliferation with a fibrous cap, and in some sections calcification, necrosis, and hemorrhage were also evident. Aortic Lesions
-
Two types of aortic lesions were evident on gross specimens in both normal and vWD pigs. The aortic arch and thoracic aorta contained flat, translucent lesions as described in previous studies.23 In contrast, the lesions found in the abdominal aorta not only contained flat, translucent lesions but also ones that were raised, pale gray, and fibrous.23 By light microscopy, some regions of the aorta were Table 3.
Group
Lpb Genotype and Diet-induced Hypercholesterolemia Lpb genotypes Genotypes
Lpbl"/'113,1/8 Lpbl15.5/8 Lpb3/8,8/8
1. 2. 3.
-
Serum cholesterol (mg/dl)* n
19 10 4
Mean + SE 777.8 + 43.8 866.1 ± 64.0t 542.5 ± 56.2
Average of mean of monthly values for the groups while receiving the atherogenic diet. t P = 0.0343, Kruskal-Wallis test for cholesterol across the three Groups of Lpb Genotypes. *
Minimum 475 620 432
Maximum 1080 1319 699
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Figure 1. Diet-induced atherosclerosis of abdominal aorta in a von Willebrand disease pig uith Lpb'8genotype (pig number 165, Table 2, dietfedfor 25 weeks). Morphometric anallsis indicated the atheromatous plaque area was 0.938 mmu?' or 6.6% of medial area. The mean values of these measurements for the z'WD pigs in group 1 on Table 7 considered separately was 0.811 + 0.201 mm2 and 8.2 + 2.3% (n 8). For comparison, the mean 'alues of these measurements for normal pigs in group 1 on Table 7 considered separately uwas 1.854 + 0.723 mm' and 20.5 + 8.4% (n = 4). Pigs with the Lpb' allele developed an intermediate amount of coronary and aortic atherosclerosis relative to pigs with other alleles (group 1 on Tables 5 and 7); (A) is a whole mount; (B) is a x4.6 magnification which includes lesion. Aorta opened and immersion fixed Verhoeff-van Gieson. Bar = 1 mm in both (A) and (B). =
cause group 3 only contained four pigs. These differences in coronary atherosclerosis between pigs in groups 1, 2, and 3 were similarly evident in these regression analyses when serum cholesterol was taken into ac-
count. Also with Lpb genotype taken into account, neither serum cholesterol nor presence or absence of vWD was significantly associated with severity of coronary atherosclerosis. When analyzed without adjustment for Lpb ge-
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Figure 2. Diet-induced atherosclerosis of abdominal aorta in a Willebrand disease pig with Lph5 8 genotype (pig number 1 54M, Table 2, dietfedfor 16 weeks). The atheromatous plaque contains lipid-ladet foam cells and afibrous cap encroaching on the lumen ofthe vessel Morphometric analysis indicated intinmal plaque area uwas 4.445 mm-' or 34% of medial area. The mean values of these measurements for the vWD pigs in group 2 on Table 7 considered separately u'as 1.886 ± 0.6738 mm12 and 21.6 ± 9.4% (n = 3). For comparison, the mean values of these measurenmentsfor the normalpigs in group 2 on Table 7 considered separatelly uas 1.478 0.445 mm2 and 15.5 -+- 3. 1% (n = 4); Pigs with the Lpbs allele developed the most severe atherosclerosis (group 2 on Tables 5 and 7); (A) is a whole mount of the cross section; (B) is x3 magnffication uhich includes lesion. Aorta pressure perfused. Methblene blue and basic fuscin, bar = 1 nim in both (A) and (B). ion
notype, serum cholesterol was significantly correlated with the three criteria used to describe coronary atherosclerosis, as has been previously reported4: for A), r = 0.39, P = 0.045; for B), r = 0.31, P = 0.091; for C), r = 0.39, P = 0.035.
Analyses were performed to determine if there was an effect of 1 % versus 2% cholesterol diet and the duration of diet feeding (16 versus 22 to 26 weeks) on the severity of coronary atherosclerosis. Analyses that adjusted for these variables supported the principal conclusions con-
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Figure 3. Diet-induced atherosclerosis of abdominal aorta in a von Willebrand diseasepig uith Lpt' genotype (pig number 86R, Table 2, diet fed for 25 uweeks). Morphometric analysis indicated lesion area uwas 0.0672 mm or 1% of medial area. The mean value of these measurements for the vWD pig in group 2 on Table 7 considered separately was 0.121 mm and 1. 1% (n = 1). For comparison, the mean value of these measurements for the normal pig in group 3 on Table 7 considered separately was 0.1-35 mm2 and 2.1% (n = 1); (A) is a uhole mount; (B) is a x 4 magnification which includes lesion. Pigs in this group ofLpb Genotvpes developed the least severe coronary and aortic atherosclerosis (group .3 on Tables 5 and 7). Aorta opened and immersion fixed. Verhoeff-van Gieson, bar = 1 mm in both (A) and
(B).
cerning Lpb genotypes, as did analyses that did not adjust for these variables (data not shown). To determine the effect of including diet and balloon injury methods of inducing coronary atherosclerosis, analyses were performed that excluded the subset of coronary arteries that were ballooned. There was no change in the association of severity of coronary atherosclerosis with Lpb genotype when the ballooned coronary artery was excluded (data not shown).
Aortic Atherosclerosis: Effect of vWD and Lpb Genotypes and Serum Cholesterol The extent of aortic atherosclerosis, expressed as both the intimal area in square millimeters and the mean intimal area as a percentage of media, tended to be lower in the vWD pigs when compared with normal pigs (Table 6). The differences were not significant considering either
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Table 4. Coronary Atherosclerosis Morphometry in Normal and vWD Swine Normal and vWD swine genotypes
Normal vWD P valuet
Coronary
atherosclerosis
morphometry
n
Percent luminal narrowing (A)
Number CACS 15% (B)
Percent CACS ,15% (C)
14 13
14.44 ± 2.68% 13.97 ± 3.22% 0.628
4.14 ± 1.01 3.92 ± 0.93
34.05 ± 7.91% 33.90 ± 8.95%
0.752
0.950
Summary statistics of the coronary atherosclerosis morphometry are reported for the normal and vWD pigs as mean ± SE. The severity of coronary atherosclerosis is reported by three criteria: A) mean percent luminal narrowing by intima by genotype; B) the mean number of coronary artery cross sections (CACS) per genotype with a15% luminal narrowing by intima; and C) the mean percent of the total number of coronary artery cross sections per genotype with 2 15% luminal narrowing by intima. The individual values were calculated for each pig and were averaged, and then the result was averaged with those for all other pigs with normal or vWD genotypes. Coronary atherosclerosis morphometry data and calculations are from the formulas reported in references 4 and 5. t P values are from Wilcoxon rank sum test. *
the entire abdominal aorta or the distal half (P 0.201, Wilcoxon rank sum test, Table 6). For the Lpb genotypes, the correlation of the Lpb5 allele with severity of atherosclerosis approached significance for the entire abdominal aorta (for mean intimal area in square millimeters, r = 0.35, P = 0.120; for intimal area as a percentage of medial area, r = 0.38, P = 0.086). Because most of the raised fibrous plaques appeared in the distal aorta, as has been noted before,23 the distal half of the abdominal aorta was evaluated separately. The Lpb5 allele was significantly correlated with the development of atherosclerosis in the distal half (for mean intimal area in square millimeters, r = 0.450, P = 0.041; and for intimal area as a percent medial area: r = 0.434, P = 0.0495) (Table 7, Figure 2). The degree of diet-induced hypercholesterolemia was significantly correlated with aortic atherosclerosis expressed as intimal area as a percentage of media for both the entire abdominal aorta and the distal half (respectively, r = 0.42, P = 0.058; and r = 0.44, P = 0.045). The same trend was suggested when aortic atherosclerosis was considered as intimal area in square millimeters (for the entire abdominal aorta, r = 0.31, P = 0.165; for the distal half, r = 0.39, P = 0.08). -
Multiple regression analyses for analysis of variance (ANOVA) were used also for both measures of abdominal aortic atherosclerosis to analyze relationships between severity of atherosclerosis and combinations of Lpb alleles and genotypes as well as presence or absence of von Willebrand disease. In addition, these analyses were made with and without adjustment for cholesterol. The correlation of the Lpb5 allele or serum cholesterol with the amount of aortic atherosclerosis in these analyses tended to be significant when considered separately (P < 0.05, Wilcoxon rank sum test) or at least suggested trends. With adjustment for serum cholesterol, however, differences in the degree of atherosclerosis between Lpb genotypes were no longer significant (P 3 0.15, ANOVA); these differences were also not significant when there was adjustment for 1% versus 2% cholesterol diet and duration of diet feeding (16 versus 22 to 26 weeks) (data not shown). Similarly serum cholesterol was no longer significant with adjustment for Lpb status. These latter findings could be mainly due to the small sample size for analysis of aortic atherosclerosis. Importantly, comparisons of severity of aortic atherosclerosis based on the absence or presence of von Willebrand disease were clearly not significant (P 3 0.15, Wilcoxon rank sum test).
Table 5. Lpb Genotype and Coronary Atherosclerosis Morphometry Lpb genotypes Coronary atherosclerosis morphometry* n Group Genotypes Percent luminal narrowing (A) Number CACS >15% (B) Percent CACS a15% (C) it Lpbl/1"1/3,1/8 14 12.9 + 2.4% 3.5 t 0.8 31.5 ± 8.1%
2t
Lpbl/5t5t./8
9
3t
Lpb3/8a8/8
4
(0.1, 29.4)
(0,8)
21.6 t 3.3% (9.0, 39.7)
6.4 t 1.1 (2, 13)
(0, 4.8)
0.3 ± 0.3 (0, 1)
2.3 ± 1.2%
(0,100) 50.8 t 8.5% (13.3, 92.9) 2.1 t 2.1%
(0, 8.3)
Summary statistics of the severity of coronary atherosclerosis are reported for the three groups of Lpb genotypes as mean ± SE with min and max values listed below in parenthesis. Coronary atherosclerosis is reported for each group with three criteria as defined in Table 4 and
in Materials and Methods. The individual values were calculated for each pig and were averaged, and then the result was averaged with those for all other pigs in a given group. Calculations are from the formulas reported in references 4 and 5. t 0.002 - P < 0.007 for the three criteria for coronary atherosclerosis for group 2 versus group 3 and 0.026 - P - 0.107 for group 2 versus group 1 in analysis of variance. t The Lpb5 allele correlates with all three criteria for coronary atherosclerosis: for A) r = 0.52, P = 0.005; for B) r = 0.48, P = 0.01 1; and for C) r = 0.46, P s 0.015.
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Table 6. Abdominal Aortic Atherosclerosis Morphomety in Normal and vWD Swine Abdominal aortic atherosclerosis* Normal and vWD swine genotypes Normal vWD P valuet
Entire Intimal area
Distal half Intimal area
n
mm2
% of media
mm2
% of media
9 12
1.495 ± 0.391 1.022 ± 0.260 0.320
16.24 ± 4.16% 10.99 ± 3.16% 0.227
2.358 ± 0.569 1.510 ± 0.436 0.201
25.09 ± 6.46 16.14 ± 4.98 0.256
* Aortic atherosclerosis was defined as the mean intimal area expressed as percent of media and the mean intimal area in mm2 for 10-12 sequential sections per pig for both the entire abdominal aorta and the distal half (see Materials and Methods, "Aortic Atherosclerosis"). The values were calculated for each pig and were averaged, and then the result was averaged with those for all other pigs in a given group. The summary statistics are listed as mean ± SE. t P values are from Wilcoxon rank sum test.
Discussion The relationship between vWD genotype, polymorphism in Lpb genotype, and diet-induced atherosclerosis and hypercholesterolemia was studied in normal and vWD pigs. Four different Lpb alleles were identified in the these pigs: Lpbs 1, 3, 5, and 8. The presence of the Lpb5 allele correlated significantly with development of greatest amounts of coronary and aortic atherosclerosis. The Lpb5 allele occurs frequently in populations of pigs of various breeds and has been associated with atherosclerosis.12-14 In contrast, the Lpbl allele is rare and untested in studies of atherosclerosis.14 In the normal and vWD pigs, the presence of the Lpbl allele correlated with an intermediate amount of disease relative to the other alleles. Pigs with Lpb3/8 or Lpb8/8 genotypes developed the least amount of atherosclerosis. Although pigs with the Lpb5 allele tended to have higher cholesterol levels, this relationship was not as strong as that for atherosclerosis. Importantly, vWD genotype did not influence the development of either hypercholesterolemia or coronary or aortic atherosclerosis. These results suggest that apolipoprotein B100 genotype is a major factor in determining the severity of coronary and aortic atherosclerosis that develops in this porcine model.
For simplicity, the atherosclerosis and cholesterol data in the seven different genotypes found in the normal and vWD pigs were presented in three groups. It is important to note, however, that the pigs with the Lpb5 allele had the most amount of coronary and aortic atherosclerosis when analyzed before genotype grouping. In addition, these pigs developed higher cholesterol levels, particularly ones with the Lpb5/8 genotypes, which developed the highest cholesterol levels and the greatest amount of coronary atherosclerosis. This analysis then identified pigs with Lpb3/8 and Lpb8/8 genotypes as having the least amount of atherosclerosis and lowest serum cholesterol levels. All other pigs were in between these two extremes and shared the Lpbl allele. Although other groupings may be possible, the three Lpb groups were identified by having mild, moderate, or severe disease when each pig was considered individually. Previous studies with varying protocols have found that the Lpb5 allele is associated with diet-induced hypercholesterolemia and aortic atherosclerosis.14 Pigs with the Lpb5 allele fed a cholesterol-free, high-fat diet developed both higher mean serum cholesterol levels and aortic intimal lipidosis than pigs with the Lpb8 allele. In a separate study, Thompson miniature swine with the Lpb5 allele developed hypercholesterolemia when fed a
Table 7. Lpb Genotype and Abdominal Aortic Atherosclerosis Morphomety Abdominal aortic atherosclerosis* Entire Distal half Intimal area Lpb genotypes Intimal area n mm2 % media mm2 % media Group genotypes 1. 12 Lpbl /11/3l1/8 1.159 ± .0294 12.3 ± 3.4% 1.589 ± 0.438 17.9 ± 5.4% (0.049, 3.771) (0.8, 41.9) (0.085, 5.616) (1.4, 65.9) 7 2. Lpbl/5t.5t/8 1.65 ± 0.375 18.1 ± 4.1% 2.826 ± 0.574t 28.5 t 6.4%t (0.442, 2.877) (4.8, 37.3) (0.706, 4.768) (6.5, 58.0) 2 3. 0.128 ± 0.007 1.6 + 0.5% 0.195 t 0.47 2.6 t 0.3% Lpb3/8 (0.121, 0.135) (1.1, 2.1) (0.147, 0.242) (2.3, 2.9) Aortic atherosclerosis is reported as defined in the legend to Table 6. The summary statistics are listed as mean + SE with min and max values listed below in parenthesis. t The Lpb5 allele correlates with the degree of aortic atherosclerosis in the distal half of the abdominal aorta (for mean intimal area in mm2, r = 0.450, P = 0.041; and for intimal area as a percent medial area: r = 0.434, P = 0.0495).
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2% cholesterol, 25% fat diet, whereas pigs with the Lpb3/3 genotype were nonresponders.12 No data on the degree of hypercholesterolemia or atherosclerosis found in these animals were reported. The present study confirms and extends these associations of Lpb genotypes and degree of atherosclerosis to normal and vWD male pigs fed a 1% to 2% cholesterol, high-fat diet. Pigs with the Lpb5 allele not only developed the most severe aortic atherosclerosis and highest cholesterol levels but also the most severe coronary atherosclerosis. In addition, the normal and vWD pigs with the Lpb3/8 and Lpb8/8 genotypes developed the smallest rise in serum cholesterol and the least amount of coronary and aortic atherosclerosis. It should be noted that this group contained only four pigs: one received 2% cholesterol in the diet for 16 weeks and three received 1% or 2% cholesterol for 22 to 25 weeks. The influence of Lpb genotype on diet-induced atherosclerosis is further supported by the additional new finding of an intermediate amount of disease in pigs with the rare Lpb1 allele. These findings have identified groups of normal and vWD pigs with specific Lpb genotypes suited for further study of graded susceptibility to diet-induced atherosclerosis. How different Lpb genotypes might influence lipid metabolism and atherogenesis is understood in part. One strain of pigs with a mutant Lpb5 allele phenotypically exhibits spontaneous hypercholesterolemia and accelerated atherosclerosis.2325 These Lpb5 mutant pigs have normal LDL receptor binding activity but have abnormal low density lipoproteins and defective LDL catabolism, resulting in hypercholesterolemia even while receiving a cholesterol-free diet.23 24 Also a direct relationship exists between the mutant Lpb5 genotype and the concentration and composition of LDL subfractions.26 Although there appears to be considerable homology between human and porcine LDL molecules at the proposed LDL receptor binding sites, LDL from pigs with Lpb2 and Lpb4 alleles has a higher affinity than human LDL for both the pig and human receptor.27 Interestingly the LDL receptor binding regions of alleles Lpb2,3,7 and mutant Lpb5 do not have major differences by DNA sequence analysis.28 The differences in the phenotypic expression of these alleles, then, may result from differences outside of the LDL receptor binding region. These findings are also consistent with the observation in this present study that the correlation of Lpb5 allele with amount of atherosclerosis was greater than that found for degree of hypercholesterolemia. These studies indicate, then, that the polymorphism at the apo-B locus exerts a strong influence on lipid metabolism and atherogenesis in pigs. It is possible that the various Lpb genotypes produce important differences in cholesterol distribution among different lipoproteins, lipid particle size, or lipoprotein
clearance, similar to what has been found in the spontaneously hypercholesterolemic mutant Lpb5 pig.242629 Such differences would not have been detected by measurement of total cholesterol as reported in this study. Further study is needed to document whether or not the Lpb 1, 3, 5, and 8 alleles alter specific aspects of lipoprotein metabolism and atherogenesis in this model of dietinduced atherosclerosis. Lpb genotypes as determinants of the severity of coronary and aortic atherosclerosis may provide insight into the wide range of differences in the degree of atherosclerosis noted in the earlier studies.1'- In this study, vWD pigs with severe hypercholesterolemia, male gender, and especially the Lpb5 allele developed the most severe coronary and aortic atherosclerosis, which was not significantly different from normal pigs that also had the Lpb5 allele. With different Lpb genotypes, both normal and vWD pigs still developed comparable degrees of coronary and aortic atherosclerosis but at graded levels of severity. It is clear that polymorphism in Lpb genotypes could have affected the results of the atherogenesis experiments in the vWD stock, confusing the interpretation of the results either to cause apparent enhancement of the perceived effect of vWF or to mask a more important effect of vWF. It seems likely that any effect of vWF on the development of atherosclerosis is part of a complex array of variables affecting atherogenesis, including the important contribution of lipids and lipoproteins. A mechanism linking vWF with atherogenesis would most likely involve support by vWF of platelet adhesion and activation on arterial walls. Von Willebrand factor functions as a ligand binding platelets to exposed subendothelium, particularly under high shear conditions3033 and in the development of occlusive arterial thrombosis.6.3.3 Platelets thus attached to the vessel wall and activated could release platelet-derived growth factor (PDGF) and thereby promote atherogenesis.3536 It is probable that PDGF can be delivered to the vessel wall by a vWF-independent fashion or that other in vivo factors play a significant role in the proliferative response of smooth muscle cells to injury.37-42 Data from our laboratory have shown that both pseudopod formation and spread of platelets adhering to injured arterial walls is impaired in normocholesterolemic, vWD pigs.543 Interestingly the number of adherent platelets in these injured coronary arteries is not significantly influenced by the presence or absence of vWF.543 Reddick et al, however, have recently found that platelets adhering to ulcerated or damaged coronary artery plaques in vWD pigs fed a high-cholesterol diet are spread or activated to 73% of the degree found in pigs with normal levels of vWF.5 This raises the possibility that the Lpb5 allele leads to a greater uptake of lipid by the vessel wall and that the lipid-rich vessel wall enhances platelet activation by a mechanism
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that bypasses the need for vWF as demonstrated by Reddick et al.5 Such a series of interactions would explain to some degree the loss of protection from atherosclerosis in vWD pigs when Lpb5 and hypercholesterolemia are present.
11.
12.
Acknowledgments The authors thank Mr. Jeff Sigman for excellent technical assistance and Ms. Robin Raymer, manager of the Francis Owen Blood Research Laboratory.
References 1. Brinkhous KM, Reddick RL, Griggs TR: Arterial thrombosis, atherosclerosis, and the factor VlIl/vWF complex, The Role of Factor VIII and von Willebrand Factor. Edited by TS Zimmerman, ZN Ruggeri. New York, Marcel Dekker, 1989, pp 283-303 2. Griggs TR, Reddick RL, Sultzer D, Brinkhous KM: Susceptibility to atherosclerosis in aortas and coronary arteries of swine with von Willebrand's disease. Am J Pathol 1981, 102:137-145 3. Sultzer DL, Brinkhous KM, Reddick RL, Griggs TR: Effects of carbon monoxide on atherogenesis in normal pigs and pigs with von Willebrand's disease. Atherosclerosis 1982,
43:303-319 4. Griggs TR, Bauman RW, Reddick RL, Read MS, Koch GG, Lamb MA: Development of coronary atherosclerosis in swine with severe hypercholesterolemia. Lack of influence of von Willebrand factor or acute intimal injury. Arteriosclerosis 1986, 6:155-165 5. Reddick RL, Read MS, Brinkhous KM, Bellinger DA, Nichols TC, Griggs TR: Coronary atherosclerosis in the pig: Induced-plaque injury and platelet response. Arteriosclerosis
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1990,10:541-550 6. Nichols TC, Bellinger DA, Tate DA, Reddick RL, Read MS, Koch GG, Brinkhous KM, Griggs TR: von Willebrand factor and occlusive arterial thrombosis: A study in normal and von Willebrand's disease pigs with diet-induced hypercholesterolemia and atherosclerosis. Arteriosclerosis 1990, 10:449461 7. Bowie EJW, Fuster V, Owen CA Jr, Brown AL: Resistance to the development of spontaneous atherosclerosis in pigs with von Willebrand's disease. Thromb Diathes Haemorrh 1975, 34:599 8. Fuster V, Bowie EJW, Lewis JC, Fass DN, Owen Jr CA, Brown AL: Resistance to arteriosclerosis in pigs with von Willebrand's disease. J Clin Invest 1978, 61:722-730 9. FusterV, Fass DN, Kaye MP, Josa M, Zinsmeister AR, Bowie EJW: Arteriosclerosis in normal and von Willebrand pigs: Long-term prospective study and aortic transplantation study. Circ Res 1982, 51:587-593 10. Badimon L, Steele P, Badimon JJ, Bowie EJW, Fuster V: Aortic atherosclerosis in pigs with heterozygous and von Willebrand disease. Comparison with homozygous von
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