Tohoku J. exp. Med.

, 1979, 129, 9-16

Vitamin D-Induced Coronary Atherosclerosis

in Normolipemic Swine : Comparison with
Human Disease

SEIYA TAURA, MICHIKO TAURA, AKINORI KAMIO* and FRED

A. KUMMEROW•õ

Harlan E. Moore Heart Research Foundation Champaign, IL

61820, USA, *Second Department of Pathology, School of

Medicine, University of Fukuoka, Fukuoka 814, and •õthe

Burnsides Research Laboratory, University of Illinois, Urbana,

IL 61801, USA

TAURA, S., TAURA, M., KAMIO, A. and KUMMEROW, F.A. Vitamin D-

Induced Coronary Atherosclerosis in Normolipenic Swine: Comparison to Human
Disease. Tohoku J. exp. Med., 1979, 129 (1), 9-16-Coronary atherosclerosis
developed in normolipemic swine fed a basal ration supplemented with 125,000
IU, 62,500 IU and 31,250 IU of vitamin D,/kg of diet for 3 months and
subsequently only the basal ration for the following 3 months. Lesions consisted
of intimal atheromata and calcified internal elastics and caused luminal narrow-
ing. The incidence of atherosclerotic lesions was proportional to the vitamin
D, doses. The present experimentally induced lesions had many morphological
features resembling those in coronary arteries from human subjects.
atherosclerosis; coronary artery; vitamin D; calcification

A crucial step in studying the pathogenesis of human coronary atherosclerosis

involves the duplication of lesions which resemble those of man. In coronary
atherosclerosis in man, Lansing et al. (1948) have shown that arterial calcification
occurred previous to intimal atheromata formation, and that not a single case of
atherosclerosis was observed without a calcified lesion associated with elastic fibers.
It has been shown that hypervitaminosis D results in severe medial calcification
and fibromuscular intimal thickening in the arteries of experimental animals
(Eisenstein and Zerulois 1964; Hass et al. 1958; Kent et al. 1958).
The current study was undertaken to determine what would happen in the
calcified foci of coronary artery when animals were maintained on a basal diet for
an extended period of time. The calcified foci of artery were induced by
hypervitaminosis D3. By this procedure, we produced coronary atherosclerosis in
normolipemic swine in 3 months after the elimination of vitamin D supplements.
These lesions were comparable to coronary atherosclerosis in man.

Received for publication, August 29, 1978.

9
10 S. Taura et al.

MATERIALS AND METHODS

Forty-six Yorkshire swine (barrows and gilts), approximately 2 months of age, were
used. The animals were divided into four groups. Group 1 (control group) consisted of
ten animals which were fed a basal ration of corn, soybean meal and a vitamin and mineral
premix containing 387 IU of vitamin D3 and 6.3 g of calcium per kg of diet. Five
animals were sacrificed at 5 months of age and the rest after 8 months of age. Group 2 (3
months D3 supplemented group) consisted of fifteen animals (see Table 2) which were
divided into five subgroups and were fed the basal ration supplemented with 125,000 IU,
62,500 IU, 31,250 IU, 6,250 IU and 1,250 IU of vitamin D3 per kg of diet for 3 months,
and then sacrificed at 5 months of age. Group 3 (6 months D3 supplemented group)
consisted of three animals which were fed 125,000 IU vitamin D3 throughout the experi
mental period and sacrificed at 8 months of age. Group 4 (vitamin D3 withdrawal group)
consisted of eighteen animals (see Table 3). They were fed the same amounts of vitamin D3
as the five subgroups of group 2 for the same time period, and subsequently received only
the basal ration for the following 3 months. Animals in group 4 were sacrificed at 8
months of age. Blood samples were taken for all animals except for group 3 at the time of
sacrifice for determination of serum cholesterol and calcium. An average of 30 coronary
artery samples (10 samples each for the three major branches) were taken from each heart for
histological evaluation.

TABLE1. Serum cholesterol and calcium values (mg/100 ml)

Mean•}s.E.M.

TABLE 2. Calcific lesions and complications with intimal plaque in group 2

TABLE 3. Coronary atherosclerotic lesions in group 3

 Coronary Atherosclerosis in Normolipemic Swine 11

Eighty-three samples of human coronary artery were obtained from 15 autopsied

subjects, ranging from 43 to 68 years of age, who died of various causes including cancer,
accidents and myocardial infarction. The coronary artery samples from swine and humans
were fixed in 1.4% glutaraldehyde in phosphate buffer, pH 7.4. For light microscopy,
frozen sections were stained with hematoxylin and oil red 0, and paraffin embedded
sections were stained with Von Kossa, and hematoxylin and eosin. Tissues for electron
microscopy were postfixed in osmium tetroxide, dehydrated in acetone, embedded in epoxy
resin, and sectioned with glass knives on an ultramicrotome. Thick sections were stained
with alkaline-toluidine-blue or Von Kossa. Thin sections were stained with uranyl acetate
and lead citrate and examined on an electron microscope. Unstained thick sections were
used for x-ray microanalysis.

RESULTS

The serum cholesterol and calcium levels of the experimental swine are
summarized in Table 1. The animals of 2 or 3 highest vitamin D3 fed groups in
group 2 had slightly depressed cholesterol levels and abnormally high calcium levels.
No difference in serum cholesterol and calcium levels was observed between group
4 and the control animals.
No abnormal areas of intimal thickening or calcified lesions were demonstrable
in the control group. In group 2, calcified lesions were noted in the coronary
arteries of animals fed 125,000 IU, 62,500 IU and 31,250 IU of vitamin D3 as
shown in Table 2. These calcified lesions were located in the internal elastica and
were frequently accompanied by fibromuscular intimal thickening (Fig. 1). In the
animals fed 6,250 IU and 1,250 IU of vitamin D3, intimal thickening or calcified
lesions were not observed. In group 3, prominent calcified lesions and fibromus
cular intimal thickening were noted in the coronary arteries. The incidence of
calcified lesions in the arteries was 58/98 or 51%. No lipid deposits were present
in the coronary arteries of animals from group 2 or 3.
In group 4, coronary atherosclerosis was produced in all animals initially fed
125,000 IU, 62,500 IU and 31,250 IU of vitamin D3, but in none of the animals
fed 6,250 IU or 1,250 IU of vitamin D3. The incidence of atherosclerotic lesions
was proportional to the dosage of vitamin D3 and more frequently involved the
left anterior descending artery, as shown in Table 3. An atherosclerotic intima
was always observed above the calcified internal elastica (Fig. 2). The size of the
atheromata paralleled the extent of calcification in the internal elastica; the larger
atheromata had more prominent calcified deposits in the internal elastica than
small ones. The largest atheroma was seen in the left anterior descending artery
and had the usual criteria for full-fledged atheromata having abundant lipid,
cholesterol crystals, foam cell formation, necrosis, pools of glycosaminoglycans,
fibroelastosis and calcification. The incidence of calcified lesions in the internal
elastica of samples from group 4 (Table 3) was less than that of group 2 (Table 2),
which received the same doses of vitamin D3. No intramural hemorrhage, ulcera
tion or luminal thrombosis was observed in the arteries, and no evidence of
myocardial infarction was noted in the myocardium.
Electron microscopic observations revealed many foam cells in the athero-
 12 S. Taura et al.

Fig. 1. Swine, 125,000 IU vitamin D3 supplemented group (group 2), uranyl acetate and
lead citrate stains. Fibromuscular intimal thickenning (FIT) and calcified
internal elastica (Ca) are noted. Calcification is restricted in the internal elastica.
x 3600.
Fig. 2. Swine, 125,000 IU vitamin Da withdrawal group, oil red 0 and hematoxylin . Lipid
rich atheromata (A) are located above the calcified internal elastica (Ca) and show
luminal narrowing. X 200.

sclerotic plaques. Extremely dense particles were observed in the extracellular

spaces. They were voluminously aggregated around the internal elastica (Fig . 3).
Microprobe analysis disclosed these dense particles contained calcium and
phosphorus. Similar dense particles were also noted in the macrophages (Fig. 4).
The human subjects varied from 125 to 256 mg/100 ml in serum cholesterol
 Coronary Atherosclerosis in Normolipemic Swine 13

Fig. 3. Swine, 62,500 IU vitamin D, withdrawal group. Electron dense deposits are
noted around the internal elastica (IE). Many foam cells (F) are seen in the intima.
x 3600.
Fig. 4. Swine, 125,000 IU vitamin D, withdrawal group (group 4). Electron dense
particles are noted in the extracellular space and in the macrophage (M). Intracellular
dense particles are accompanied with lysosomal activity (L) in the surroundings. x 9000.
 14 S. Taura et al.

levels ; 71 out of 83 coronary artery specimens contained atherosclerotic lesions.

The severity of atherosclerotic lesions varied from case to case and from specimen
to specimen. Atherosclerotic lesions taken from people who died of myocardial
infarction were always more severe than those of individuals dying from any other
disease, and frequently were complicated with ulceration, intramural hemorrhage
or thrombus formation in the coronary arteries.
All 71 human atherosclerotic specimens contained calcified deposits of various
degrees. In mild to moderate cases calcification was limited to the internal elastica

Fig. 5. Human, 51 years old, Von Kossa and H-E stains. Calcified deposits are noted in
the internal elastica (IE). X 200.
Fig. 6. Human, 65 years old, unstained Epon specimen. Extremely dense particles
(arrows) are noted in the endothelium and intimal cells (arrows). X 3600.
 Coronary Atherosclerosis in Normolipemic Swine 15

(Fig. 5), while in severe cases calcification also involved the intima and media.
Atherosclerotic involvement tended to be severe in the calcified areas. In general,
severe atherosclerotic lesions of humans were more extreme than those in the
experimental animals. But the mild to moderate atherosclerotic lesions in man had
many morphological features which resembled those in the experimental animals.
In such cases, atherosclerotic involvement was only noted above the calcified
internal elastica. Upon electron microscopic examination, extremely dense
particles were noted around the internal elastica and in the foam cells and
endothelium (Fig. 6). Microprobe analysis showed calcium to be contained within
these dense particles.

DISCUSSION

We induced full-fledgedatheromata in the coronary arteries of normolipemic

swine. These atheromata protruded into the lumen and caused luminal narrowing.
Duguid (1954)pointed out that cholesterolfeedingto the rabbit induced dilatation
of the aortic lumen and increased thickness of the aortic wall, but did not produce
luminal narrowing. The luminal narrowing due to atherosclerotic lesion in the
coronary artery has been believed to be the major factor responsiblefor causing
myocardial ischemia (Friedberg 1968; Bulkley and Roberts 1976). Such coronary
arterial lesions have been produced in experimental animals by vitamin D and
cholesterol diets (Bajwa et al. 1971), epinephrine, and thyroxine administration
with cholesterol cotton oil diet (Constantinidies1958), a balloon endothelial cell
denudation procedure, a cholesterol and butter fat diet (Lee and Lee 1975) and
vitamin D, and cholesteroldiets and total thyroidectomy (Shenk et al. 1965). We
noted similar pathological findings in the coronary arteries of swine without
dietary fat additives, mechanical or surgical procedures. To date, no other
investigators have produced coronary atherosclerosisin normolipemic animals.
Recently, several investigators have shown that mechanicalinjury to the aorta
(Bjorkerud and Bondjers 1973; Moore 1973; Woolf et al. 1973) resulted in
atherosclerosisin normolipemicanimals. These authors concluded that prolonged
endothelial damage or thrombus formation may promote atherosclerotic involve
ment. We suggest that the calcifiedinternal elastica may act as another sourceof
arterial injury. The current study verifiedthat the calcifiedfoci were more sensitive
to the atherosclerotic involvement than the uncalcifiedareas. A similar concept
was initially introduced by Constantinidieset al. (1958)in the rabbit aortae under
hypercholesterolemia. Sincethe group 2 or 3 animalsdid not contain atherosclerotic
lesions, the return to a normal cholesterollevel after the withdrawal of D3 might
have been responsible for developmentof atherosclerosisin group 4.
It is well known that the left anterior descendingartery is most frequently and
severely affected by atherosclerosisin man (Friedberg 1968; Bulkley and Roberts
1976). Although the present experimentally induced atherosclerosis showed no
signs of ulcer, intramural hemorrhage or thrombus formation which have been
frequent in the coronary arteries of myocardial infarction, the morphological
 16 S. Taura et al.

features closely resembled those of mild to moderate atherosclerotic cases in man.

In both swine and human, atherosclerotic lesions had the usual criteria for full-
fledged atheroma and luminal narrowings were distributed predominantly in the
left anterior descending artery. Also, atheromata were complicated by a calcified
internal elastica. Finally, calcified deposits were noted in the endothelium, foam
cells and extracellular space. We feel that the experimental model we presented
is useful because the atherosclerotic lesions resembled those in humans and were

produced by simply feeding the vitamin D3 for a short period of time and then
withdrawing it from the diet.

Acknowledgment

A part of this work was presented in the 11th International Congress of Angiology at
Prague, Czechoslovakia, July 2-8,1978. We wish to thank Drs. Ben T. Williams, Fernando
B. Toledo and Sandhya Sarwate, Mercy Hospital, Urbana, IL 61801, for the specimens of
coronary arteries. This study was supported by the NHLI Grant $15504-05, the Wallace
Genetic Foundation, the Illinois Heart Association, the National Dairy Council and the
American Egg Board.

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