Production of arteriosclerosis by homocysteinemia.

12 Pages·2007·1.49 MB·English

The American Journal of
PATHOLOGY
October 1970 * Volume 61, Number 1
Production of Arteriosclerosis by Homocysteinemia
Kilmer S. McCully, MD and Bruce D. Ragsdale, MD
ACCELERATED kRTERIosciLERosis was discovered to be asso-
ciated with homocysteinemia in two individuals with different enzy-
matic disorders of sulfur amino acid metabolism.1 Cells cultured from
the skin of individuals with one of these disorders, cvstathionine syn-
thetase deficiency, were found to produce an abnormal proteoglycan
substance that is granular, aggregated and flocculent; addition of homo-
cysteine to cultures ofnormal skin cells resulted in replacement of some
of the normal fibrillar proteoglycans by the same granular substance.2
These findings suggested that homocysteinemia produced accelerated
arteriosclerosis in the children with enzyme deficiency by altering the
normal fibrillar structure of arterial wall proteoglycan molecules and
that a similar process may occur in individuals without enzyme deficien-
cies.
In experiments designed to test this hypothesis, homocysteinemia
was produced in rabbits, and the resulting vascular lesions, to be de-
scribed in this report, were found to reproduce the essential features of
vascular lesions found in individuals with homocysteinemia1.3 and those
found in individuals with early arteriosclerosis. Mforeover, addition of
cholesterol to the diet of the homocvsteinemic rabbits resulted in de-
position of lipid in the aortic lesions, suggesting that lipid accumula-
tion is a secondarv complication of the primanr vascular alteration.
Supported by Grant 924 from the Greater Boston Chapter of the Massachusetts Heart
Association and Grant CA-11119 from the US Public Health Service.
From the Department of Pathology, Harvard Medical School, and the James Homer
W'right Pathology Laboratories, Massachusetts General Hospital, Boston, Mass.
Acceptedforpublication June 18, 1970.
Address for reprint requests: Dr. Kilmer S. McCullv, Department of Pathology, The
Massachusetts General Hospital, Boston, Mass 02114.
2 McCULLY AND RAGSDALE American Journal
of Pathology
Since the only significant source of homocysteine in the human diet is
the methionine content of protein, the demonstration that experimental
homocysteinemia produces arteriosclerotic plaques suggests that diets
containing high concentrations of methionine have the potential for
producing arteriosclerosis in man. The molecular interactions respon-
sible for the histopathologic features of the homocysteine-produced le-
sions, the relationship of the homocysteine theory to other factors in
human arteriosclerosis, and dietary recommendations for the preven-
tion of human arteriosclerosis are discussed.
Materials and Methods
Production of Homocysteinemia
Thirty milligrams of dl-homocysteine thiolactone (Calbiochem), 1 mg/ml in
5% dextrose-water, were administered subcutaneously with 1.0 ,uCi of carboxyl-14C
1-homocysteine thiolactone (Calbiochem) to a 3-kg New Zealand white rabbit.
Serum samples taken at 7 hr and 12 hr were deproteinized with sulfosalicylic acid
and chromatographed on a 60-cm sulfonated polystyrene resin column, Aminex
A-6 (Bio-Rad), at 50 C, pH 3.25, pH 4.25 and pH 7.0.4 Aliquots of the fractions
were counted in a Packard liquid scintillation counter using Bray's solution, and
the concentration of dl-homocysteine thiolactone in serum was found to be 1.54
,umole/ml at 7 hr, 0.74 ;pmole/ml at 12 hr and 0.00 ,umole/ml at 24 hr.
Dietary Maintenance and HomocysteineAdministration
Seven yearling (2.5-3.0 kg) and 17 weanling (1.0-1.5 kg) New Zealand white
rabbits were studied. Four yearlings were fed rabbit chow (Agway) and injected
subcutaneously with 10 mg/kg dl-homocysteine thiolactone, 1 mg/ml in 5%
dextrose-water, every 12 hr for 20 days (Group I), and 3 control yearlings were
injected with 5% dextrose-water on the same schedule (Group II).
Three each of the weanlings were placed on various diets and injected sub-
cutaneously with 25 mg/kg dl-homocysteine thiolactone, 1 mg/ml in 5% dextrose-
water (D/W) every 24 hr for 35 days, according to the following schedule:
Group Diet
III 5% D/W, rabbit chow
IV homocysteine thiolactone, rabbit chow
V 5%D/W, rabbitchow + 2%cholesterol
VI homocysteinethiolactone, rabbitchow + 2%cholesterol
VII 5%D/W, pyridoxine-deficient diet (2animals only)
VIII homocysteinethiolactone, pyridoxine-deficient diet
The diets contained the following concentrations of methionine: rabbit chow
(Agway), 0.29% and pyridoxine-deficient diet (Nutritional Biochem), 0.56%.
Preparation ofTissues for Microscopy
The animals were killed by air embolism; heart, lungs, liver, kidneys, aorta, and
brain were examined, and samples of each were fixed in 10% formalin with 0.5%
cetyl pyridinium chloride for light microscopy or buffered glutaraldehyde for elec-
Vol. 61, No. 1 ARTERIOSCLEROSIS AND HOMOCYSTEINEMIA 3
October 1970
tron microscopy. The paraffin-embedded sections of aortas and the major organs
were stained with H&E, Verhoeff-Van Gieson, toluidine blue and PAS. Frozen sec-
tions of aorta were stained with Sudan, and selected samples of aorta were em-
bedded in Epon and examinedbvelectron microscopy.
Results
The aortas of all 13 of the animals injected with homocvsteine were
moderatelv thickened, compared to the controls. Focal elevated tan
circular granular plaques were found in the arch of the aorta and
adjacent to the ostia of major branches in 5 of 13 of the homocvsteine-
injected animals. The aortas of all of the animals on cholesterol and
homocvsteine (VI) thickened, and yellow-tan. Small
were opaque,
intimal lipid streaks were found in the aortas in only two animals, one
each from Group IV (homocysteine) and Group VI (cholesterol and
homocysteine). One of the yearling controls (II) had chronic
numerous
calcified and ossified plaques in the arch of the aorta. One of the wean-
ling controls (III), of the animals cholesterol alone (V), and
one on
of the pyridoxine-deficient animals (VII) had earlv granular
one
plaques in the arch of the aorta.
The distribution of plaques found bv microscopy in the aortas and
arteries of other organs in the various groups is shown in Table 1. All
the aortas of the homocysteine groups and the pyridoxine-deficient
group were found to have early fibrous arteriosclerotic plaques, both
in the arch and in the abdominal aorta. Fibrous plaques were also found
in the arteries of the major organs of 12 of 13 animals injected with
Table 1. Production ofArteriosclerotic Plaques by Homocysteinemia
Aortic plaques
Arterial plaques
Group Conditions Fibrous Fibrolipid Chronic in majororgans
I Homocysteine 4/4 0/4 0/4 4/4
11 Control 0/3 0/3 1/3 0/3
111 Control 1/3 0/3 0/3 1/3
IV Homocysteine 3/3 0/3 0/3 2/3
V 2% cholesterol 1/3 1/3 0/3 0/3
VI 2%cholesterol + 3/3 3/3 0/3 3/3
homocysteine
VII Pyridoxine-deficient 2/2 0/2 0/2 1/2
VilI Pyridoxine-deficient 3/3 0/3 0/3 3/3
+ homocysteine
The number of animals in each group with arteriosclerotic plaques identified micro-
scopically in sections oftheaorta and otherorgans is listed with thetotal numberin each
group. Groups I and 11 wereyearlings (2.5-3.0 kg) injected twice daily with a total dose of
20 mg/kg/day homocysteine thiolactone for 20 days, and Groups III-VIII were weanlings
(1.0-1.5 kg) injected dailywith 25 mg/kg/day homocysteine thiolactone for35 days.
4 McCULLY AND RAGSDALE American Journal
of Pathology
homocysteine and 1 of the 2 pyridoxine-deficient animals. The plaques
were formed by focal fragmentation, splitting and destruction of the
internal elastic membrane of peripheral arteries (Fig 1 and 2) and the
inner elastic lamellae of the aorta (Fig 3) with associated hyperplasia
of adjacent myointimal cells, some of which were in mitosis. Many of
the hyperplastic myointimal cells had abundant vacuolated cytoplasm
containing granular or fibrillar metachromatic substance (Fig 3 and 4).
The wall of the aorta was thickened by small pools of metachromatic
substance between elastic lamellae (Fig 4) and by increased numbers
of fine metachromatic fibrils closely associated with elastic fibers
throughout the wall of the aorta. The most marked splitting of elastic
tissue and cellular hyperplasia were found in the intima of peripheral
arteries and in the inner layers of the aortic intima and media. In-
creased numbers of fine irregular collagen fibers were identified by
Van Gieson staining and by electron microscopy within the inter-
lamellar spaces and within the intimal plaques both in aorta and in
the peripheral arteries.
Fibrolipid plaques were found in the aortas of all the animals on
cholesterol and homocysteine; in these aortas many small fat droplets
were found deposited in the interlamellar space, within the cytoplasm
of myointimal cells, and extracellularly oriented along elastic fibers,
in a distribution very similar to that of the metachromatic substance
(Fig 5). Fibrolipid plaques were found in the aorta of 1 of the 3
animals on cholesterol alone. A few fine fat droplets were found in
the cytoplasm of the myointimal cells of the aortas of animals on
homocysteine alone.
Earlyfibrous plaques were found in only one aorta from the 6 animals
in the control groups, and only 1 of the 9 animals in the control and
cholesterol groups had scattered fibrous plaques in the arteries of the
major organs. One aorta from the yearling control group (II) had
chronic calcified plaques, but none of the other animals had similar
lesions.
Discussion and Conclusions
The demonstration that homocysteinemia rapidly produces wide-
spread arteriosclerotic plaques in rabbits confirms experimentally the
previous conclusion that an elevated concentration of homocysteine
or a derivative of homocysteine was the factor responsible for acclerated
arteriosclerosis in the children with different enzymatic disorders of
sulfur amino acid metabolism.' The experimental lesions reproduce the
essential features of the vascular pathology associated with homocys-
Vol. 61, No. 1 ARTERIOSCLEROSIS AND HOMOCYSTEINEMIA 5
October 1970
teinemia.1- Furthermore, the distribution of the experimental lesions,
located in the arch and abdominal aorta, adjacent to ostia of major
arterial branches and within coronarn branches and other arteries, as
well as the histopathologic changes, are characteristic of the early-
fibrous plaques encountered in human arteriosclerosis.
Homoc,vsteine is produced by demethvlation of methionine, an es-
sential amino acid derived from dietary proteins: no other source of
homocvsteine has vet been identified. The dose of homoevsteine used
in these experiments is comparable to the recommended daily intake
of methionine for human adults. The activity of cvstathionine svnthe-
tase, which catalyzes the conversion of homocvsteine and serine to
cvstathionine, is dependent upon pvridoxal phosphate; pvridoxine de-
ficiencv- in rabbits results in exeretion of homocv-stinej presumably by
reducing the activity of cvstathionine svnthetase. The present finding
of fibrous arteriosclerotic plaques in pyTidoxine-deficient rabbits con-
firms the original observation in primates,6 and the findings in the
homocvsteine-injected animals suggest that homocvsteinemia was the
metabolic abnormality that produced the lesions in the experiments
using primates. The presence of scattered fibrous plaques in several
of the control animals may be explained by assuming that an increased
amount of homocvsteine is formed from the relativelv high methionine
content of the standard rabbit chow (0.29rf), compared to the amount
of homocvsteine formed from the low methionine content of the strict
vegetarian dietthat wvild rabbits normally consume.
Animal proteins are relatively abundant in methionine compared to
plant proteins, and many experimental atherogenic diets contain high
concentrations of methionine as well as cholesterol and other lipids.
The present experiments, demonstrating that homocvsteinemia pro-
duces arteriosclerotic plaques in rabbits, suggest that consumption of
foods rich in methionine can lead to arteriosclerosis in experimental
animals and in normal human adults, presumably- by increased homo-
cv-steine synthesis and the consequent production of abnormal sulfated
proteoglvcans in arterial walls.2 This interpretation of the dietary origin
of arteriosclerosis correlates very well with the data on consumption
of methionine-rich foods by various socioeconomic groups wvith a high
incidence of cardiovascular disease, and "significant atherosclerosis is
rare in peoples whose diet over the life span is predominantl1 vege-
tarian . . .n Dietary methionine restriction has been shown to result
in a decreased concentration of homocvstine in the plasma and urine
of children with cvstathionine svnthetase deficiencv,v but no infomia-
tion is available on whether methionine restriction reduces the inci-
6 McCULLY AND RAGSDALE American Journal
of Pathology
dence or severity of arteriosclerosis, either in these homocystinuric
childrenorinnormalhumanpopulations.
In these relatively short-term experiments, cholesterol feeding re-
sulted in lipid deposition in the fibrous plaques produced by homo-
cysteine. Fine lipid droplets were found in the same distribution as
the increased metachromatic substance produced by homocysteinemia,
suggesting that hypercholesterolemia produced by dietary cholesterol
increased the amount of binding of serum lipoprotein molecules to
aortic sulfated proteoglyeans. From the results of these experiments,
the variability in the correlation between hyperlipidemia and experi.-
mental or clinical atherosclerosis may be explained by assuming varia-
tion in the degree of vascular damage produced by homocysteine be-
cause of differences in dietary methionine consumption or differences
in the rates of methionine and homocysteine metabolism in the in-
dividual. Thus, the susceptibility to lipid deposition may depend not
only upon the plasma lipid concentration and the rate of plasma flow
through the vessel wall but upon the degree of vascular damage pro-
ducedby homocysteine.
Since homocysteine leads to the production of abnormal proteo-
glycans by cultured cells, probably by altering the sulfate content of the
carbohydrate envelope of the molecule,2 the histopathology of the le-
sions produced in the arteries in these experiments can be interpreted,
in part at least, on a molecular level. The accumulation of sulfate-rich
proteoglyeans, appearing as metachromatic substance in close asso-
ciation with elastic fibers (Fig 3 and 4), presumably leads to frag-
mentation and splitting of the elastic fiber structure by ionic binding
between proteoglyean sulfate groups and the E-amino groups of the
desmosine and isodesmosine moieties necessary for normal cross link-
ing of elastin fibers.9 The accumulation of fine lipid droplets in close
association with metachromatic substance of the aortic wall (Fig 5),
probably comprising filtered serum lipoprotein molecules,10 presumably
results from the ionic binding between proteoglycan sulfate groups
and the quartenary ammonium group of lecithin and sphingomyelin,
which are important constituents of serum lipoprotein. The formation
of increased numbers of fine collagen fibrils in close association with
metachromatic substance in the aortic wall suggests that the abnormal
sulfated proteoglyean produced by homocysteine administration in-
creased the deposition of collagen. Purified sulfated proteoglycan pre-
parations have been observed to increase the rate of polymerization
of purified tropocollagen in vitro.1" The calcification observed in hu-
man lesions associated with homocysteinemia 1 or in chronic fibrous
Vol. 61, No. 1 ARTERIOSCLEROSIS AND HOMOCYSTEINEMIA 7
October 1970
arteriosclerotic plaques may result in part from ionic binding betw"een
calcium ion and the excess sulfate groups of proteoglvcan molecules.
The observation of cellular hyperplasia in the fibrous plaques in these
experiments may be related to a change in contact inhibition of
mvointimal cells resulting from the production of abnormal sulfated
proteoglvcan. Homocvsteinemia would be expected to affect the struc-
ture of sulfated proteoglycans produced bv cells of other tissues than
vessel wall, possiblv explaining increased platelet stickiness in homo-
cvsteinemia 5.12,13 and the predisposition for vascular lesions to be-
come complicated by thrombosis.3
The homocvsteine theorv of the pathogenesis of arteriosclerosis does
not preclude the importance of other factors that influence the de-
velopment of the disease.4 Mfanv of these phvsiologic, pathologic and
biochemical factors mav be shown bv future work to be explained by
their influence on homocvsteine metabolism and the consequent in-
fluence upon the sulfated proteoglvcan molecules of vessel walls.
Summary
The association of accelerated arteriosclerosis with homocvsteinemia
in individuals with different enzymatic disorders of sulfur amino acid
metabolism and the production of abnormal sulfated proteoglvcans by
cultured human skin cells exposed to homocysteine suggested that simi-
lar vascular lesions would be found in animals with experimental homo-
cvsteinemia. Rabbits were injected for 3-5 weeks with a physiologic
dose of homocysteine thiolactone, and the resulting arteriosclerotic
lesions were found to reproduce the essential features of the vascular
lesions found in individuals with homocvsteinemia and in individuals
with early arteriosclerosis. Addition of cholesterol to the diet of the
homocysteinemic rabbits resulted in deposition of lipid in the aortic
lesions, suggesting that lipid accumulation is a secondarv complication
of the primary vascular alteration produced bv homocvsteinemia. The
molecular interactions resulting in the elastic tissue damage, lipid dep-
osition, myointimal cell hvperplasia, collagen deposition and calcifica-
tion found in the homocysteine-produced lesions are related to the
accumulation of sulfated proteoglvcan molecules within the arterial
wall. Since the onlv significant source of homocvsteine in the human
diet is the methionine content of protein, the findings explain the
known correlation of human arteriosclerosis with the consumption of
methionine-rich foods and suggest that dietary methionine restriction
mav prevent human arteriosclerosis.
8 McCULLY AND RAGSDALE American Journal
of Pathology
References
1. McCully, KS: Vascular pathology of homocysteinemia: Implications for
the pathogenesis of arteriosclerosis. Amer J Path 56:111-128, 1969
2. McCully KS: Importance of homocysteine-induced abnormalities of pro-
teoglycan structure in arteriosclerosis. Amer J Path 59:181-193, 1970
3. Gibson JB, Carson NAJ, Neill DW: Pathological findings in homocystinuria.
J Clin Path 17:427-437, 1964
4. Moore S, Spackman DH, Stein WH: Chromatography of amino acids on
sulfonated polystyrene resins. Anal Chem 30:1185-1206, 1958
5. Barber GW, Spaeth GL: The successful treatment of homocystinuria with
pyridoxine. J Pediat 75:463-478, 1969
6. Rinehart JF, Greenberg LD: Arteriosclerotic lesions in pyridoxine-deficient
monkeys. AmerJ Path 25:481-491, 1949
7. Katz LN, Stamler J, Pick R: Nutrition and Atherosclerosis. Philadelphia,
Leaand Febiger, 1958, p20
8. Perry TL, Hansen S, Love DL, Crawford LE, Tischler B: Treatment of
homocystinuria with a low methionine diet, supplemental cystine, and a
methyldonor. Lancet 1:474-478, 1968
9. Thomas J, Elsden DF, Partridge SM: Partial structure of two major degrada-
tion products from the cross-linkages in elastin. Nature 200:651-652, 1963
10. Walton KW, Williamson N: Histological and immunofluorescent studies on
the evolution of the human atheromatous plaque. J Atheroscler Res 8:599-
624, 1968
11. Mathews MB: The interaction of collagen and acid mucopolysaccharides:
A model for connective tissue. Biochem J 96:710-716, 1965
12. McDonald L, Bray C, Field C, Love F, Davies B: Homocystinuria, throm-
bosis and the blood-platelets. Lancet 1:745-746, 1964
13. Cusworth DC, Dent CE: Homocystinuria. Brit Med Bull 25:42-47, 1969
14. Sandler M, Bourne GH: Atherosclerosis and its Origins. New York, Aca-
demic Press, 1963
Dr. J. B. Caulfield is gratefully acknowledged for examination of the aortas by elec-
tron microscopy. The excellent technical assistance of R. Corrigan is also acknowledged.
A preliminary summary of this work was presented at the meeting of the American
Society for Experimental Pathology held in Atlantic City, NJ on April 14, 1970.
:~~~~~~~~~~~~~~~~~~~~~~~~~~~
=.e-. ~m'
L~wS_AM
Fg 1. Branch of coronary artery of homocysteine-treated yearling is narrowed
by intimal plaque formed by fragmentation and splitting of internal elastic mem-
brane with associated hyperplasia of myointimal cells. Verhoeff-Van Gieson.
x 160.
Fig 2. Small intimal plaque in medium-size artery of homocysteine-treated year-
ling is formed by splitting of intemal elastic membrane and hyperplasia of
myointimal cells. Verhoeff-Van Gieson. x 750.
Fig 3. Fibrous intimal plaque in thoracic aorta of homocysteine-treated yearling
contains fragmented elastic fibers and hyperplastic myointimal cells containing
granular and fibrillar metachromatic substance in cytoplasm. Toluidine blue.
X 750.
Fig 4. Thoracic aorta of homocysteine-treated yearling is thickened by pools of
granular and fibrillar metachromatic substance lying between elastic lamellae.
Toluidine blue. x 750.