Hyperhomocysteinemia
causing atherogenesis in rabbits' aorta. An experimental model
(Portuguese
PDF version)
Henrique
Jorge Stahlke Júnior1, Luís Henrique Gil França2,
Paulo Henrique Stahlke3,Paulo Sérgio Stalhke3
1. Associate
professor, Supervisor of the Undergraduate Course of Vascular Surgery, Hospital
de Clínicas, Universidade Federal do Paraná, Brazil.
2.
Vascular surgeon. Graduate student in Clinical Surgery, Hospital de Clínicas,
Universidade Federal do Paraná, Brazil.
3. Vascular surgeon.
Hospital de Clínicas, Universidade Federal do Paraná, Brazil.
Correspondence:
Henrique Jorge Stahlke Jr.
Hospital de Clínicas UFPR - Disciplina
de Cirurgia Vascular
Rua General Carneiro, 181
CEP 80060-000 - Curitiba,
PR, Brazil.
Phone: +55 (41) 360.1800 R.: 6518
ABSTRACT
Objective:
This study aims at evaluating the effects of hyperhomocysteinemia
in the formation of atherosclerotic plaques in the aortas of rabbits.
Methods:
An experimental comparative study was performed with two homogenous groups of
rabbits during 60 days. Twenty New Zealand rabbits were divided into two groups
of 10: control group (C) and methionine group (M). All rabbits received the same
solid diet and 500 ml of water. Rabbits in group M received 2 ml of a methionine
solution at 200 mg/ml every 24 hours. Blood samples were collected for analyses
of total cholesterol, triglycerides, HDL, LDL and homocysteine concentrations
on the 0, 30th and 60th days. Euthanasia was performed by use of a lethal dose
of anesthetic on the 60th day. Thoracic and abdominal aortas were removed for
anatomicopathological study.
Results: There was no significant change in total cholesterol,
triglycerides, HDL and LDL concentrations in either group. There
was no change in plasma homocysteine levels in group C on the 0,
30th and 60th days. In group M, plasma homocysteine levels ranged
from an average value of 3.65 µmol/l on the 0 day to 11.10
µmol/l on the 30th day, and 16.19 µmol/l on the 60th
day (P < 0.0001). Microscopic examinations of the aortas
of group C did not evidence any pathological changes, being similar
to each other in all stainings, with hyperplastic intima, preserved
endothelium and presence of subendothelial deposits. Plaques were
formed by foam macrophages, but there were no smooth muscle cells,
cholesterol crystals or inflammatory cells. Tunica media presented
intact internal elastic lamina.
Conclusion:
We concluded that homocysteinemia caused atherogenesis in the aortas of rabbits.
Key-words:
homocysteine, aorta, rabbits, experimental design.
Palavras-chave:
homocisteína, aorta, coelhos, desenho experimental.
J
Vasc Br 2004;3(1):20-30
Throughout
the 20th century, the control of communicable diseases and the improvement
of the population's living conditions lead to an almost two-fold increase
in the life expectancy in developed countries.1
This improvement in longevity made degenerative diseases of the circulatory
system the major problem of public health in the end of the last century
and in the beginning of the current century.2
therosclerosis is the main cause of deaths in Western countries.1
According to the World Health Organization, circulatory diseases will
become the main health problem in this century. In the United States,
approximately 5.4 million people are diagnosed with coronary heart diseases
each year, and over 550,000 deaths are attributed to coronary atherosclerosis.3
In Brazil, cardiovascular diseases are also the leading cause of deaths,
killing approximately 300,000 patients a year. The three Brazilian capitals
with the highest mortality rates due to ischemia are, in decreasing
order of incidence, Porto Alegre, Rio de Janeiro and Curitiba.4
Among the
most usual risk factors for atherosclerosis are arterial hypertension,
diabetes mellitus, obesity, sedentary habits, heart disease in
the family, male sex, smoking and elevated plasma cholesterol.1,5
However, many patients who were affected by clinical disease did not
present any of those risk factors.6 During
the last years, other factors were identified, such as apolipoproteins
AI, AII, B, E and LP (a), fibrinogen and, more recently, homocysteine,
which may contribute to a better understanding of pathophysiological
mechanisms of atherosclerosis and allow for the development of new prophylactic
or therapeutic procedures.
In
1969, McCully2 reported the case of children affected
by homocystinuria who died due to an inborn deficiency in the metabolism of cobalamine
and deficiency of cystathionine beta-synthase. These children had severe atherosclerosis
and the only metabolic change diagnosed was elevated plasma homocysteine level.
Thus, McCully suggested that atherosclerosis could be attributed to hyperhomocysteinemia.
A more
recent study by Wilcken & Wilcken (1976) confirmed that elevated
plasma homocysteine was an independent risk factor for atherosclerosis.7
Such a relation was also confirmed by Nehler & Taylor (1997)8
for patients with carotid disease, coronary heart disease and lower
extremity arterial diseases.
Stampfer
at al. followed 14,916 physicians for five years, and concluded that
the risk for myocardial infarction was 3.1 higher in the group of physicians
who had higher plasma homocysteine concentrations.9
Studies by Taylor et al. (1991 and 1999) evidenced a greater incidence
of elevated plasma homocysteine in patients with peripheral arterial
disease than in the asymptomatic control group. These studies also evidenced
the progression of peripheral arterial disease in patients with hyperhomocysteinemia
(prospective study), with a mean follow-up of 37 months.10,11
Some
experimental studies showed that hyperhomocysteinemia or homocysteine perfusion
could lead to changes in the arterial wall. Nevertheless, none of the experimental
models included a methonine overload to induce hyperhomocysteinemia. The
aim of the present study is to evaluate the atherogenic effects of elevating plasma
homocysteine levels (through methionine overload) in rabbits' aorta. MATERIAL
AND METHOD Experimental
design Twenty
healthy New Zealand albino rabbits with an average weight of 2.000±456
g were kept at the vivarium of the Laboratory of Graduate Research in Clinical
Surgery of Universidade Federal do Paraná (Brazil). It is equipped with
a system for air exhaustion and ventilation, with temperature control, which allows
for keeping the room at a temperature of around 21ºC. All animals were weighted
and examined by a veterinarian in order to confirm their health. The rabbits were
placed in individual cages and received water and rat chow (Nutriara Company)
for seven days during the adaptation phase. The animals were selected randomly.
The present study was approved by the Committee of Bioethics in Research with
Animals of Universidade Federal do Paraná. Protocol
description
After the
adaptation phase, rabbits were randomly divided into two groups of 10
each, kept in individual cages and were assigned numbers ranging from
1 to 10 in the control group (group C) and from 11 to 20 in the methionine
group (group M). Rabbits in group C were fed with rabbit chow (Nutriara
Company) and drinking water ad libitum. Rabbits in group M were
fed with the same chow and, every 24 hours, received 500 ml of water
to which 2 ml of a methionine solution at 200mg/ml were added. All animals
(groups C and M) were fed with this diet for 60 days.
Plasma
assays Blood
samples were collected for analyses of total cholesterol, triglycerides, HDL,
LDL and homocysteine concentrations on the 0, 30th and 60th days. Total
cholesterol, triglycerides, LDL, HDL analyses were made through the enzymatic-colorimetric
method. Homocysteine was determined through HPLC (high performance liquid chromatographic)
method. Statistical
analysis In
order to compare the groups on each day and in relation to each variable, the
equal-means null hypothesis was tested against an alternative unequal-means hypothesis.
These hypotheses were verified through covariance analysis; we controlled the
weights of rabbits and checked homogeneity of variances using Cochran's test.
For the
comparison of days, the equal-means null hypothesis was tested against
an alternative unequal-means hypothesis. Thus, we used Students' t
test for paired samples. For all tests, significance level was 5%.12
Anatomicopathological
study Euthanasia
was performed by use of a lethal dose of ethyl sodium thiopental (Thionembutal®) and pancuronium (Pavulon®). Thoracic and abdominal aortas were removed and fixed
in a 10% formaldehyde solution, and were sent to anatomicopathological study.
A macroscopic examination was performed to evaluate length, diameter and maximum
thickness of the wall of the aorta. Distal, medial and proximal segments of the
aortas were sent for microscopic study. Preparation An
automatic tissue processor (JUNG - Histokinette - 2000) was used to prepare the
material, which was later embedded in paraffin wax. The blocks were sectioned
with American Optical 820 microtome (4-7 m of thickness), and sections were placed
over glass slides coated with albumin. Slides were stained with haematoxylin and
eosin, and special stains were made for elastic fibers with Weigert stain and
Mallory trichrome stain. After, the sections were mounted with balsam and cover
slip. Histological
evaluation All
sections of aortas were evaluated, both from groups M and C, in slides stained
with haematoxylin and eosin for analysis of collagenous fibers. Tunica intima
was analyzed in terms of its thickness, endothelial lining, presence or absence
of subendothelial deposits and type of cells. Tunica media was analyzed in terms
of its thickness, proportion between elastic and collagenous fibers and their
integrity, presence or absence of deposits, types of cells and architectural changes.
Tunica adventitia was analyzed in terms of its cells and architectural changes. RESULTS
Rabbits'
weight Rabbits
were weighted on the 0 and 60th days. At initial evaluation, on the day of the
rabbits' arrival to the vivarium, groups C and M did not present any significant
difference. On the 60th day of the study, there was not any significant difference
between the groups either. Thus, in terms of their initial and final weight, rabbits
from the two groups were alike. Table 1 shows the average weight of rabbits and
the standard deviation of these weights during the experiment.
 Table
1 - Rabbits' weights during the experiment (in grams)
|
|
|
Day |
Group |
Average |
|
|
|
0 day |
Control |
2,255.50 |
|
Methionine |
2,252.40 |
|
30th day |
Control |
2,260.80 |
|
Methionine |
2,270.00 |
|
60th day |
Control |
2,294.60 |
| Methionine |
2,299.00 |
|
|
Biochemical
analysis Plasma
levels of HDL and LDL Analyses
for plasma levels of HDL and LDL were performed simultaneously with total cholesterol
and triglycerides analyses. No significant difference was found in plasma levels
of lipoproteins between the two groups (Tables 2 and 3).
Table
2 - Levels of HDL (mg/dl)
|
|
|
Day |
Group |
Average |
P |
Standard
deviation |
P |
|
|
|
0 day |
Control |
12.42 |
0.0029 |
2.4075 |
0.3715 |
|
Methionine |
15.69 |
1.7685 |
|
30th day |
Control |
12.51 |
0.0059 |
2.3073 |
0.3290 |
|
Methionine |
15.40 |
1.6465 |
|
60th day |
Control |
13.23 |
0.0247 |
2.5939 |
0.2698 |
| Methionine |
15.74 |
1.7696 |
|
|
Table
3 - Levels of LDL (mg/dl)
|
|
|
Day |
Group |
Average |
P |
Standard
deviation |
P |
|
|
|
0 day |
Control |
71.40 |
0.4160 |
15.3637 |
0.2167 |
|
Methionine |
76.30 |
10.0006 |
|
30th day |
Control |
71.30 |
0.5411 |
15.3627 |
0.2266 |
|
Methionine |
75.10 |
10.0935 |
|
60th day |
Control |
73.20 |
0.6373 |
14.5511 |
0.3244 |
| Methionine |
76.00 |
10.3495 |
|
|
Plasma
levels of total cholesterol
There was
no significant difference in the levels of total cholesterol between
the two groups (Table 4).
Table
4 - Levels of total cholesterol (mg/dl)
|
|
|
Day |
Group |
Average |
P |
Standard
deviation |
P |
|
|
|
0 day |
Control |
81.60 |
0.3341 |
5.0155 |
0.4929 |
|
Methionine |
79.10 |
6.3500 |
|
30th day |
Control |
80.50 |
0.2293 |
4.0893 |
0.2694 |
|
Methionine |
77.80 |
5.9963 |
|
60th day |
Control |
81.10 |
0.4804 |
3.2128 |
0.0599 |
| Methionine |
79.50 |
6.2583 |
|
|
Plasma
levels of triglycerides There
was no statistically significant difference between groups C and M in terms of
plasma levels of triglycerides (Table 5).
Table
5 - Plasma levels of triglycerides (mg/dl)
|
|
|
Day |
Group |
Average |
P |
Standard
deviation |
P |
|
|
|
0 day |
Control |
84.68 |
0.2836 |
17.8002 |
0.2639 |
|
Methionine |
76.95 |
12.0841 |
|
30th day |
Control |
84.90 |
0.1894 |
17.3874 |
0.3376 |
|
Methionine |
75.50 |
12.4833 |
|
60th day |
Control |
86.50 |
0.1865 |
17.8216 |
0.2483 |
| Methionine |
77.10 |
11.9392 |
|
|
Plasma
homocysteine levels (µmol/l)
Comparing
group M, which was fed a diet rich in methionine, to group C, the control group,
a significant change in the levels of homocysteine was found during the experiment.
The increase in plasma homocysteine was already found on the 30th day, and plasma
levels continued to increase until the last analysis, on the 60th day.
The establishment
of normal levels of plasma homocysteine for animals in experiments (such
as the rabbits included in the present study) is still lacking.13
Rabbits in groups M and C were fed the same solid diet, including vitamins
B6 and B12. We considered that the normal level was the average of plasma
homocysteine levels of the control group, to which the plasma homocysteine
levels of group M would be compared. In the control group, there was
no change in the levels of homocysteine during the eight weeks of the
study (average values: 3.67 µmol/l on the 0 day; 3.65 µmol/l
on the 30th day; 3.70 µmol/l on the 60th day). In group M, the
average value for plasma homocysteine levels was 3.65 µmol/l on
the 0 day, reaching the mean values of 11.10 µmol/l on the 30th
day, and 16.19 µmol/l on the 60th day (Table 6). Thus, plasma
homocysteine in group M reached a level approximately five times higher
than the control group during the period of 60 days. The levels of triglycerides,
total cholesterol, HDL and LDL were not significantly altered, evidencing
that they did not influence the final results.
Table
6 - Plasma homocysteine levels ( µmol/l)
|
|
|
Day |
Group |
Average |
P |
Standard
deviation |
P |
|
|
|
0 day |
Control |
3.67 |
0.1181 |
0.4347 |
0.8687 |
|
Methionine |
3.65 |
0.4601 |
|
30th day |
Control |
3.65 |
<
0.0001 |
0.4378 |
0.0326 |
|
Methionine |
11.10 |
1.6633 |
|
60th day |
Control |
3.70 |
<
0.0001 |
0.4163 |
0.9524 |
| Methionine |
16.19 |
0.9267 |
|
|
In
the histological evaluation of aortas of the control group, tunica intima is thin,
with no endothelial deposits, defined internal elastic lamina and absence of inflammatory
cells (Figures 1 and 2).
Figure
1 - Photomicrograph of an aorta of group C. Elastic fibers stained with
Weigert stain, 40 times magnified. The figure shows endothelium (E)
and defined internal elastic lamina, normal muscle lining, irregular
adventitia.
Figure
2 - Photomicrography of an aorta of group C. Haematoxylin and eosin
staining, 200 times magnified. The figure shows well defined endothelium
(E), smooth muscle cells (SMC) with elongated, blunt-ended nucleus,
and diffuse cytoplasm. Irregular adventitia.
In group M, histological sections evidenced protrusion of foam subendothelial
macrophages into the lumen of the vessel, with no signs of calcium, differently
from arterial lesions due to or related to dyslipidemia, confirming the hypothesis
of atherogenesis caused by hyperhomocysteinemia (Figures 3, 4 and 5).
Figure
3 - Photomicrography of an aorta of group M. Haematoxylin and eosin
staining, 200 times magnified. The figure shows atherosclerotic plaque
formed by protrusion of macrophages (foam cells) into the lumen of the
vessel.
Figure
4 - Photomicrography of an aorta of group M. Weigert staining, 200 times
magnified. The figure shows endothelium with atherosclerotic plaque,
with macrophages (foam cells). Well defined internal elastic lamina.
Figure
5 - Photomicrography of an aorta of group M. Mallory trichrome staining,
40 times magnified. The figure shows large endothelium with atherosclerotic
plaque formed by foam cells (diffuse nuclei and cytoplasm).
DISCUSSION
During
the last 50 years, several factors have been acknowledged as risk factors
for atherosclerosis.1,14
However, at least 50% of patients who develop clinical disease do not
present any of these factors.6 The identification
of other risk factors, which would increase the risk for atherosclerosis,
can improve our understanding of the pathophysiological mechanisms of
this disease and allow for the development of new prophylactic or therapeutic
procedures. Among these new factors are lipoprotein (a), fibrinogen,15
states of hypercoagulation, known as thrombophilia1,14
and homocysteinemia.
The theory
that atherosclerisis would be attributable to homocysteine was first
proposed by McCully & Wilson, in 1975. Clarke at al., von Eckardstein
et al. and Graham et al. concluded that homocysteine is an independent
risk factor for arterial disease.16-19
Since the
original description by McCully, in 1969, after having observed patients
with a rare syndrome, familial homocysteinemia, with atherosclerotic
consequences, the possibility of homocysteine as a risk factor for atherosclerosis
began to be investigated.2 Later, it was
observed that, among patients with levels of homocysteine within the
range considered normal, those who presented the highest levels would
have higher risk of having the disease, similarly to the cases of high
cholesterol levels.
Several
prospective, case-control and longitudinal studies evidenced that: patients
who developed atherosclerosis had considerably higher levels of homocysteine
than patients who did not have the disease;20
levels of homocysteine higher than 15 µmol/l double the risk for
vascular diseases;21 plasma
homocysteine levels are associated with the following risk factors:
old age, male sex, smoking, high blood pressure, elevated cholesterol
level and lack of exercise;22
however, even after controlling for these other factors, the role of
homocysteine as a risk factor prevails;23
carotid atherosclerosis24 and deep-vein
thrombosis,25 as well as coronary heart
disease, are more prevalent in patients with higher levels of homocysteine.
Several
mechanisms explain the possible atherogenic effects of homocysteine.
The direct toxic effects of homocysteine over endothelial cells have
already been described,26 and it can possibly
inhibit the synthesis of prostacyclin as well. Its effect on platelets,
increasing their adherence and aggregration and favoring thrombosis,
has also been suggested. Furthermore, some studies have also demonstrated
the effects of homocysteine on clotting factors, favoring thrombophilia
by activating V factor.23
The crucial
issue of how homocysteine a sulfur amino acid affects the biochemical
processes of cells and tissues in arterial walls, leading to the production
of atherosclerotic plaques, is still today an active field for research.
Initial studies with cell cultures collected from children affected
by homocysteinuria revealed a new biochemical pathway through which
the sulfur atom of homocysteine thiolactone is oxidated and converted
into phosphoadenosine-phosphosulphate, the coenzyme which forms sulfate
glycosaminoglycans of atherosclerotic plaques.27
Other experiments with endothelial and smooth muscles cell cultures
included homocysteine in the formation of hydrogen peroxide and in the
control of cell growth due to its effects over the insulin-like growth
factor,28 the platelet-derived growth factor
and the formation of cyclin, leading to endothelial degeneration and
hyperplasia of smooth muscle cells of the atherosclerotic plaques.29
In hyperhomocysteinemic
animals, aortic elastase activity explains the typical degeneration
of elastin in atherosclerotic plaques.29
From the several reports available in medical literature, which were
referred in the present report, one can conclude that the association
between small elevations in plasma homocysteine levels and the cardiovascular
disease is a frequent phenomenon, and not only a rare inborn deficiency
of metabolism of homocysteine. This fact may be due to two aspects:
(1) enzymes which metabolize homocysteine are dependent on three nutrients:
folate, vitamins B6 and B12, which may be lacking, thus leading to elevation
in the level of homocysteine; (2) enzymes are also target of abnormal
genetic variations, which have been commonly found in the populations
studied.
There is
also an association between homocysteinemia and female hormones, because
women present premenopausal levels of homocysteine 20% lower than men,
and, during menopause, there is a clear elevation in the level of homocysteine.
This fact is under investigation. Even with estrogen replacement therapy
for postmenopausal women, there was no significant reduction in plasma
homocysteine levels.30,31 Nephropathy patients
presented elevation in the level of homocysteine, which suggests that
the kidney controls plasma homocysteine through a still unknown mechanism.
Diabetic patients also present elevated homocysteine due to their renal
insufficiency.32,33 For smoking patients,
nicotine antagonizes pyridoxal-phosphate and elevates plasma homocysteine,
thus explaining its role as a risk factor for cardiovascular diseases.22
Studies
reveal that a diet rich in fruits and vegetables, vitamin B6 supplement
(10mg/day), vitamin B12 supplement (0.1 mg/day) and folate supplement
(1 mg/day) reduces the level of homocysteine.34
US Food
and Drug Administration (FDA) recently launched a program requiring
manufacturers to add folate to breads in the United States, in the hope
of reducing the risk for vascular diseases among the most needy population.29
In a prospective
study relating plasma homocysteine levels to the progression of symptoms
of peripheral arterial disease, it was concluded that hyperhomocysteinemia
is associated with death from cardiovascular diseases, cerebrovascular
diseases and with the progression of lower extremity occlusive diseases.11
Several studies have showed that plasma homocysteine levels are inversely
related to levels of folate, vitamin B6 and vitamin B12.35-37
Vitamin therapy is not adequate for the treatment of atherosclerosis
because there is a difference between association and cause, and not
every associated factor and cause can be changed through therapy.
Experimental
studies have showed the effect of certain chemical substances in the
development of atherosclerosis, as Steiner did in 1938, showing the
effects of choline in preventing atherosclerosis in rabbits' aortas,37
and as Hartroft did in 1952, showing atheromatous changes in aorta,
carotid and coronary arteries of rats fed with choline deficient diet.38
In 1995, Rolland et al. have already observed elevated plasma concentrations
of homocysteine and lesions to the elastic membrane of arteries of pigs
fed with a diet rich in methionine. Recently,40
the mitogenic and cytotoxic effects of homocysteine on pig carotid arteries
after angioplasty were demonstrated with a perfusion culture model.
Stead observed that, when rat hepatocytes were incubated with methionine,
these cells exported more homocysteine, which suggested that the liver
has a significant role in the regulation of plasma homocysteine levels.41
In mice, elevated plasma homocysteine impairs endothelium-dependent
vasodilatation and contributes to inactivating nitric oxide.42
It was found that, in mice with a genetic deficience in cystathionine
beta-synthase and fed a diet deficient in folic acid, hyperhonocysteinemia
altered the endothelial function.43
In 1998,
Southern was the first to associate carotid endarterectomy and hyperhomocysteinemia
in rats. They were fed homocysteine-supplemented rat chow and postoperative
intimal hyperplasia was observed.13
In 1970, McCully fed a diet with 2% cholesterol to a group of rabbits
and the same diet deficient in vitamin B6 and subcutaneous injection
of homocysteine to the other group. It was observed that atherosclerotic
lesions were larger in the group which received homocysteine.27
It is clear
that the experimental studies reported above used homocysteine-supplemented
diets, intravenous homocysteine, or homocysteine perfusion models, in
which homocysteine affects endothelial cells directly. When homocysteine
is applied directly on endothelial cells, it has a highly citotoxic
effect.40
In
the present study, we aimed at evidencing the atherogenic effects of homocysteine
in its most usual way of intake: as part of a diet rich in methionine, which is
part of our daily diet. Methionine is metabolized in the liver and turned into
homocysteine, and may have different plasma levels. We aimed at evidencing that
there is no association between atherogenesis caused by hyperhomocysteinemia and
atherosclerosis related to hyperlipidemia, with cholesterol and calcium deposits
in the arterial wall. We
observed experimentally that elevated homocysteine is a predisposing factor to
atherogenesis. It is reasonable to suppose that controlling for this factor in
humans can be beneficial to patients, reducing the symptoms or the progression
of atherosclerosis. Whereas some factors cannot be controlled, such as male sex
and diabetes, others, such as daily diet and smoking can, either using medicines
or not, although they could have unwanted side effects, similar to drugs used
for controlling arterial hypertension and plasma lipids. When it is caused by
dietary problems, homocysteinemia can be controlled with folate supplementation
and an intake of fruits and vegetables easily available.
Ever since
the seminal study by McCully, research on the effects of hyperhomocysteine
in animals is performed with injected homocysteine thiolactone, and
artery perfusion models use homocysteine thiolactone as well, which
has well-known citotoxic effects.40 Homocysteine
results from methionine metabolism. Thus, if we feed a diet rich in
methionine to a group of rabbits, we can observe the effects of homocysteinemia
in its most natural way of progressing, that it, through daily food
intake, once methionine in an essential amino acid, which is derived
from the continuous catabolism of proteins. Therefore, we can say that
our study is an unprecedented model, in which atherogenesis in rabbit's
aortas was induced through a diet rich in substances to elevate plasma
homocysteine levels, and original, because we did not use injecting
amino acids.
CONCLUSION
We
concluded that elevated plasma homocysteine due to an overload of methionine consistently
lead to atherogenesis in rabbit's aortas. REFERENCES
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