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Focal [3h]cholesterol uptake in the pig aorta Part 2. Distribution of [3h]cholesterol across the aortic wall in areas of high and low uptake in vivo
A thevosclerosis Elsevier Publishing
FOCAL
Company,
Amsterdam
[aH]CHOLESTEROL
UPTAKE
IN THE
PART 2. DISTRIBUTION OF [sH]CHOLESTEROL AREAS OF HIGH AND LOW UPTAKE IN VIVO*
J. B. SOMER**
PIG AORTA ACROSS
THE
AORTIC
WALL
IN
AND C. J. SCHWARTZ
Department of Pathology, Faculty of Medicine, Hamilton, Ontario (Canada) (Received
377
- Printed in The Netherlands
McMaster
University and The Chedoke Hospitals,
May 20th, 1971)
SUMMARY
normal
Focal areas of increased uptake of [aH]cholesterol ilz vivo by the macroscopically young pig aorta have been identified by their ability to accumulate the
protein-binding azo dye, Evans blue. The distribution of labelled cholesterol across the aortic wall in areas of dye uptake (blue areas) and no dye uptake (white areas) was determined at intervals from 10 min to 24 h after the injection Labelled distributed
free cholesterol
non-uniformly
of label.
represented
most
across the aortic
of the label in the aorta.
It was
wall, with most label concentrated
in
the intima and inner media. Labelled cholesterol ester represented only a small proportion of the aortic label and was distributed evenly through the vessel wall. Free cholesterol activity and free cholesterol specific activity were greater in blue areas than in white areas. These differences were confined to the intima and inner media and also the adventitia. Free cholesterol distribution aortic wall were similar
and
DNA
content
and distribution
across
the
in blue and white areas,
It is suggested that the increased focal uptake of labelled free cholesterol by blue areas reflects a higher free cholesterol turnover in these areas, and that labelled plasma cholesterol enters the pig aorta mainly through the intima in vivo.
Key words:
Cholesterol
- Focal
Uptake - Aortic
Wall - Cholesterol
Distribution
- DNA
- Atherogenesis
This work was supported by the Medical Research Council of Canada, Grants MA. 3067 and MT. 3067. * Presented in part to The International Academy of Pathology, March 1971. ** Research Fellow of the Ontario Heart Foundation. Atherosclerosis,
1972, 16: 377-388
J. B. SOMER,
378
C. J. SCHWARTZ
INTRODUCTION
Cholesterol mainly
in both
from plasmal-6,
cholesterol
enters
normal
and atherosclerotic
and evidence
the aorta principally
The focal aortic accumulation and pig12J4. In previous blue
obtained
accumulation
reports
exhibited
appears
to be derived
and rat indicates
that
from the lumen6-9.
of Evans blue has been shown in doglo, rabbitllJ2 it was shown in the pig, that focal areas of Evans
an increased
[3H]cholesterol
with adjacent areas of no dye uptakel3J4. In the experiments described below across the aortic wall was measured in. viva to determine
aortas
in dog, rabbit
uptake,
the distribution
when compared
of labelled
cholesterol
in focal areas of high and low cholesterol
(a) any differences
in labelled
the aortic wall in these areas and (b) the direction
cholesterol of entry
distribution
uptake across
of cholesterol
into the
pig aorta. MATERIALS
AND
Animals Yorkshire
METHODS
pigs, 8-12 weeks old and weighing
tized diet (Purina
Hog Chow, Ralston-Purina
30-50 lbs, maintained
Co., Canada)
Irzjection of labelled plasma and further treatment of animals Generally labelled [3H]cholesterol (Schwarz BioResearch, was checked
for radiopurity
diethyl
ether-glacial
acetic
greater
than 99%. Pigs were lightly
by thin-layer acid
(72:28:1,
anaesthetized
chromatography by vol.);
Orangeburg,
U.S.A.)
in petroleum
ether-
radiopurity
by the intravenous
on a pelle-
were used.
injection
was found of sodium
to be pento-
barbital (Diabutal, Diamond Laboratories, Des Moines, U.S.A.). A jugular vein was cannulated and 25 ml of blood withdrawn into 3.8 o/o (w/v) trisodium citrate solution (1 part anticoagulant:
9 parts of blood). Platelet-poor
plasma was prepared
by centri-
fugation for 15 min at 1500 x g; 1.5 mCi of [sH]cholesterol, approximately 150 pg, dissolved in 1.0 ml of ethanol, was added dropwise to the platelet-poor plasma with thorough
mixing.
The labelled
plasma
was injected
through
the cannula
approxima-
tely 45 min after collection of the blood sample. Pigs were killed by intravenous injection of sodium pentobarbital 10 min, 1 h, 4 h, and 1 day later. Evans blue was injected 3 h prior to killing, the aortas removed, washed, and blue and white segments were mapped and excisedId. Each aortic segment was serially sectioned from intima to adventitia at intervals of 100 pm on an Ames Lab-Tek Cryostat (Lab-Tek Instruments Co., Westmont, U.S.A.). Sections were numbered from intima to adventitia. Sections from blue areas having the same number were pooled; the same was done with sections from white areas having the same number. The influence of the route of administration of label on the focal pattern of Atherosclerosis,
1972,
16: 377-388
FOCAL j3H]CHOLESTEROL
[sH]cholesterol
uptake
were administered
UPTAKE IN THE PIG AORTA, PART
was studied
in two experiments.
379
2
3 mCi of [aH]cholesterol
orally and the pigs killed 3 days later, 3 h after injection
of Evans
blue. Free cholesterol specific activity was greater in blue than in white areas, findings similar to those obtained when the labelled cholesterol was injected intravenouslyi4. Lipid
methods
and DNA
determination
Pooled sections were extracted
with hot chloroform-methanol
(2:1, v/v);
15 ml
at 65°C for 1 h. Extracts were evaporated to dryness, 10 ml of toluene scintillation fluid (0.3 g of 1,4-bis-[2-(4-methyl-5phenyloxazolyl)l benzene and 5 g of PPO in 1 1 of toluene) added and the radioactivity liquid scintillation spectrometer. Quench the external standardization method. The extracts porated to dryness ml was taken
measured using an Intertechnique SL-40 corrections were made automatically by
from two animals killed 1 day after injection of label were evaand each lipid residue was dissolved in 1.0 ml of chloroform; 0.5
for measurement
of radioactivity,
and the remaining
0.5 ml applied
to a thin-layer chromatoplate and chromatographed in petroleum ether-diethyl ether-glacial acetic acid (72:2&l, v/v/v); bands corresponding to free cholesterol and cholesterol
ester were scraped into fluted filter papers, having
been visualized
under
ultraviolet light after spraying with a 0.2% solution of 2’,7’-dichlorofluorescein ethanol, eluted with 10 ml of toluene scintillation fluid and the radioactivity
in was
measured. After determination of post-extraction dry weight the DNA content of the tissue was determined by the method of DISCHE 15 after extraction with 5 o/otrichloroacetic
acidI6.
Determination
of [3H]cholesterol
specific
activity
and cholesterol
content
acY0s.s the aortic
wall im blue and white areas
Pigs were injected and killed
1 day later.
with autologous Each
aorta
plasma containing
was removed
1.5 mCi of [aH]cholesterol
and 100 pm
serial
sections
were
prepared from blue and white segments as described. Pooled sections were extracted with 15 ml of chloroform-methanol (2:1, v/v) for 1 h at 65°C and re-extracted with 15 ml of ethanol-petroleum ether (3:1, v/v) for 1 h at 65°C. Extracts were pooled and evaporated to dryness. At this stage, extracts from Sections 1, 2, 3, and n were treated individually while extracts from Sections 4 to (n-1) were combined and subsequently treated as a single entity. The lipid residues were chromatographed; free and esterified cholesterol were eluted with chloroform radioactivity as described. Cholesterol was determined Analysis
(3 x 4 ml) and measured for by an automated methodiT.
of data
Measurements obtained for sections across the vessel wall were treated follows: Sections 1, 2, and 3 were considered individually, the mean was taken
as for
values obtained for Sections 4 to n-l, and the last or adventitial section (n) was again considered individually. This treatment was considered justifiable since activity Atherosclerosis,
1972, 16: 377-388
380
J. B. SOMER,
TABLE
C. J. SCHWARTZ
1
FOCAL DISTRIBUTION INJECTION OFLABEL
OF [SHICHOLESTEROL
IN AORTAS
Idensity of Evans blue staining
[3H]Cholesterol
Blue
2207
White
(3) 1366 & 134
(3) 2812
&
F<
!x
0.05
PIGS AT VARIOUS
TIMES AFTER
activity (dpmlmg DNA)
after [3H]cholesterol 10 min lh
Time
f
OF YOUNG
342*
0.05
injection: 4h
5865 f
1046
9303
258
(3) 6020
1 day +
1324
13 164 *
1048
486
(6) 10 302 &
706
f
P (3) = 0.08
P<
0.05
*
Mean f S.E. Numbers in parentheses
indicate
number
of animals.
across the vessel wall from Section 4 to the adventitia was usually constant in each experiment (see below). In statistical
analysis of data for corresponding sections from blue or white
areas at each specific time interval, the paired t-test was used. RESULTS
[3H]Cholesterol uptake in blue and white ayeas of pig aorta at various times after injection of label Table 1 shows [sH]cholesterol activity, expressed as a function of tissue DNA content, in blue and white areas at 10 min, 1 h, 4 h, and 1 day after injection of label. Activity
in blue areas was consistently
differences being statistically
greater than activity
in white areas, the
significant at 10 min, 1 h and 1 day. Activity in both
blue and white areas increased with time after injection. Aortic DNA content and distribution
in blue and white areas
The DNA content of blue and white areas and its distribution through the vessel wall are shown in Table 2. There was no significant difference across the vessel wall, nor between corresponding blue and white sections. [3H]Cholesterol distribution across the aortic wall in blue and white ayeas at various times after injection of label As early as 10 min after injection of label, there was detectable radioactivity throughout the vessel wall in both blue and white areas (Fig. 1). At this time, activity in blue areas was greater than activity in white areas in all sections but the last (Table 3). 1 h after injection of label, a general increase in activity throughout the aortic wall in both blue and white areas was observed (Fig. 2, Table 3). Activity in blue areas was greater than activity in white areas in Sections 1, 2, and 3 (intima and inner media) (Fig. 2, Table 3). Atherosclerosis,
1972,
16: 377-388
FOCAL [3H]CHOLESTEROL TABLE DNA
UPTAKE IN THE PIG AORTA, PART
381
2
2
CONTENT
AND
DISTRIBUTION
ACROSS
THE AORTIC
WALL
IN BLUE
AND
WHITE
AREAS
IN YOUNG
PIGS
SectionNo.
DNA
content
(rag D.VA/g dry tissue weight) E’hite
Blue 1 2 3 Mean 4&(n-1) n Whole aorta
11.3 (17) 11.5 (18) 11.5 (18) 12.5 (18) 12.8 (18) 11.6 (11)
+ o.s7*
12.6 (17) 13.5 (18) 14.1 (18) 13.0
+ 0.87 & 0.78 += 0.87
* 0.85 + 1.06 + 1.25 * 0.97
(18) 12.7 & 0.82 (18) 13.3 & 1.11 (11)
+ 0.78 + 0.72
* Mean f S.E. Numbers in parentheses indicate number of animals.
At 4 h blue and white vessel wall, particularly
areas showed
in the intima
most of the vessel wall, the activities the first two sections adventitia)
(intima
was activity
a further
increase
in activity
across the
and inner media (Fig. 3, Table 3). Throughout in blue and white areas were similar.
and inner media),
and the last section
Only in
(outer media and
in blue areas greater than that in white areas (Fig. 3, Table 3).
B 1 SCCTION NUYBCR
5
5
7
9
11
1J
1
I5
I7
89
SCCTION NUYBCR
Fig. 1. Distribution of [sH]cholesterol activity across the aortic wall in blue areas (A) and white areas (B) 10 min after injection of label. Sections (100 pm) are numbered from intima to adventitia. Results obtained in three animals shown. Atherosclerosis,
1972, 16: 377-388
Y ;” ;
6 .
5 s
F 2 2. P
a
0
-i
?
b
3
3)
4 h (n =
* Mean
f
1 day (n =
3)
=
1 h(n
6)
3)
S.E.;
10 min (H =
Time after injection of label
N.S.,
+
f
&
5
&
20 632 2004
17 838 f 3521 0.065
non significant.
P < 0.01
38 320 4319
P =
43 532 13002
5 508 * 1616 P < 0.05
19 905 2470
2 727 863
DNA)
f
*
N.S.
+
P = 0.08
17 090 4867
P < 0.05
7 592 1343
20 325 5625
6 372 1451
2 445 484
189
879
White
+ P = 0.065
645
2 734
17 604 & 2286
f
*
i:
Blue
2
activity (dpnlmg
White
& P < 0.05
8810 1465*
BlUl?
1
Section No.
[3H]Cholesterol
f
12 682 + 731 * N.S.
4 270 + 529 & N.S.
* P < 0.05
9541 1571
4 175 1081
1736 333
125
431
White
P < 0.05
223
1022
3 059 & 599
&
Blue
3
+
&
*
8718 1706 N.S.
4313 873 N.S.
3 598 711 N.S.
&
&
&
8 280 763
4 020 642
2 139 251
108
1069
White
+ P < 0.01
_ir 121
1286
Blue
4-(%-l)
D~~TRIBUTI~N~F[~H]CHOLESTEROLA~TIVITYACROSSTHEAORTI~WALLINBLUEANDWHITEAREASINYOUNGPIGSATVARIOU~ TION OFLABEL
TABLE
&
f
f
f
N.S.
N.S.
f
94
4 930 303
3251 193
f
2 186
White
INJEC-
7 391 * 747 P < 0.001
9 999 580
+ P < 0.05
7 307 699
3 909 384
206
1871
Blue
1z
TIMES AFTER
q
FOCAL [~H]CHOLESTEROL
UPTAKE IN THE PIG AORTA, PART 2
383
30-
30-
SECTION
N”Y.tl
SECTION
NUYILR
Fig. 2. Distribution of [sH]cholesterol activity across the aortic wall in blue areas (A) and white areas (B) 1 h after injection of label. Sections (100 pm) are numbered from intima to adventitia. Results obtained in three animals shown.
;
30-
30-
0‘ 0
?
I
1 I I
‘o-
. 30-
A 1
3
0
7
SLCTION
9 NIlMIll
11
13
1s
B 1
3
3
7
9
SICTION
II
I3
I3
NUYW”
Fig. 3. Distribution of [aH]cholesterol activity across the aortic wall in blue areas (A) and white areas (B) 4 h after injection of label. Sections (100 pm) are numbered from intima to adventitia. Results obtained in three animals shown. Atherosclerosis,
1972, 16: 377-388
J. B. SOMER, C. J. SCHWARTZ
384
One day after injection of label, activity in the first two sections in blue areas had not changed, whereas activity in the remainder of the vessel wall showed a further increase (Fig. 4, Table 3). In white areas, activity in the first section was relatively unchanged, with the remainder of the vessel wall showing a further increase (Fig. 4, Table 3). At this time, there was significantly greater activity in only the first and last sections of blue areas in relation to white areas, while throughout the remainder of the vessel wall activities were similar (Fig. 4, Table 3). Distribution
of aH-free cholesterol and [3H]cholesterol
ester awoss the aortic wall 1 day
after injection of label The distribution of labelled free and esterified cholesterol across the vessel wall in blue and white areas 1 day after injection of label is shown in Fig. 5.
I.,,*,,,, *....,, A
B
??
1
,
I
7
SICTION
9
II
NUYIER
Ia
1s
1
9
s
7
9
,I
II
I9
SCCTION N”Y.99
Fig. 4. Distribution of [sH]cholesterol activity across the aortic wall in blue areas (A) and white areas (B) 1 day after injection of label. Sections (100 pm) are numbered from intima to adventitia. Results obtained in six animals shown. Atherosclerosis,
1972, 16: 377388
FOCAL [SHICHOLESTEROL
UPTAKE IN THE PIG AORTA, PART 2
385
JC
20
1c
B I,.
1
3
,
.
.
5
7
9
.
11
I
11
1
SSCTION NUMSCR
Fig. 5. Distribution areas (A) and white activity and closed bered from intima
3
5
7
9
SECTION
11
1,
I5
NUMBER
of sH-free and sH-esterified cholesterol activity across the aortic wall in blue areas (B) 1 day after injection of label. Open s,yybols denote sH-free cholesterol symbols denote sH-esterified cholesterol activity. Sections (100 ,um) are numto adventitia. Results obtained in two animals shown.
Most label was present in the form of free cholesterol. The accumulation
of
label in the intima and inner media was due almost exclusively to an accumulation of labelled free cholesterolin this region, while labelled cholesterol ester was distributed evenly throughout the vessel wall. Further, the differences in activity through the vessel wall between blue and white areas were due to differences in sH-free cholesterol activity. There was no difference in [sH]cholesterol ester activity between blue and white areas. Distribution
of 3H-free
cholesterol
s$eci$c
activity
across
the aortic
wall
in blue and
white areas
The distribution of sH-free cholesterol specific activity across the vessel wall in blue and white areas 1 day after injection of label is shown in Table 4. Free choAtherosclerosis,
1972, 16: 377-388
J.
386 TABLE
B. SOMER,
C. J. SCHWARTZ
4
DISTRIBUTION AORTIC
WALL
OF
FREE
IN BLUE
CHOLESTEROL AND
WHITE
AND AREAS
3H-FREE IN YOUNG
CHOLESTEROL PIGS
AT
SPECIFIC
ONE
DAY
ACTIVITY
AFTERINJECTION
ACROSS
THE
OFLABEL -
Section No.
3H-Free cholesterol spec. act. (dpnlmg free cholesterol) Blue
1 2 3 4-(n-1) n
147 704 72 640 39879 28073 209444
* Mean
& S.E.
* & h j=
42 os4* 27 080 13689 10890 3457
Aortic free cholesterol content (wag/gdry tissue weight) White
Blue
White
P
71675 * 18667 30 613 j= 7 227
3.36 f 0.19 3.27 _C 0.29
2.95 $= 0.50 3.56 & 0.51
P < 0.05 P < 0.2 P < 0.05
1 586
3.29 f
0.43
3.26 h 0.41
14 975 f 15273+
3 297 2603
3.33 f 0.30 4.14 & 0.10
3.57 .& 0.16 4.07 5 0.43
P < 0.05
9 344 +
of five experiments.
lesterol specific activity was greater in blue than in white areas in the intima and inner media (Sections l-3) and in the adventitia
(Section n). Specific activity was
greatest in the intima and inner media and decreased towards the adventitia. Free
cholesterol
distribution
across the aortic w/z11 in blue and white ayeas
The distribution of free cholesterol across the vessel wall in blue and white areas is shown in Table 4. There was no significant difference between blue and white areas. Cholesterol content was greatest in the last section (outer media and adventitia) and constant through the remainder of the vessel wall. Cholesterol ester content was too small to measure in almost all sections. DISCUSSION
The present series of experiments has confirmed and extended previous studies showing an increased appearance of labelled cholesterol in areas of Evans blue dye accumulation when compared with contiguous areas of no dye accumulationrsJ4. The data show that the increase in cholesterol activity in blue areas was confined to the intima and inner media with no differences in most of the remaining media (Figs 1-5, Tables 3 and 4). The data in Table 4 indicate that the differences between blue and white areas are not due to differences in cholesterol content in the intima and inner media and suggest an increased cholesterol turnover in these regions of the aortic wall in blue areas. Results obtained with the outer or adventitial sections are more difficult to interpret. At short time periods after injection of label, there are no differences in activity between blue and white areas (Table 3); however, by one day, activity in the outer or adventitial sections from blue areas is greater than that of white areas (Tables 3 and 4). A possible explanation could invoke a comparatively small uptake of label at the adventitial side, greater in blue areas than in white areas. A small amount of entrapment of blood in the vasa vasorum might mask these differences at earlier time periods. Atherosclerosis,
1972,
16: 377-388
FOCAL [~H~CHOLESTEROLUPTAKE IN THE PIG AORTA, PART 2 These cholesterol
experiments uptake
confirm
our
earlier
observations
the aortic wall. The highest proportion
the
1 and 2 (Fig. 1, Table 3). This pattern
after
of label,
(Figs 2-4,
Table
difference
in
in the appear-
radioactivity
was present
of label was located in the intimal
side in Sections injection
that
in blue and white areas is due solely to a difference
ance of labelled free cholesterol (Fig. 5, Table 4, ref. 14). As early as 10 min after injection of [3H]cholesterol, throughout
387
persisted
at 1 h, 4 h and 1 day
3). The distribution
of free cholesterol
specific activity showed a similar pattern. Specific activity was highest in the first section (in blue areas, approximately two thirds of the terminal serum total cholesterol specific activity, in white areas approximately towards the adventitia (Table 4). These results provide of the labelled
cholesterol
crossed the intima
one third) and decreased strong evidence that most
with part of it being transported
out-
ward to the adventitia. The distribution of labelled cholesterol across the aortic wall has been studied in a number of laboratories 6- 9. Evidence that cholesterol enters mainly across the intima
has been obtained
in normal
and atherosclerotic
pigeons, normal
rat7, and normal and cholesterol-fed rabbit7-9. Our results indicate true in the macroscopically normal young pig aorta. either
dog6, normal
that the same is
It would appear that in the pig, the contribution of label from the vasa vasorum, from entrapment of blood in the capillaries or from actual uptake, plays but
a small part in the appearance of labelled cholesterol in the aorta. Free cholesterol specific activity of the outer media and adventitia is only a fraction of the free cholesterol specific activity in the first or intimal section, which itself is less than the terminal serum cholesterol specific activity. If a considerable amount of entrapment of blood in and/or uptake of label from the vasa vasorum were to occur, one could reasonably expect a much higher free cholesterol specific activity in the outer portion of the vessel. Free and esterified
labelled
cholesterol
exhibit
marked
differences
in distribu-
tion across the vessel wall, with free cholesterol activity showing a maximum in the intima and inner media, while labelled cholesterol ester is distributed evenly throughout the vessel wall in both blue and white areas (Fig. 5). These observations could be explained by postulating a small uptake and uniform distribution of lipoprotein across the vessel wall, equal in blue and white areas, and sufficient to account for the relatively small amount of [3H]cholesterol ester activity observed. Alternatively, the uniform distribution of labelled cholesterol ester could reflect a small amount of cholesterol esterifying Cholesterol esterifying specieslsJ9.
enzyme activity distributed evenly throughout activity has been demonstrated in the aortas
the aortic wall of a number of
The distribution of free cholesterol across the aortic wall of the young, normal pig is not uniform, with the outer media and adventitia showing a higher free cholesterol content than the remainder of the vessel wall. Similar observations were made by DUNCAN et al.6 in the normal dog aorta. Evidence has been Fresented to indicate that the differences in uptake of Atherosclerosis,
1972, 16: 377-388
J. B. SOMER, C. J. SCHWARTZ
333 labelled
cholesterol
between
blue and white
areas are not uniform
throughout
the
vessel wall but are confined to the intima and inner both blue and white areas labelled cholesterol entered
media and the adventitia. In primarily through the intimal
surface.
These findings
observations
aortais
not homogeneous
the handling
confirm
and extend
previous
in some of its properties
including
protein
that
the normal
uptakelo-is
and
of cholesterolis*i4.
ACKNOWLEDGEMENTS
The authors and Biostatistics,
thank Mr M. Gent of the Department of Clinical Epidemiology McMaster University, for advice concerning the statistics used,
Dr F. P. Bell for many segments
helpful
and Mrs G. Couture
discussions,
Mr J. Korthuis
for able technical
for sectioning
arterial
assistance.
REFERENCES 1
2
3
4
5
3
7
3
9
10
11
12
13
14
NEWMAN, H. A. I. AND D. B. ZILVERSMIT, Quantitative aspects of cholesterol flux in rabbit atheromatous lesions, J. Biol. Chew, 1962, 237: 2078. CHRISTENSEN, S., Transfer of labelled cholesterol across the aortic intimal surface of normal and cholesterol-fed cockerels, J. Atheroscler. Res., 1964, 4: 151. BELL, F. P., H. B. LOFLAND AND N. A. STOKES, Cholesterol flux in vifro in aortas of cholesterolfed and non-cholesterol-fed pigeons, Atherosclerosis, 1970, 11: 235. LOFLAND, H. B. AND T. B. CLARKSON, The bi-directional transfer of cholesterol in normal aorta, fatty streaks and atheromatous plaques, Proc. Sot. Exe. Biol. Med., 1970, 133: 1. SCHWENK, E. AND D. F. STEVENS, Deposition of cholesterol in experimental rabbit atherosclerosis, Proc. Sot. Exp. Biol. Med., 1960, 103: 614. DUNCAN, L. E. AND K. BUCK, Passage of labelled cholesterol into the aortic wall of the normal dog, Circ. Res., 1959, 7: 765. ADAMS, C. W. M., 0. B. BAYLISS, A. N. DAVISON AND M. Z. M. IBRAHIM, Autoradiographic evidence for the outward transport of [sH]cholesterol through rat and rabbit aortic wall, J. Pathol. Bact., 1964, 87: 297. ADAMS, C. W. M. AND R. S. MORGAN, Autoradiographic demonstration of cholesterol filtration and accumulation in atheromatous rabbit aorta, Nature, 1966, 210: 175. ADAMS, C. W. M., S. VIRhG, R. S. MORGAN AND G. C. ORTON, Dissociation of [sH]cholesterol and issI-labelled plasma protein influx in normal and atheromatous rabbit aorta, J. Atheroscler. Res., 1968, 8: 679. MCGILL, H. C., J. C. GEER AND R. L. HOLMAN, Sites of vascular vulnerability in dogs demonstrated by Evans blue, Arch. Pathol., 1957, 64: 303. FRIEDMAN, M. AND S. 0. BYERS, Endothelial permeability in atherosclerosis, Avch. Pathol., 1963, 76: 111. PACKMAN, M. A., H. C. ROWSELL, L. JBRGENSENAND J. F. MUSTARD, Localized protein accumulation in the wall of the aorta, Exp. Mol. Pathol., 1967, 7: 214. SCHWARTZ, C. J., E. E. NISHIZAWA, J. B. SOMER AND J. F. MUSTARD, Focal [3H]- and [14C]cholesterol accumulation in the pig aorta, Circulation, 1968, 38: Suppl. 6: 22. SOMER, J. B. AND C. J. SCHWARTZ,Focal [sH]cholesterol uptake in the pig aorta, Atherosclerosis, 1971, 13: 293.
15
16 17
13
19
DISCHE, Z., Color reactions of nucleic acid components, In: E. CHARGAFF AND I. N. DAVIDSON (Eds.), The Nucleic Acids, Vol. 1, Academic Press, New York, 1955, p. 285. SCHNEIDER, W. C., Phosphorus compounds in animal tissues, J. Biol. Chem., 1955, 161: 293. BLOCK, W. D., K. J, JARRET AND J. B. LEVINE, An improved automated determination of serum total cholesterol with a single color reagent, Clin. Chew, 1966, 12: 681. ABDULLAH, Y. H., C. C. ORTON AND C. W. M. ADAMS, Cholesterol esterification in human and experimental atheromatous lesions, J. Atheroscler. Res., 1968, 8: 967. ST. CLAIR, R. W., H. B. LOFLAND AND T. B. CLARKSON, Influence of duration of cholesterol feeding on esterification of fatty acids by cell-free preparation of pigeon aorta, Circ. Res., 1970, 27: 213.
Atherosclerosis,
1972, 16: 377-388
FOCAL
Company,
Amsterdam
[aH]CHOLESTEROL
UPTAKE
IN THE
PART 2. DISTRIBUTION OF [sH]CHOLESTEROL AREAS OF HIGH AND LOW UPTAKE IN VIVO*
J. B. SOMER**
PIG AORTA ACROSS
THE
AORTIC
WALL
IN
AND C. J. SCHWARTZ
Department of Pathology, Faculty of Medicine, Hamilton, Ontario (Canada) (Received
377
- Printed in The Netherlands
McMaster
University and The Chedoke Hospitals,
May 20th, 1971)
SUMMARY
normal
Focal areas of increased uptake of [aH]cholesterol ilz vivo by the macroscopically young pig aorta have been identified by their ability to accumulate the
protein-binding azo dye, Evans blue. The distribution of labelled cholesterol across the aortic wall in areas of dye uptake (blue areas) and no dye uptake (white areas) was determined at intervals from 10 min to 24 h after the injection Labelled distributed
free cholesterol
non-uniformly
of label.
represented
most
across the aortic
of the label in the aorta.
It was
wall, with most label concentrated
in
the intima and inner media. Labelled cholesterol ester represented only a small proportion of the aortic label and was distributed evenly through the vessel wall. Free cholesterol activity and free cholesterol specific activity were greater in blue areas than in white areas. These differences were confined to the intima and inner media and also the adventitia. Free cholesterol distribution aortic wall were similar
and
DNA
content
and distribution
across
the
in blue and white areas,
It is suggested that the increased focal uptake of labelled free cholesterol by blue areas reflects a higher free cholesterol turnover in these areas, and that labelled plasma cholesterol enters the pig aorta mainly through the intima in vivo.
Key words:
Cholesterol
- Focal
Uptake - Aortic
Wall - Cholesterol
Distribution
- DNA
- Atherogenesis
This work was supported by the Medical Research Council of Canada, Grants MA. 3067 and MT. 3067. * Presented in part to The International Academy of Pathology, March 1971. ** Research Fellow of the Ontario Heart Foundation. Atherosclerosis,
1972, 16: 377-388
J. B. SOMER,
378
C. J. SCHWARTZ
INTRODUCTION
Cholesterol mainly
in both
from plasmal-6,
cholesterol
enters
normal
and atherosclerotic
and evidence
the aorta principally
The focal aortic accumulation and pig12J4. In previous blue
obtained
accumulation
reports
exhibited
appears
to be derived
and rat indicates
that
from the lumen6-9.
of Evans blue has been shown in doglo, rabbitllJ2 it was shown in the pig, that focal areas of Evans
an increased
[3H]cholesterol
with adjacent areas of no dye uptakel3J4. In the experiments described below across the aortic wall was measured in. viva to determine
aortas
in dog, rabbit
uptake,
the distribution
when compared
of labelled
cholesterol
in focal areas of high and low cholesterol
(a) any differences
in labelled
the aortic wall in these areas and (b) the direction
cholesterol of entry
distribution
uptake across
of cholesterol
into the
pig aorta. MATERIALS
AND
Animals Yorkshire
METHODS
pigs, 8-12 weeks old and weighing
tized diet (Purina
Hog Chow, Ralston-Purina
30-50 lbs, maintained
Co., Canada)
Irzjection of labelled plasma and further treatment of animals Generally labelled [3H]cholesterol (Schwarz BioResearch, was checked
for radiopurity
diethyl
ether-glacial
acetic
greater
than 99%. Pigs were lightly
by thin-layer acid
(72:28:1,
anaesthetized
chromatography by vol.);
Orangeburg,
U.S.A.)
in petroleum
ether-
radiopurity
by the intravenous
on a pelle-
were used.
injection
was found of sodium
to be pento-
barbital (Diabutal, Diamond Laboratories, Des Moines, U.S.A.). A jugular vein was cannulated and 25 ml of blood withdrawn into 3.8 o/o (w/v) trisodium citrate solution (1 part anticoagulant:
9 parts of blood). Platelet-poor
plasma was prepared
by centri-
fugation for 15 min at 1500 x g; 1.5 mCi of [sH]cholesterol, approximately 150 pg, dissolved in 1.0 ml of ethanol, was added dropwise to the platelet-poor plasma with thorough
mixing.
The labelled
plasma
was injected
through
the cannula
approxima-
tely 45 min after collection of the blood sample. Pigs were killed by intravenous injection of sodium pentobarbital 10 min, 1 h, 4 h, and 1 day later. Evans blue was injected 3 h prior to killing, the aortas removed, washed, and blue and white segments were mapped and excisedId. Each aortic segment was serially sectioned from intima to adventitia at intervals of 100 pm on an Ames Lab-Tek Cryostat (Lab-Tek Instruments Co., Westmont, U.S.A.). Sections were numbered from intima to adventitia. Sections from blue areas having the same number were pooled; the same was done with sections from white areas having the same number. The influence of the route of administration of label on the focal pattern of Atherosclerosis,
1972,
16: 377-388
FOCAL j3H]CHOLESTEROL
[sH]cholesterol
uptake
were administered
UPTAKE IN THE PIG AORTA, PART
was studied
in two experiments.
379
2
3 mCi of [aH]cholesterol
orally and the pigs killed 3 days later, 3 h after injection
of Evans
blue. Free cholesterol specific activity was greater in blue than in white areas, findings similar to those obtained when the labelled cholesterol was injected intravenouslyi4. Lipid
methods
and DNA
determination
Pooled sections were extracted
with hot chloroform-methanol
(2:1, v/v);
15 ml
at 65°C for 1 h. Extracts were evaporated to dryness, 10 ml of toluene scintillation fluid (0.3 g of 1,4-bis-[2-(4-methyl-5phenyloxazolyl)l benzene and 5 g of PPO in 1 1 of toluene) added and the radioactivity liquid scintillation spectrometer. Quench the external standardization method. The extracts porated to dryness ml was taken
measured using an Intertechnique SL-40 corrections were made automatically by
from two animals killed 1 day after injection of label were evaand each lipid residue was dissolved in 1.0 ml of chloroform; 0.5
for measurement
of radioactivity,
and the remaining
0.5 ml applied
to a thin-layer chromatoplate and chromatographed in petroleum ether-diethyl ether-glacial acetic acid (72:2&l, v/v/v); bands corresponding to free cholesterol and cholesterol
ester were scraped into fluted filter papers, having
been visualized
under
ultraviolet light after spraying with a 0.2% solution of 2’,7’-dichlorofluorescein ethanol, eluted with 10 ml of toluene scintillation fluid and the radioactivity
in was
measured. After determination of post-extraction dry weight the DNA content of the tissue was determined by the method of DISCHE 15 after extraction with 5 o/otrichloroacetic
acidI6.
Determination
of [3H]cholesterol
specific
activity
and cholesterol
content
acY0s.s the aortic
wall im blue and white areas
Pigs were injected and killed
1 day later.
with autologous Each
aorta
plasma containing
was removed
1.5 mCi of [aH]cholesterol
and 100 pm
serial
sections
were
prepared from blue and white segments as described. Pooled sections were extracted with 15 ml of chloroform-methanol (2:1, v/v) for 1 h at 65°C and re-extracted with 15 ml of ethanol-petroleum ether (3:1, v/v) for 1 h at 65°C. Extracts were pooled and evaporated to dryness. At this stage, extracts from Sections 1, 2, 3, and n were treated individually while extracts from Sections 4 to (n-1) were combined and subsequently treated as a single entity. The lipid residues were chromatographed; free and esterified cholesterol were eluted with chloroform radioactivity as described. Cholesterol was determined Analysis
(3 x 4 ml) and measured for by an automated methodiT.
of data
Measurements obtained for sections across the vessel wall were treated follows: Sections 1, 2, and 3 were considered individually, the mean was taken
as for
values obtained for Sections 4 to n-l, and the last or adventitial section (n) was again considered individually. This treatment was considered justifiable since activity Atherosclerosis,
1972, 16: 377-388
380
J. B. SOMER,
TABLE
C. J. SCHWARTZ
1
FOCAL DISTRIBUTION INJECTION OFLABEL
OF [SHICHOLESTEROL
IN AORTAS
Idensity of Evans blue staining
[3H]Cholesterol
Blue
2207
White
(3) 1366 & 134
(3) 2812
&
F<
!x
0.05
PIGS AT VARIOUS
TIMES AFTER
activity (dpmlmg DNA)
after [3H]cholesterol 10 min lh
Time
f
OF YOUNG
342*
0.05
injection: 4h
5865 f
1046
9303
258
(3) 6020
1 day +
1324
13 164 *
1048
486
(6) 10 302 &
706
f
P (3) = 0.08
P<
0.05
*
Mean f S.E. Numbers in parentheses
indicate
number
of animals.
across the vessel wall from Section 4 to the adventitia was usually constant in each experiment (see below). In statistical
analysis of data for corresponding sections from blue or white
areas at each specific time interval, the paired t-test was used. RESULTS
[3H]Cholesterol uptake in blue and white ayeas of pig aorta at various times after injection of label Table 1 shows [sH]cholesterol activity, expressed as a function of tissue DNA content, in blue and white areas at 10 min, 1 h, 4 h, and 1 day after injection of label. Activity
in blue areas was consistently
differences being statistically
greater than activity
in white areas, the
significant at 10 min, 1 h and 1 day. Activity in both
blue and white areas increased with time after injection. Aortic DNA content and distribution
in blue and white areas
The DNA content of blue and white areas and its distribution through the vessel wall are shown in Table 2. There was no significant difference across the vessel wall, nor between corresponding blue and white sections. [3H]Cholesterol distribution across the aortic wall in blue and white ayeas at various times after injection of label As early as 10 min after injection of label, there was detectable radioactivity throughout the vessel wall in both blue and white areas (Fig. 1). At this time, activity in blue areas was greater than activity in white areas in all sections but the last (Table 3). 1 h after injection of label, a general increase in activity throughout the aortic wall in both blue and white areas was observed (Fig. 2, Table 3). Activity in blue areas was greater than activity in white areas in Sections 1, 2, and 3 (intima and inner media) (Fig. 2, Table 3). Atherosclerosis,
1972,
16: 377-388
FOCAL [3H]CHOLESTEROL TABLE DNA
UPTAKE IN THE PIG AORTA, PART
381
2
2
CONTENT
AND
DISTRIBUTION
ACROSS
THE AORTIC
WALL
IN BLUE
AND
WHITE
AREAS
IN YOUNG
PIGS
SectionNo.
DNA
content
(rag D.VA/g dry tissue weight) E’hite
Blue 1 2 3 Mean 4&(n-1) n Whole aorta
11.3 (17) 11.5 (18) 11.5 (18) 12.5 (18) 12.8 (18) 11.6 (11)
+ o.s7*
12.6 (17) 13.5 (18) 14.1 (18) 13.0
+ 0.87 & 0.78 += 0.87
* 0.85 + 1.06 + 1.25 * 0.97
(18) 12.7 & 0.82 (18) 13.3 & 1.11 (11)
+ 0.78 + 0.72
* Mean f S.E. Numbers in parentheses indicate number of animals.
At 4 h blue and white vessel wall, particularly
areas showed
in the intima
most of the vessel wall, the activities the first two sections adventitia)
(intima
was activity
a further
increase
in activity
across the
and inner media (Fig. 3, Table 3). Throughout in blue and white areas were similar.
and inner media),
and the last section
Only in
(outer media and
in blue areas greater than that in white areas (Fig. 3, Table 3).
B 1 SCCTION NUYBCR
5
5
7
9
11
1J
1
I5
I7
89
SCCTION NUYBCR
Fig. 1. Distribution of [sH]cholesterol activity across the aortic wall in blue areas (A) and white areas (B) 10 min after injection of label. Sections (100 pm) are numbered from intima to adventitia. Results obtained in three animals shown. Atherosclerosis,
1972, 16: 377-388
Y ;” ;
6 .
5 s
F 2 2. P
a
0
-i
?
b
3
3)
4 h (n =
* Mean
f
1 day (n =
3)
=
1 h(n
6)
3)
S.E.;
10 min (H =
Time after injection of label
N.S.,
+
f
&
5
&
20 632 2004
17 838 f 3521 0.065
non significant.
P < 0.01
38 320 4319
P =
43 532 13002
5 508 * 1616 P < 0.05
19 905 2470
2 727 863
DNA)
f
*
N.S.
+
P = 0.08
17 090 4867
P < 0.05
7 592 1343
20 325 5625
6 372 1451
2 445 484
189
879
White
+ P = 0.065
645
2 734
17 604 & 2286
f
*
i:
Blue
2
activity (dpnlmg
White
& P < 0.05
8810 1465*
BlUl?
1
Section No.
[3H]Cholesterol
f
12 682 + 731 * N.S.
4 270 + 529 & N.S.
* P < 0.05
9541 1571
4 175 1081
1736 333
125
431
White
P < 0.05
223
1022
3 059 & 599
&
Blue
3
+
&
*
8718 1706 N.S.
4313 873 N.S.
3 598 711 N.S.
&
&
&
8 280 763
4 020 642
2 139 251
108
1069
White
+ P < 0.01
_ir 121
1286
Blue
4-(%-l)
D~~TRIBUTI~N~F[~H]CHOLESTEROLA~TIVITYACROSSTHEAORTI~WALLINBLUEANDWHITEAREASINYOUNGPIGSATVARIOU~ TION OFLABEL
TABLE
&
f
f
f
N.S.
N.S.
f
94
4 930 303
3251 193
f
2 186
White
INJEC-
7 391 * 747 P < 0.001
9 999 580
+ P < 0.05
7 307 699
3 909 384
206
1871
Blue
1z
TIMES AFTER
q
FOCAL [~H]CHOLESTEROL
UPTAKE IN THE PIG AORTA, PART 2
383
30-
30-
SECTION
N”Y.tl
SECTION
NUYILR
Fig. 2. Distribution of [sH]cholesterol activity across the aortic wall in blue areas (A) and white areas (B) 1 h after injection of label. Sections (100 pm) are numbered from intima to adventitia. Results obtained in three animals shown.
;
30-
30-
0‘ 0
?
I
1 I I
‘o-
. 30-
A 1
3
0
7
SLCTION
9 NIlMIll
11
13
1s
B 1
3
3
7
9
SICTION
II
I3
I3
NUYW”
Fig. 3. Distribution of [aH]cholesterol activity across the aortic wall in blue areas (A) and white areas (B) 4 h after injection of label. Sections (100 pm) are numbered from intima to adventitia. Results obtained in three animals shown. Atherosclerosis,
1972, 16: 377-388
J. B. SOMER, C. J. SCHWARTZ
384
One day after injection of label, activity in the first two sections in blue areas had not changed, whereas activity in the remainder of the vessel wall showed a further increase (Fig. 4, Table 3). In white areas, activity in the first section was relatively unchanged, with the remainder of the vessel wall showing a further increase (Fig. 4, Table 3). At this time, there was significantly greater activity in only the first and last sections of blue areas in relation to white areas, while throughout the remainder of the vessel wall activities were similar (Fig. 4, Table 3). Distribution
of aH-free cholesterol and [3H]cholesterol
ester awoss the aortic wall 1 day
after injection of label The distribution of labelled free and esterified cholesterol across the vessel wall in blue and white areas 1 day after injection of label is shown in Fig. 5.
I.,,*,,,, *....,, A
B
??
1
,
I
7
SICTION
9
II
NUYIER
Ia
1s
1
9
s
7
9
,I
II
I9
SCCTION N”Y.99
Fig. 4. Distribution of [sH]cholesterol activity across the aortic wall in blue areas (A) and white areas (B) 1 day after injection of label. Sections (100 pm) are numbered from intima to adventitia. Results obtained in six animals shown. Atherosclerosis,
1972, 16: 377388
FOCAL [SHICHOLESTEROL
UPTAKE IN THE PIG AORTA, PART 2
385
JC
20
1c
B I,.
1
3
,
.
.
5
7
9
.
11
I
11
1
SSCTION NUMSCR
Fig. 5. Distribution areas (A) and white activity and closed bered from intima
3
5
7
9
SECTION
11
1,
I5
NUMBER
of sH-free and sH-esterified cholesterol activity across the aortic wall in blue areas (B) 1 day after injection of label. Open s,yybols denote sH-free cholesterol symbols denote sH-esterified cholesterol activity. Sections (100 ,um) are numto adventitia. Results obtained in two animals shown.
Most label was present in the form of free cholesterol. The accumulation
of
label in the intima and inner media was due almost exclusively to an accumulation of labelled free cholesterolin this region, while labelled cholesterol ester was distributed evenly throughout the vessel wall. Further, the differences in activity through the vessel wall between blue and white areas were due to differences in sH-free cholesterol activity. There was no difference in [sH]cholesterol ester activity between blue and white areas. Distribution
of 3H-free
cholesterol
s$eci$c
activity
across
the aortic
wall
in blue and
white areas
The distribution of sH-free cholesterol specific activity across the vessel wall in blue and white areas 1 day after injection of label is shown in Table 4. Free choAtherosclerosis,
1972, 16: 377-388
J.
386 TABLE
B. SOMER,
C. J. SCHWARTZ
4
DISTRIBUTION AORTIC
WALL
OF
FREE
IN BLUE
CHOLESTEROL AND
WHITE
AND AREAS
3H-FREE IN YOUNG
CHOLESTEROL PIGS
AT
SPECIFIC
ONE
DAY
ACTIVITY
AFTERINJECTION
ACROSS
THE
OFLABEL -
Section No.
3H-Free cholesterol spec. act. (dpnlmg free cholesterol) Blue
1 2 3 4-(n-1) n
147 704 72 640 39879 28073 209444
* Mean
& S.E.
* & h j=
42 os4* 27 080 13689 10890 3457
Aortic free cholesterol content (wag/gdry tissue weight) White
Blue
White
P
71675 * 18667 30 613 j= 7 227
3.36 f 0.19 3.27 _C 0.29
2.95 $= 0.50 3.56 & 0.51
P < 0.05 P < 0.2 P < 0.05
1 586
3.29 f
0.43
3.26 h 0.41
14 975 f 15273+
3 297 2603
3.33 f 0.30 4.14 & 0.10
3.57 .& 0.16 4.07 5 0.43
P < 0.05
9 344 +
of five experiments.
lesterol specific activity was greater in blue than in white areas in the intima and inner media (Sections l-3) and in the adventitia
(Section n). Specific activity was
greatest in the intima and inner media and decreased towards the adventitia. Free
cholesterol
distribution
across the aortic w/z11 in blue and white ayeas
The distribution of free cholesterol across the vessel wall in blue and white areas is shown in Table 4. There was no significant difference between blue and white areas. Cholesterol content was greatest in the last section (outer media and adventitia) and constant through the remainder of the vessel wall. Cholesterol ester content was too small to measure in almost all sections. DISCUSSION
The present series of experiments has confirmed and extended previous studies showing an increased appearance of labelled cholesterol in areas of Evans blue dye accumulation when compared with contiguous areas of no dye accumulationrsJ4. The data show that the increase in cholesterol activity in blue areas was confined to the intima and inner media with no differences in most of the remaining media (Figs 1-5, Tables 3 and 4). The data in Table 4 indicate that the differences between blue and white areas are not due to differences in cholesterol content in the intima and inner media and suggest an increased cholesterol turnover in these regions of the aortic wall in blue areas. Results obtained with the outer or adventitial sections are more difficult to interpret. At short time periods after injection of label, there are no differences in activity between blue and white areas (Table 3); however, by one day, activity in the outer or adventitial sections from blue areas is greater than that of white areas (Tables 3 and 4). A possible explanation could invoke a comparatively small uptake of label at the adventitial side, greater in blue areas than in white areas. A small amount of entrapment of blood in the vasa vasorum might mask these differences at earlier time periods. Atherosclerosis,
1972,
16: 377-388
FOCAL [~H~CHOLESTEROLUPTAKE IN THE PIG AORTA, PART 2 These cholesterol
experiments uptake
confirm
our
earlier
observations
the aortic wall. The highest proportion
the
1 and 2 (Fig. 1, Table 3). This pattern
after
of label,
(Figs 2-4,
Table
difference
in
in the appear-
radioactivity
was present
of label was located in the intimal
side in Sections injection
that
in blue and white areas is due solely to a difference
ance of labelled free cholesterol (Fig. 5, Table 4, ref. 14). As early as 10 min after injection of [3H]cholesterol, throughout
387
persisted
at 1 h, 4 h and 1 day
3). The distribution
of free cholesterol
specific activity showed a similar pattern. Specific activity was highest in the first section (in blue areas, approximately two thirds of the terminal serum total cholesterol specific activity, in white areas approximately towards the adventitia (Table 4). These results provide of the labelled
cholesterol
crossed the intima
one third) and decreased strong evidence that most
with part of it being transported
out-
ward to the adventitia. The distribution of labelled cholesterol across the aortic wall has been studied in a number of laboratories 6- 9. Evidence that cholesterol enters mainly across the intima
has been obtained
in normal
and atherosclerotic
pigeons, normal
rat7, and normal and cholesterol-fed rabbit7-9. Our results indicate true in the macroscopically normal young pig aorta. either
dog6, normal
that the same is
It would appear that in the pig, the contribution of label from the vasa vasorum, from entrapment of blood in the capillaries or from actual uptake, plays but
a small part in the appearance of labelled cholesterol in the aorta. Free cholesterol specific activity of the outer media and adventitia is only a fraction of the free cholesterol specific activity in the first or intimal section, which itself is less than the terminal serum cholesterol specific activity. If a considerable amount of entrapment of blood in and/or uptake of label from the vasa vasorum were to occur, one could reasonably expect a much higher free cholesterol specific activity in the outer portion of the vessel. Free and esterified
labelled
cholesterol
exhibit
marked
differences
in distribu-
tion across the vessel wall, with free cholesterol activity showing a maximum in the intima and inner media, while labelled cholesterol ester is distributed evenly throughout the vessel wall in both blue and white areas (Fig. 5). These observations could be explained by postulating a small uptake and uniform distribution of lipoprotein across the vessel wall, equal in blue and white areas, and sufficient to account for the relatively small amount of [3H]cholesterol ester activity observed. Alternatively, the uniform distribution of labelled cholesterol ester could reflect a small amount of cholesterol esterifying Cholesterol esterifying specieslsJ9.
enzyme activity distributed evenly throughout activity has been demonstrated in the aortas
the aortic wall of a number of
The distribution of free cholesterol across the aortic wall of the young, normal pig is not uniform, with the outer media and adventitia showing a higher free cholesterol content than the remainder of the vessel wall. Similar observations were made by DUNCAN et al.6 in the normal dog aorta. Evidence has been Fresented to indicate that the differences in uptake of Atherosclerosis,
1972, 16: 377-388
J. B. SOMER, C. J. SCHWARTZ
333 labelled
cholesterol
between
blue and white
areas are not uniform
throughout
the
vessel wall but are confined to the intima and inner both blue and white areas labelled cholesterol entered
media and the adventitia. In primarily through the intimal
surface.
These findings
observations
aortais
not homogeneous
the handling
confirm
and extend
previous
in some of its properties
including
protein
that
the normal
uptakelo-is
and
of cholesterolis*i4.
ACKNOWLEDGEMENTS
The authors and Biostatistics,
thank Mr M. Gent of the Department of Clinical Epidemiology McMaster University, for advice concerning the statistics used,
Dr F. P. Bell for many segments
helpful
and Mrs G. Couture
discussions,
Mr J. Korthuis
for able technical
for sectioning
arterial
assistance.
REFERENCES 1
2
3
4
5
3
7
3
9
10
11
12
13
14
NEWMAN, H. A. I. AND D. B. ZILVERSMIT, Quantitative aspects of cholesterol flux in rabbit atheromatous lesions, J. Biol. Chew, 1962, 237: 2078. CHRISTENSEN, S., Transfer of labelled cholesterol across the aortic intimal surface of normal and cholesterol-fed cockerels, J. Atheroscler. Res., 1964, 4: 151. BELL, F. P., H. B. LOFLAND AND N. A. STOKES, Cholesterol flux in vifro in aortas of cholesterolfed and non-cholesterol-fed pigeons, Atherosclerosis, 1970, 11: 235. LOFLAND, H. B. AND T. B. CLARKSON, The bi-directional transfer of cholesterol in normal aorta, fatty streaks and atheromatous plaques, Proc. Sot. Exe. Biol. Med., 1970, 133: 1. SCHWENK, E. AND D. F. STEVENS, Deposition of cholesterol in experimental rabbit atherosclerosis, Proc. Sot. Exp. Biol. Med., 1960, 103: 614. DUNCAN, L. E. AND K. BUCK, Passage of labelled cholesterol into the aortic wall of the normal dog, Circ. Res., 1959, 7: 765. ADAMS, C. W. M., 0. B. BAYLISS, A. N. DAVISON AND M. Z. M. IBRAHIM, Autoradiographic evidence for the outward transport of [sH]cholesterol through rat and rabbit aortic wall, J. Pathol. Bact., 1964, 87: 297. ADAMS, C. W. M. AND R. S. MORGAN, Autoradiographic demonstration of cholesterol filtration and accumulation in atheromatous rabbit aorta, Nature, 1966, 210: 175. ADAMS, C. W. M., S. VIRhG, R. S. MORGAN AND G. C. ORTON, Dissociation of [sH]cholesterol and issI-labelled plasma protein influx in normal and atheromatous rabbit aorta, J. Atheroscler. Res., 1968, 8: 679. MCGILL, H. C., J. C. GEER AND R. L. HOLMAN, Sites of vascular vulnerability in dogs demonstrated by Evans blue, Arch. Pathol., 1957, 64: 303. FRIEDMAN, M. AND S. 0. BYERS, Endothelial permeability in atherosclerosis, Avch. Pathol., 1963, 76: 111. PACKMAN, M. A., H. C. ROWSELL, L. JBRGENSENAND J. F. MUSTARD, Localized protein accumulation in the wall of the aorta, Exp. Mol. Pathol., 1967, 7: 214. SCHWARTZ, C. J., E. E. NISHIZAWA, J. B. SOMER AND J. F. MUSTARD, Focal [3H]- and [14C]cholesterol accumulation in the pig aorta, Circulation, 1968, 38: Suppl. 6: 22. SOMER, J. B. AND C. J. SCHWARTZ,Focal [sH]cholesterol uptake in the pig aorta, Atherosclerosis, 1971, 13: 293.
15
16 17
13
19
DISCHE, Z., Color reactions of nucleic acid components, In: E. CHARGAFF AND I. N. DAVIDSON (Eds.), The Nucleic Acids, Vol. 1, Academic Press, New York, 1955, p. 285. SCHNEIDER, W. C., Phosphorus compounds in animal tissues, J. Biol. Chem., 1955, 161: 293. BLOCK, W. D., K. J, JARRET AND J. B. LEVINE, An improved automated determination of serum total cholesterol with a single color reagent, Clin. Chew, 1966, 12: 681. ABDULLAH, Y. H., C. C. ORTON AND C. W. M. ADAMS, Cholesterol esterification in human and experimental atheromatous lesions, J. Atheroscler. Res., 1968, 8: 967. ST. CLAIR, R. W., H. B. LOFLAND AND T. B. CLARKSON, Influence of duration of cholesterol feeding on esterification of fatty acids by cell-free preparation of pigeon aorta, Circ. Res., 1970, 27: 213.
Atherosclerosis,
1972, 16: 377-388