- Email: [email protected]
Cholesterol diet and permeability of rabbit aorta
EXPISRIMICNTbL
AND
Cholesterol
YOLI.:CUL.lR
Diet
PATHOLOGY
and
VLADIMIR Department Biochemistry,
of Pathology, Boston
14, 20-29
(1971)
Permeability
STEFANOVICH
University University Received
Hospital School August
of Rabbit
Aorta”’
AND IRA GORE” and Departments
of Medicine,
Bosto,t[,
of Pathology
and
Mnssuchusetts
17,19?0
During dietary atherogenesis in rabbits the, pcrmeabilit)of the nortic wall to “‘I-labeled albumin increases to a maximum 3 vc-e&s nftcr initiating cholesterol feeding. This change which involved thr entire length of the aorta, preceded the development of grossl;v apparent intimal discuse and persisted throughoul the 12 weeks of the study. It is suggested that increasrs in 15-aopllospllolipids may be related to altered pcrmenbility. Other biochemical findings accompanying ntherogenesis were incrc-ases of acid mucopolysaccharidrs in distal srgmcxnt of rabbit aorta, with diminished content of chondroiiin sulfate B in proximal hcgment, and increases in total phospholipids.
Many believe t,he incorporation or passageof circulating lil)oprotcGus into the arterial wall to be the most critical event in the dcvelopmcnt of atherosclerosis (Page, 1964). The identification of P-lipoproteins and globulin in the :~tlicrosclerotic human aorta strongly supports this concept (Kao and Wissler, 1965; Walton and Williamson, 1968; Adams et al., 1967; IV’ntts, 1963). =2sa ~on~equc~ncc,thcarc: is a need for more quantitative studies on the passage of labclecl lipoprot~cins or other macromolecules into arterial tissue. Duncan et al. (1959 and 1962), using lCjlI-albunlin in (logs, found that t)he rutc: of entrance of albumin into the canine aortic wall decreasesad the distance from the aortic valve increases. Furt,hermorc, an increase in blood presaurc :~cccleratcs the passageof albumin into the proximal portion of the aortic intima. In hyperlipcmic rabbits, however, Okishio (1961) did not obscrvc altercld pcrmc~ability with radioioclinated albumin, but he did find an increased ratio of the transfer of radioiodinated low density lipoproteins. Usin,(7Evans blue and Trypan blue as indicators of permeability, Friedman and Byers (1963) found that the permeability of rabbit aortas increaseswith the appearance of early at8hero~cleroticplaques. Packham, et al. (1967j observed similar pat’terns of accumulation of ‘“lI-labeled serum albumin and Evans blue in t,he aortic intima of normal pigs and rabbits. The accumulation was greater in the thoracic segment than in the abdominal, but within the thoracic portion there was variability in the l)attcrn. The recent study by Adams et al. (1968) indicates higher aortic permeability in atherosclerotic rabbits. It suggests. further, that in the normal and mildly atherosclerotic rabbit 1 Part, of these data were included in a preliminary report at the annual meeting of tl,c Fedrration of American Societies for Experimental Biology, Chicago, Illinois:. 1967. n This work was sul)l)orted by Public Health Scrvicc Research Grant HE-07327. 3 Present Address: Department of Pathology. Unircrwity of Alabama Medical Ccntc,r, Birmintrhnm. .Ilahamn. 20
ATHEROGENESIS
AND AORTIC
PERMEABILITY
21
aorta, albumin and a, y-globulin mainly enter the vessel wall from the advential surface, e.g., vasa vasorum. Conversely, in some severely atherosclerotic aortas, the plasma proteins appear to leak into the vessel wall directly from the lumen. Our purpose in this study is to quantitate permeability during the development of the atherogenesis in rabbits and to relate changes in permeability to biochemical alterations at the arterial wall. Using rabbits fed a cholesterol diet, aortic permeability was determined wit’h 1311-albumin, gross intimal diseasewas evaluated, and aortic acid mucopolysaccharides and phospholipids were measured after 3, 7, or 12 weeks of dietary treatment. MATERIALS
AND
SIETHODS
Five groups of New Zealand, albino, female rabbits weighing 2.0 t’o 3.2 kg. were used. Each group consisted of 10 rabbits. The first group had been fed a stock diet (Purina Rabbit Chow, Ralston Purina Company, St. Louis) and was sacrificed at zero days of the experimental period. The second, t,hird, and fourth groups were maintained on an atherogenic diet (prepared from 1: 4: 45 w/w ratio of cholesterol, corn oil (Mazola) and Purina Rabbit Chow) administered ad Zibitum for 3, 7, and 12 weeks, respectively. A fifth group was maintained on a stock diet only, during the ent’ire experimental period (12 weeks). At the end of each experimental period levels of 1311-albumininjected 24 hours earlier, and free and esterified cholesterol in plasma were determined. Determination of aortic permeability in viva was performed in the following manner: 5 mCi of 1311-albumin (RISA, Abbot Laboratories, Chicago, Illinois) was injected into a rabbit (five rabbits were required, one for each experimental group). After 24 hours the animal was sacrificed by air injection (20 ml) and bled to yield a maximum volume of blood. Heparin (3 mg, 123 USP units/mg, The Upjohn Company, Kalamazoo, Michigan) was immediately added and plasma was then dialyzed in Visking tubing in 500-ml beaker against 400 ml distilled water with constant stirring by magnetic stirrer. Water for dialysis was changed twice, and dialysis was ended after 24 hours. The activities of the tenate and of the total dialysate were counted in a well-type scintillation counter. In this manner it was established that only 0.80 to 0.85% of radioactivity was dialysable. This activity, which could be separated by dialysis from 1311-albumin, consists of ionic iodine and iodinated amino acids. The purified plasma obtained by the above procedure (Duncan et al., 1962) and containing labeled 1311-albuminwas then injected into the ear vein of each rabbit, usually 5 ml of plasma per animal. After exactly 24 hours, 2 ml of blood were taken from the rabbit with a heparinized syringe. The animals were sacrificed by air injection (20 ml) and the aorta immediately dissected out, stripped free of adventitial tissue, opened longitudinally, washed five times with 2 ml of distilled water until washings were devoid of activity and separat’ed into the abdominal and arch-thoracic portions. The arch and thoracic segments were combined and referred as the proximal segment; the abdominal aorta, as the distal segment. TO count the radioactivity present in 0.1 ml of plasma and in the aortic segment (proximal and distal), a well-type scintillation counter with an automatic scaler was employed. Samples were counted to an accuracy of 2%. The ratio of the aortic
22
STEFAKOT’ICH
,iSD C:Ol
counts (per gram of aortic t&sue1 to t)lie counts in 0.1 ml of pl:~mw was calculatccl and expressed as an index of aortic pcrmeahility. More than 9076 of 1311-act,ivitJ in the plasma ant1 in the tissue was precipitablc with trichloracet,ic acid. The concentration of serum albumin did not change during t,he athcrogeuic diet and therefore the specific activity of l”‘I-all~uniin ~~21sidentical in c~xpc~riincntal anal control groups. The free cholesterol and cholcsteryl esters were determined in rabbit plasma in the following manner: Lipids were extracted and purified using Folch’s procedure (Folch et al.] 1957 1; free cholesterol and cholesteryl esters were separntcd by thin layer chromatography (Mangold, 1961’). Quantitative determination was accomplished by gas liquid chromatography using an F&M model 402 gas chromategraph with hydrogen flame ionization detector; for free cholestcbrol, a column with 6% SE-30 coated on Diatoport S (60-80 meshi was u~d ; column ov(‘n temperature was 23O”C, and carrier gas nitrogen at 5 psi. Cholestcryl esters were detcrmined as the fatty acid methyl esters, after transnietliylation with 10°C horoll t,rifluorjde in methanol and using a column with 6 % diethylcne glycol succinntc coated on Diat)oport S (SO-80 mesh j. Column OVC’II temperature was progranunctl from 120 to 200°C; nitrogen was 5 psi. Reference compounds: cholcstanc for frcct cholesterol and corresponding fatty acid methyl esters for choleeteryl esters were run simult.aricously with experimental ~amplcu. The percentage of the aortic surface involved with atherosclerotic lesions was evaluated planimetrically. The determination of aortic l~hosl~l~olil~id concentration was accompli:~hcd in the following manner: The aortic segments were minced with scissors aud homogenizcd at 0°C with chloroform:nic~tlianol (2: l., v/v’) in the ratio of I gm. of wet tissue to 20 ml of solvent, using a glass to glass dual action homogenizer. The homogenized sample was filtered through Whatman #l filter paper aucl w:t.shctl with distilled water saturated with a chloroform:methanol (2: 1 V/IT) mixt’urc. Ethanol-ether (3: 1 v/v), 75 ml of solvent for 1 gm. of tissue, was added to the residue which was then left in the dark for 48 hours at room temperature. Roth extracts were combined, cvaporatcd to dryness by flash cvaporalion (5O”C), rcdissolved in chloroform-methanol mixture (20 mlj , and used for determination of the phospholipids. A 10 ml aliquot of total lipid extract from the aortic sc>gment was evaporated to dryness under nitrogen and was redissolved in 0.4 ml of a chloroform-methanol solution (2: 1 v/v,. Phospholipids were separated into components in the following manner: 20 to 40 ~1 was applied on a thin layer plate coated with 0.25 mm silica gel H (Merck, A. G., Darmstadt, Germany). Silica get H was purified essentially by the method of Parker and Peterson (1965)) but in our procedure perchloric acid rather than formic acid was employed. I-sing two solvent systems for development, phospholipids were separated. System I consisted of petroleum ether, ethyl ether, and acetic acid (90: 10: 1 v/v). When the solvent front reached 15 cm, the plate was taken out, air dried for 2 to 3 minutes, and subjected to thin layer chromatography in System II. This consisted of chloroform, methanol, and water (62:25:4 v/v). Iodine vapor was employed to locate separated components, and individual phospholipids were identified using corresponding reference compounds. Silica gel containing separated components was scraped into test tubes and individual phospholipids were determined. Essentially.
ATHEROGENESIS
iZND
AORTIC
TABLE CHANGES
IN
Weight of rabbit ikg)
Diet
AORUC
PICRMK.IBILITP
Tissue weight (ma)
Athlerosclerotic involvemerit (%)
23
PERMESBILITI
1 IN
Fm
RABBITS
lyo
CHOLF:STEROL
Cont. of total ‘Tissue cholesterol in Plasma ‘311 ‘3’1 count plasma (mg/ Tissue ‘311 __ 100 ml) and 0.1 ,(~P$&, (cpm/g X 1OF “?;%eability esterified index) cholesterol (‘3%)
Prnxinml segmelll Stock Cholesterol weeks Choles-
3
terol weeks Choles-
i
terol
12.3
f
0.1+3ci
*
2.3
+
0.10 ’ 393 f
2.5
+
0.1
37 1 36
(70% 2.8
f
0.2c
2.4
zk 0.1
59.3
12
weeks Siock 12
173’2 zk 105” 25.X ((ii’; ) 01 k
0
1
3
27.9
xk 1.131.J
zk 2.01.33
+
0.03b
zk 1.921.0
&
+
0.05
1.50.75
( (615,) /
weeks
h IIifference
hetweell
stock
group
alrd
esprrimental
group
is
P < .OOl. I’ IXfference
between
stock
grorlp
nl~d
esperimelrtd
group
is.stat
hetweet)
stock
group
nuti
experimental
group
is statistically
P
< .Ol. d Ijifference
r
<
statistically istically
significant
at
significant
at
significant
at
.05.
the method of Barrlett (1959) was employed for determination of phosphorous. The results were usually expressed as micrograms of phosphorous per milligram of dry, delipidized tissue, Acid mucopolysaccharides were determined in the dry residue obtained after lipid extraction of aortic tissue by the method of Stefanovich and Gore (1967). RESULTS The d$a in Table I are relevant to the study of permeability for the proximal segment of the aorta under various expcrimcntal conditions. It is clear from this table that the body weights and the weight of the proximal segments of the aortas are significantly increased only a,fter 12 weeks of an atherogenic diet. A significant increase in permeability (the rat,io of plasma/tissue counts) is observed aft’er three weeks of cholesterol feeding. At this time, aortic involvement in an atherosclerotic lesion is rather minimal (2.3%). The increased permeability remained, however, at the same level throughout the 12 weeks of study. At the end of this period, the aortic involvement is maximal (59.3%)) but there is no significant change in permeability from 3 to 12 weeks of cholesterol feeding. All control animals showed similar levels of permeability during the experiment. In all statistical evaluations cholesterol diet groups were compared with control groups sacrificed at zero days of the experimental period.
24
STEFANOVICH
AND
Distal
Stock
2.3
Choles2.3 terol 3 weeks Choles2.5 ted 7 i weeks 2.x Cholesterol 12 weeks
+
0.1a 1109 z!z 13 lo/
& 0.1
143 zt
! x
segment
(i0
2.0
lob
GORE
‘1x19 I
+ 1 (CiO' ; )
‘16.5
f 1lXC 11.6 (7.3“;’ )
zt
0.1
172 *
I
15.0
I 1867 *
lW21.1
&
0.2
?5(i * ?3Ji
55.3
,17x2
105c “5.X
zt 1.51:3.0
f
1.10.n
f
0.W
It
f
1.91.71
l
0.15c
1.l:SS.S ’
f
0.91.3ti /
+
o.oxc
.3
*
1.01.x5
f
0.W
0.311!).!) I
f
1ooc’i )( ~ 1 +
+
47.3
b 1)ifferencr P < .Ol.
between
stock
group
alltl
experirrlrlltnl
gruup
is stat islically
signific:~llt
at
d I)ifferenre P < .05.
between
stock
group
atld
experimental
group
is slxtistically
signifimllt
at
In the distal segment’ (Table II) the observations were similar to those seen in the proximal segment,. The permeability index however at 7 weeks was lower than at 3 weeks and at 12 weeks. The difference in the permeability of the dist’al segment between 7 and 12 weeks of atherogenic diet was statistically significant (P < .OOl). When the permeability indices at 12 weeks at atherogenic diet are compared for distal and proximal segments, the former shows statistically significant increase in permeability (P < .OOl). It is possible to suggest &at this increased permeability is related to the relatively high degree of aortic atherosclerotic involvement for the distal segment (55.3%) which was in our study rat,hcr close to the involvement of the proximal segment (59.3%). Usually in rabbits on atherogenic diet, the proximal aortic segment is involved in the atherosclerotic lesion to a significantly higher degree than the distal segment. The degree of atherosclerotic involvement for the distal segment was similar to t#hat observed for the proximal part. The degree of atherosclerotic involvement for the distal segment was similar. The proximal segment (Table III) showed a highly significant increase in phospholipid concentrations (calculated as ,LL g of phosphorous per 100 mg of lipids) after 7 or 12 weeks of cholesterol feeding. This increase was also observed after 12 weeks of stock diet, especially in the distal aortic segment. In the atherogenic diet groups, however, there appeared a significant concentration of lysolecithin and
ATHEROGEKESIS
AND AORTIC TABLE
AORTIC
PHOSPHOLIPIDS
Phosphorus bg/100 mg wet tissue)
Diet
IN
Phospholipids (Irg of P/100 mg of lipids)a
III
RABBITS
24.8 25.0 33.7 18.3 28.9
z!r ;t * f f
2.05b 3.2 4.Y 3.2# 1.2
a P = phosphorus b Mean zt SE of mean. ’ Difference between stock group d Difference between stock group ’ Difference between stock group
366 372 651 1175 552
3z zt f f zk
69 88 86d
12od SSd
Llnlecithinl
‘$F$fi-
and experimental and experimental and experimental
Diet
-I
PHOSPHOLIPIDS
Phosphorus OIgjlOO mg wet tissue)
group group group
IN
a b c d c
11.4 27.4 18.0 15.7 17.0
zt f zt f *
P = phosphorus. Mean zt SE of mean. Difference between stick Difference between stock Difference between stock
I.+ 2.4c 3.2 5.2 2.2’
group group group
26.0 21.3 37.0 26.4 25.0
i f ;f; i zt
15 1’ 15* 16d 10d
and experimental and experimental and experimental
(‘%)
Lecithin
I ,,“iziin
1
Cephalin
zk f * * zk
6.6 1.6 l.gd 4.4 1.8
37.0 40.0 13.3 27.0 32.0
f * zt * f
significant significant significant
8.3 3.4 z.2d 2..+ 1.5
3.2 6.3 14.3 17.0
0 3~ z!z f f
24.6 22.6 20.4 18.0 25.0
0.4 0.8 0.8 1.1
z!z f f f f
11.5 2.1 1.6 z.ad 0.7
at P < .05. at P < ,001. at P < .Ol.
IV FJGD
IT,
CHOLESTJGROL
Phospholipids
Phospholi ids Gg of P?lOO me of lipids) Lysolecithin
173 408 462 552 482
/
is statistically is statistically is statistically
RABBITS
Distal Stock Cholesterol 3 weeks Cholesterol 7 weeks Cholesterol 12 weeks Stock 12 weeks
CHOLESTEROL
segment
0 10.5 zk 1.4 21.0 A 2.2 19.8 j, 2.4 0
TABLE AORTIC
1%
FED
Phospholipids
Proximal Stock Cholesterol 3 weeks Cholesterol 7 weeks Cholesterol 12 weeks Stock 12 weeks
25
PERMEABILITY
“gpg-
(
(%)
Lecithin
( ,t$&
1
Cephalin
segment
0 6.5 zt 2.8 4.4 It 0.9 1.8 i 1.8 0
group group gulp
27.4 27.5 28.0 22.0 24.4
+ 2.3 f 1.1 * 1.9 f 4.3 f 2.ge
is statistically is statistically is statistically
44.6 39.2 45.0 25.7 41.6
significant significspt significant
* zt zk zt 4~
1.3 2.4 1.8 4.0c 3.0c
5.5 4.0 17.0 11.0
0 f * f f
0.7 0.9 0.9 1.3
20.6 21.3 14.8 18.0 22.5
f f f f zt
2.3 1.3 2.0’ 1.4 2.8
at P < ,001. at P < .Ol. at P < .05.
lysocephalin, even after 3 weeks of diet. A similar situation was observed in the distal segment (Table IV) where the relative increase in phospholipids was observed after 3 weeks of atherogenic diet and continued up to 12 weeks. In both proximal and distal segments of the aorta of rabbits fed for 12 weeks on the stock diet, phospholipids were increased and some amounts of lysocephalin were noted. In regard to lysolecithin, there was always a significantly higher concentration of this phospholipid in the proximal segment then in the distal segment and at 7 and 12 weeks of cholesterol diet this difference in lysolecithin content between the proximal segment and the distal segment was statistically significant (P < .OOl) . This could indicate perhaps a somewhat different metabolism in the distal than in the proximal segment. There was a significant decrease in aortic lecithin in the proximal segment particularly at 7 weeks and also at 12 weeks of the cholesterol diet. In the distal segment a significant decrease in lecithin was observed only after 12 weeks of the cholesterol diet, but not after 7 weeks. This data could suggest that the proximal segment is more susceptible toward at,herosclerosis than
26
STEFANOVICH
Die1
.UIPS acid
AX-D
GORE
as uranic img;‘g)
the distal segment. It also agrcc~ with t,he higher concentration of lysolec~ithin obscrvetl in the proximal scgmcnt at 7 and 12 weeks of the atherogcnic dkt. Total acid mucopolysaccharide contcnnt (determined a:: uranic acid) did not change during atherogriiic lwriods in the prosimal segment (Tahlc VI, but :t significant increase \vas obscrrcd in the distal part for the 3%and 12%wek groups (Tnblc VI). Thcrc VW a constant clecrcnw in the concentration of choutlroitiu sulfat,c B in proximal wgnwntP iu rabbits ftid cholesterol. However. a d~crraw of cliondroitin ,sulfatc B was also noted in tlios:c rabljits fchclstock clkt for 12 w.c~~ki.
It is gvnerally argued that’ the accun~ulation of certain lipiclr in the intima of diseawtl nrteriw is partially due to increased ent,ry of p-lipoprotcin~. Clinical obxcrrations indicat.c the importaucc of thcl plasma lipid rontcnt. It is also possiblc that the altered permc~ahility of arterial endothelium contributes to these changes. Although t#lle molecular weight of human serum albumin used in the present study is lower than that of P-lipoprotein [69 x lo4 for human wrum albumin and 5-20 X 10” for l)rc-P-lipoprot,~iil 6 0.98-1.002 (Putnam, 1965 )] they are both significantly large macromolecules and, it can be presumed for purposed of this st,udy, that similar mechanisms arc involved for traversing the endothelium. It is pertincnt in this respect, moreover, that some lipids (e.g., lyaolccithin and free fatty acids) are bound and transported by albumin 18witzer and Etlcr, 1965; Fredrickson and Gordon, Jr., 1958). Our results indicate that the permeability (ratio of pla.wla/‘nortic tissue+ lwlIalbumin concentration) of the aortic wall increased significantly after only 3 wcck~
BTHEROGENESIS
.4&-D
AORTIC
TABLE Ac~o Diet stock Cholesterol 3 weeks Cholesterol 7 weeks Cholesterol 13 weeks Stock 12 weeks
MUCOPOLYS.~CCHARIDES
AMPS acid
as uranic (w/d
IN
VI SEGMJ
PROSIM.ZL
HS (y;)b
HS (%ja
27
PERMEABILITY
OF R.~~F~ITs CS-B
(s,)<
AORT.~ cs-c(y~y~
3.44 4.31
f l
0.15e 0.21r
13.0 14.9
f f
0.4 1.1
36.4 32.4
+ *
0.7 l.lQ
26.1 25.6
f *
1.1 0.x
24.5 27.0
+ f
1.2 0.x
3.47
f
0.12
12.1
+
0.7
34.4
*
0.8
2Y.8
f
0.9
24.7
+
1.1
3.99
*
0.13Q
13.x
+
1.2
29.5
+
1.3*
29.4
+
1.7
27.3
f
1.5
4.25
f
0.W
15.1
f
1.4
31.3
*
0.5*
20.4
zt
1.08
34.3
f
1.1
n HA = hyaluronic acid. 6 HS = heparitin sulfate. c CS-B = choudroitiu sulfate B. r( CS-C = chondroitin sulfate C. p ,Mean and SE of mean. ’ Difference between stock group P < .Ol. g Difference between stock group I’ < .05. h 1)ifferenre between stock group P < .OOl.
arLd experimental
grollp
is statistically
significant
at
and
experimental
grollp
is statistically
significant
at
and
experimental
group
is statistically
significaut
at,
of cholesterol feeding. By this time only minor observable gross atherosclerotic changes had developed in the aorta. It is known that some minor changes can appear in the arterial wall after 2 weeks of an atherogenic diet, as observed by light and electron microscopy (Parker and Odland, 1966). Still, it is important to note that in our present experiment significantly increased permeability precedes any dramatic damage to the aortic wall in rabbits fed the atherogenic diet. The permeability, rather than increasing progressively with the continuation of the diet for the additional 9 weeks, appears to stay approximately at the same level regardless of the significant increase in involvement of the aorta by atherosclerotic lesions (e.g., 2.3 vs. 59.3% in the proximal segment at 3 and 12 weeks of cholesterol diet respectively). It should also be noted that in our experiment the atherosclerotic involvement of rabbit aortas was similar for both the proximal and distal parts. The appearance of lysolecithin and lysocephalin seems to be the most significant and outstanding chemical change resulting from only 3 weeks of cholesterol feeding. One can speculate that the appearance of lysolecithin and lysocephalin, which are more water soluble than the corresponding original compounds, increase osmotic pressure within the cell. The experimental results of Kellaway and Saunders (1967) may be pertinent here. They found that the quantity of progesterone solubilized in an ultrasonically irradiated aqueous solution increased when lysolecithin was included. It is possible that the increased concentration of lysolecithin favors both solubilization and dispersion of the other lipids present in the cells of arterial tissue, thus further increasing the osmotic pressure within the cell. This, in
28
STEFANOT’ICH
AND
GORE
turn, may cause water-carrying macromolecules to permea,t,e such cells.* Furthermore, the membrane lecithin may be hydrolyzed to water soluble lysolecithin which may provide abnormal “openings” in the membrane, facilitating the rupture of such membranes. The release of p-glucuronidase attached to the membrane was observed in lysosome granules by Weissman et al. (1964) with concentrations of lysolecithin as low as 2 X low6 M. The origin of lysolecithin and lysocephalin in the aorta tissues of rabbits fed an atherogenic diet remains unresoh-ed. However, another view can be proposed. This explains that at least the appearance of lysolecithin may be due to the action of the lecithin-cholesterol acyhraneferase. Such a mechanism for cholesterol est’erification in rabbit aortic tissue was recently reported (hbdula et al., 1968) ..i An hypothesis for the etiology of dietary atherosclerosis can be proposed from the above discussion: /?-lipoproteins, carrying free cholesterol, enter the intimal layer of the aortic wall. Some quantity of cholesterol becomes esterified at the cell membranes. For this esterification, lecithin (and perhaps cephalini serves as a donor of fatty acids, simultaneously producing water soluble lysolecithin which is then ‘keleased” from the cell membrane. This may cause the disruption of the membrane. Lysolecithin may also produce an increase in osmotic pressure due to its solubility and its ability to solubilize other lipids. The final result may be an increased influx of ,&lipoproteins, carrying the excess cholesterol which is then continually esterified, ultimately causing an abnormal accumulation of cholesteryl esters within the cell. Such an hypothesis is consistent with the pathological manifestations of the early disease process. ACKNOWLEDGMENT We are indebted technical assistance
to Dr. L. C. Fillios for providing several of Mr. D. Shapiro is greatly appreciated.
helpful
comments.
The
excellent
REFERENCES T. transacylation 973.
ABDULLA,
ADAMS,
arterial ADAMS,
H.,
ORTEN,
in human
C. C., and ADAXS, and experimental
C. W. M. atheromatous
(1968). Cholesterol esterificntion by lesions. J. dtheroscler. Res. 8, 96i-
M., BAYLISS, 0. B.. and ORTOS. C. C. (1967). Plasma prokin accumulation in degeneration. J. Atheroscler. Res. 7,473-489. C. W. M.. VIRM. S., MORGAS. R. S.. and OSTOS, C. C. (1968). Dissociation of 3HC. W.
’ It should also be understood that there appears to be a relative increase in the tritiated water influx in atherosclerotic rabbit aortas compared with normal aortas. Two rabbits, one healthy and one atherosclerotic were injected with the same amount of tritiated water. Both rabbits were sacrificed and the aortas were immersed into separate beakers of distilled water. Water was changed three times and radioactivity in the water counted. The norta of the atherosclerotic rabbit “released” 92% more tritiated water than the aorta of the normal rabbit in respect to 1 gm of wet tissue. 5 In our experiment when employing lecithin with 1-l”C-labeled oleic acid in the C-2 position of lecithin, 4% (mean of three experiments) of the labeled acid was recovered as a cholesteryl ester after incubation with an homogenate of rabbit aorta. The homogenate was prepared by homogenizing rabbit aortas (1 gm./lO ml) in Tris buffer (0.1 M, pH 8.0). The supernatant obtained after centrifugation at 1OOOg. for 15 minutes was discarded and the residue in 0.1 M, pH 6.8 phosphate buffer (1 gm./l ml) was incubated for 18 hours at 37” C with labeled lecithin.
ATHEROGENESIS
AND
AORTIC
PERMEABILITY
29
cholesterol and l9-labeled plasma protein influx in normal and atheromatous rabbit aorta. J. Atheroscler. Res. 8, 67S696. BARTLETT, G. R. (1959). Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466471. DCNCAN, L. E. JR.! CORSFIELD, J.: and BUCK. K. (1959). Circulation of labeled albumin through the aortic wall of the dog. Circ. Res. 7,390-397. DUNCAN, L. E. JR., CORNFIELD, J., and Bucrc. K. (1962). The effect of blood pressure on the passage of labeled plasma albumin into canine aortic wall. J. Clin. Invest. 41, 1537-1545. FRIEDMAN, M., and Byers, S. 0. (1963). Endothelial permeability in atherosclerosis. Arch. Pathol. 76,99-105. FREDRICKSON, D. S.. and GORDON, R. S. JR. (1958). Transport of fatty acids. Physiol. Rev. 38,585X30. FOLCH, J., LEES, M., and SLONE-STANLEI., G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226,497-509. GORE, I., and LARKEY. B. J. (1960). Functional activity of aortic mucopolysaccharides. J. Lab. Clin. Med. 56, 839-846. KAO, V. C. Y., and WISSLER, R. W. (1965). A study of the immunohistochemical localization of serum lipoproteins and other plasma proteins in human atherosclerotic lesions. Ez~. Mol. Pathol. 4,465479. KELLAWAY, I. W., and SAUNDERS. L. (1967). The solubilization of some steroids by phosphatidyl choline and lysophosphatidyl choline. Biochim. Biophys. Acta 144, 145-148. MANGOLD, H. K. (1961). Thin-layer chromatography of lipids. J. Amer. Oil Chem. Sot. 38, 708-727. OKISHIO, T. (1961). Study on the transfer of ‘“‘I-labeled serum lipoproteins into the aorta of rabbits with experimental atherosclerosis. Med. J. Osaka Univ. 11, 367-381. PACKHAM, M. A., ROSWELL, H. C., JORGENSEN, L., and MUSTARD. J. F. (1967). Localized protein accumulation in the wall of the aorta. Exp. Mol. Pathol. 7,214-232. PAGE, I. H. (1954). Atherosclerosis: an introduction. Circulation 10, l-27. PARKER, F., and PETERSOS, N. F. (1965). Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin layer chromatograms. J. Lipid Res. 6,455-460. PARKER, F., and ODLAXD, G. F. (1966). A correlative histochemical, biochemical, and electron microscopic study of experimental atherosclerosis in rabbit aorta with specific reference to the myointimal cell. Amer. J. Pathol. 48, 197-239. PUTPI’AM, F. W. (1965). Structure and function of the plasma proteins. In “The Proteins” (H. Neuoth. ed.), 2nd ed., Vol. III. Academic Press, New York: N. Y. STEFANOVICH, V., and GORE, I. (1967). A micromethod for the determination of acid mucopolysaccharides in vascular tissue. J. Chromntog. 31,473-478. SWITZER, S., and EDER, H. A. (1965). Transport of lysolecithin hg albumin in human and rat plasma. J. Lipid Res. 6,506511. WALTON, K. W.. and WILLIAMSON, N. (1968). Histological and immunofluorescent studies on the evolution of the human atheromatous plaque. J. Atheloscler. Res. 8,599-624. WATTS. H. F. (1963). Role of lipoproteins in the formation of atherosclerotic lesions. In “Evolution of the Atherosclerotic Plaque” (R. J. Jones, ed.). Chicago University Press. Chicago, Ill. WEISWAN, G., BECKER. B.. and THOWS, L. (1964). Studies on lysosomes V. The effects of streptolysins and other hemolytic agents on isolated leurocyte granules. J. Cell Biol. 22,
115-126.
AND
Cholesterol
YOLI.:CUL.lR
Diet
PATHOLOGY
and
VLADIMIR Department Biochemistry,
of Pathology, Boston
14, 20-29
(1971)
Permeability
STEFANOVICH
University University Received
Hospital School August
of Rabbit
Aorta”’
AND IRA GORE” and Departments
of Medicine,
Bosto,t[,
of Pathology
and
Mnssuchusetts
17,19?0
During dietary atherogenesis in rabbits the, pcrmeabilit)of the nortic wall to “‘I-labeled albumin increases to a maximum 3 vc-e&s nftcr initiating cholesterol feeding. This change which involved thr entire length of the aorta, preceded the development of grossl;v apparent intimal discuse and persisted throughoul the 12 weeks of the study. It is suggested that increasrs in 15-aopllospllolipids may be related to altered pcrmenbility. Other biochemical findings accompanying ntherogenesis were incrc-ases of acid mucopolysaccharidrs in distal srgmcxnt of rabbit aorta, with diminished content of chondroiiin sulfate B in proximal hcgment, and increases in total phospholipids.
Many believe t,he incorporation or passageof circulating lil)oprotcGus into the arterial wall to be the most critical event in the dcvelopmcnt of atherosclerosis (Page, 1964). The identification of P-lipoproteins and globulin in the :~tlicrosclerotic human aorta strongly supports this concept (Kao and Wissler, 1965; Walton and Williamson, 1968; Adams et al., 1967; IV’ntts, 1963). =2sa ~on~equc~ncc,thcarc: is a need for more quantitative studies on the passage of labclecl lipoprot~cins or other macromolecules into arterial tissue. Duncan et al. (1959 and 1962), using lCjlI-albunlin in (logs, found that t)he rutc: of entrance of albumin into the canine aortic wall decreasesad the distance from the aortic valve increases. Furt,hermorc, an increase in blood presaurc :~cccleratcs the passageof albumin into the proximal portion of the aortic intima. In hyperlipcmic rabbits, however, Okishio (1961) did not obscrvc altercld pcrmc~ability with radioioclinated albumin, but he did find an increased ratio of the transfer of radioiodinated low density lipoproteins. Usin,(7Evans blue and Trypan blue as indicators of permeability, Friedman and Byers (1963) found that the permeability of rabbit aortas increaseswith the appearance of early at8hero~cleroticplaques. Packham, et al. (1967j observed similar pat’terns of accumulation of ‘“lI-labeled serum albumin and Evans blue in t,he aortic intima of normal pigs and rabbits. The accumulation was greater in the thoracic segment than in the abdominal, but within the thoracic portion there was variability in the l)attcrn. The recent study by Adams et al. (1968) indicates higher aortic permeability in atherosclerotic rabbits. It suggests. further, that in the normal and mildly atherosclerotic rabbit 1 Part, of these data were included in a preliminary report at the annual meeting of tl,c Fedrration of American Societies for Experimental Biology, Chicago, Illinois:. 1967. n This work was sul)l)orted by Public Health Scrvicc Research Grant HE-07327. 3 Present Address: Department of Pathology. Unircrwity of Alabama Medical Ccntc,r, Birmintrhnm. .Ilahamn. 20
ATHEROGENESIS
AND AORTIC
PERMEABILITY
21
aorta, albumin and a, y-globulin mainly enter the vessel wall from the advential surface, e.g., vasa vasorum. Conversely, in some severely atherosclerotic aortas, the plasma proteins appear to leak into the vessel wall directly from the lumen. Our purpose in this study is to quantitate permeability during the development of the atherogenesis in rabbits and to relate changes in permeability to biochemical alterations at the arterial wall. Using rabbits fed a cholesterol diet, aortic permeability was determined wit’h 1311-albumin, gross intimal diseasewas evaluated, and aortic acid mucopolysaccharides and phospholipids were measured after 3, 7, or 12 weeks of dietary treatment. MATERIALS
AND
SIETHODS
Five groups of New Zealand, albino, female rabbits weighing 2.0 t’o 3.2 kg. were used. Each group consisted of 10 rabbits. The first group had been fed a stock diet (Purina Rabbit Chow, Ralston Purina Company, St. Louis) and was sacrificed at zero days of the experimental period. The second, t,hird, and fourth groups were maintained on an atherogenic diet (prepared from 1: 4: 45 w/w ratio of cholesterol, corn oil (Mazola) and Purina Rabbit Chow) administered ad Zibitum for 3, 7, and 12 weeks, respectively. A fifth group was maintained on a stock diet only, during the ent’ire experimental period (12 weeks). At the end of each experimental period levels of 1311-albumininjected 24 hours earlier, and free and esterified cholesterol in plasma were determined. Determination of aortic permeability in viva was performed in the following manner: 5 mCi of 1311-albumin (RISA, Abbot Laboratories, Chicago, Illinois) was injected into a rabbit (five rabbits were required, one for each experimental group). After 24 hours the animal was sacrificed by air injection (20 ml) and bled to yield a maximum volume of blood. Heparin (3 mg, 123 USP units/mg, The Upjohn Company, Kalamazoo, Michigan) was immediately added and plasma was then dialyzed in Visking tubing in 500-ml beaker against 400 ml distilled water with constant stirring by magnetic stirrer. Water for dialysis was changed twice, and dialysis was ended after 24 hours. The activities of the tenate and of the total dialysate were counted in a well-type scintillation counter. In this manner it was established that only 0.80 to 0.85% of radioactivity was dialysable. This activity, which could be separated by dialysis from 1311-albumin, consists of ionic iodine and iodinated amino acids. The purified plasma obtained by the above procedure (Duncan et al., 1962) and containing labeled 1311-albuminwas then injected into the ear vein of each rabbit, usually 5 ml of plasma per animal. After exactly 24 hours, 2 ml of blood were taken from the rabbit with a heparinized syringe. The animals were sacrificed by air injection (20 ml) and the aorta immediately dissected out, stripped free of adventitial tissue, opened longitudinally, washed five times with 2 ml of distilled water until washings were devoid of activity and separat’ed into the abdominal and arch-thoracic portions. The arch and thoracic segments were combined and referred as the proximal segment; the abdominal aorta, as the distal segment. TO count the radioactivity present in 0.1 ml of plasma and in the aortic segment (proximal and distal), a well-type scintillation counter with an automatic scaler was employed. Samples were counted to an accuracy of 2%. The ratio of the aortic
22
STEFAKOT’ICH
,iSD C:Ol
counts (per gram of aortic t&sue1 to t)lie counts in 0.1 ml of pl:~mw was calculatccl and expressed as an index of aortic pcrmeahility. More than 9076 of 1311-act,ivitJ in the plasma ant1 in the tissue was precipitablc with trichloracet,ic acid. The concentration of serum albumin did not change during t,he athcrogeuic diet and therefore the specific activity of l”‘I-all~uniin ~~21sidentical in c~xpc~riincntal anal control groups. The free cholesterol and cholcsteryl esters were determined in rabbit plasma in the following manner: Lipids were extracted and purified using Folch’s procedure (Folch et al.] 1957 1; free cholesterol and cholesteryl esters were separntcd by thin layer chromatography (Mangold, 1961’). Quantitative determination was accomplished by gas liquid chromatography using an F&M model 402 gas chromategraph with hydrogen flame ionization detector; for free cholestcbrol, a column with 6% SE-30 coated on Diatoport S (60-80 meshi was u~d ; column ov(‘n temperature was 23O”C, and carrier gas nitrogen at 5 psi. Cholestcryl esters were detcrmined as the fatty acid methyl esters, after transnietliylation with 10°C horoll t,rifluorjde in methanol and using a column with 6 % diethylcne glycol succinntc coated on Diat)oport S (SO-80 mesh j. Column OVC’II temperature was progranunctl from 120 to 200°C; nitrogen was 5 psi. Reference compounds: cholcstanc for frcct cholesterol and corresponding fatty acid methyl esters for choleeteryl esters were run simult.aricously with experimental ~amplcu. The percentage of the aortic surface involved with atherosclerotic lesions was evaluated planimetrically. The determination of aortic l~hosl~l~olil~id concentration was accompli:~hcd in the following manner: The aortic segments were minced with scissors aud homogenizcd at 0°C with chloroform:nic~tlianol (2: l., v/v’) in the ratio of I gm. of wet tissue to 20 ml of solvent, using a glass to glass dual action homogenizer. The homogenized sample was filtered through Whatman #l filter paper aucl w:t.shctl with distilled water saturated with a chloroform:methanol (2: 1 V/IT) mixt’urc. Ethanol-ether (3: 1 v/v), 75 ml of solvent for 1 gm. of tissue, was added to the residue which was then left in the dark for 48 hours at room temperature. Roth extracts were combined, cvaporatcd to dryness by flash cvaporalion (5O”C), rcdissolved in chloroform-methanol mixture (20 mlj , and used for determination of the phospholipids. A 10 ml aliquot of total lipid extract from the aortic sc>gment was evaporated to dryness under nitrogen and was redissolved in 0.4 ml of a chloroform-methanol solution (2: 1 v/v,. Phospholipids were separated into components in the following manner: 20 to 40 ~1 was applied on a thin layer plate coated with 0.25 mm silica gel H (Merck, A. G., Darmstadt, Germany). Silica get H was purified essentially by the method of Parker and Peterson (1965)) but in our procedure perchloric acid rather than formic acid was employed. I-sing two solvent systems for development, phospholipids were separated. System I consisted of petroleum ether, ethyl ether, and acetic acid (90: 10: 1 v/v). When the solvent front reached 15 cm, the plate was taken out, air dried for 2 to 3 minutes, and subjected to thin layer chromatography in System II. This consisted of chloroform, methanol, and water (62:25:4 v/v). Iodine vapor was employed to locate separated components, and individual phospholipids were identified using corresponding reference compounds. Silica gel containing separated components was scraped into test tubes and individual phospholipids were determined. Essentially.
ATHEROGENESIS
iZND
AORTIC
TABLE CHANGES
IN
Weight of rabbit ikg)
Diet
AORUC
PICRMK.IBILITP
Tissue weight (ma)
Athlerosclerotic involvemerit (%)
23
PERMESBILITI
1 IN
Fm
RABBITS
lyo
CHOLF:STEROL
Cont. of total ‘Tissue cholesterol in Plasma ‘311 ‘3’1 count plasma (mg/ Tissue ‘311 __ 100 ml) and 0.1 ,(~P$&, (cpm/g X 1OF “?;%eability esterified index) cholesterol (‘3%)
Prnxinml segmelll Stock Cholesterol weeks Choles-
3
terol weeks Choles-
i
terol
12.3
f
0.1+3ci
*
2.3
+
0.10 ’ 393 f
2.5
+
0.1
37 1 36
(70% 2.8
f
0.2c
2.4
zk 0.1
59.3
12
weeks Siock 12
173’2 zk 105” 25.X ((ii’; ) 01 k
0
1
3
27.9
xk 1.131.J
zk 2.01.33
+
0.03b
zk 1.921.0
&
+
0.05
1.50.75
( (615,) /
weeks
h IIifference
hetweell
stock
group
alrd
esprrimental
group
is
P < .OOl. I’ IXfference
between
stock
grorlp
nl~d
esperimelrtd
group
is.stat
hetweet)
stock
group
nuti
experimental
group
is statistically
P
< .Ol. d Ijifference
r
<
statistically istically
significant
at
significant
at
significant
at
.05.
the method of Barrlett (1959) was employed for determination of phosphorous. The results were usually expressed as micrograms of phosphorous per milligram of dry, delipidized tissue, Acid mucopolysaccharides were determined in the dry residue obtained after lipid extraction of aortic tissue by the method of Stefanovich and Gore (1967). RESULTS The d$a in Table I are relevant to the study of permeability for the proximal segment of the aorta under various expcrimcntal conditions. It is clear from this table that the body weights and the weight of the proximal segments of the aortas are significantly increased only a,fter 12 weeks of an atherogenic diet. A significant increase in permeability (the rat,io of plasma/tissue counts) is observed aft’er three weeks of cholesterol feeding. At this time, aortic involvement in an atherosclerotic lesion is rather minimal (2.3%). The increased permeability remained, however, at the same level throughout the 12 weeks of study. At the end of this period, the aortic involvement is maximal (59.3%)) but there is no significant change in permeability from 3 to 12 weeks of cholesterol feeding. All control animals showed similar levels of permeability during the experiment. In all statistical evaluations cholesterol diet groups were compared with control groups sacrificed at zero days of the experimental period.
24
STEFANOVICH
AND
Distal
Stock
2.3
Choles2.3 terol 3 weeks Choles2.5 ted 7 i weeks 2.x Cholesterol 12 weeks
+
0.1a 1109 z!z 13 lo/
& 0.1
143 zt
! x
segment
(i0
2.0
lob
GORE
‘1x19 I
+ 1 (CiO' ; )
‘16.5
f 1lXC 11.6 (7.3“;’ )
zt
0.1
172 *
I
15.0
I 1867 *
lW21.1
&
0.2
?5(i * ?3Ji
55.3
,17x2
105c “5.X
zt 1.51:3.0
f
1.10.n
f
0.W
It
f
1.91.71
l
0.15c
1.l:SS.S ’
f
0.91.3ti /
+
o.oxc
.3
*
1.01.x5
f
0.W
0.311!).!) I
f
1ooc’i )( ~ 1 +
+
47.3
b 1)ifferencr P < .Ol.
between
stock
group
alltl
experirrlrlltnl
gruup
is stat islically
signific:~llt
at
d I)ifferenre P < .05.
between
stock
group
atld
experimental
group
is slxtistically
signifimllt
at
In the distal segment’ (Table II) the observations were similar to those seen in the proximal segment,. The permeability index however at 7 weeks was lower than at 3 weeks and at 12 weeks. The difference in the permeability of the dist’al segment between 7 and 12 weeks of atherogenic diet was statistically significant (P < .OOl). When the permeability indices at 12 weeks at atherogenic diet are compared for distal and proximal segments, the former shows statistically significant increase in permeability (P < .OOl). It is possible to suggest &at this increased permeability is related to the relatively high degree of aortic atherosclerotic involvement for the distal segment (55.3%) which was in our study rat,hcr close to the involvement of the proximal segment (59.3%). Usually in rabbits on atherogenic diet, the proximal aortic segment is involved in the atherosclerotic lesion to a significantly higher degree than the distal segment. The degree of atherosclerotic involvement for the distal segment was similar to t#hat observed for the proximal part. The degree of atherosclerotic involvement for the distal segment was similar. The proximal segment (Table III) showed a highly significant increase in phospholipid concentrations (calculated as ,LL g of phosphorous per 100 mg of lipids) after 7 or 12 weeks of cholesterol feeding. This increase was also observed after 12 weeks of stock diet, especially in the distal aortic segment. In the atherogenic diet groups, however, there appeared a significant concentration of lysolecithin and
ATHEROGEKESIS
AND AORTIC TABLE
AORTIC
PHOSPHOLIPIDS
Phosphorus bg/100 mg wet tissue)
Diet
IN
Phospholipids (Irg of P/100 mg of lipids)a
III
RABBITS
24.8 25.0 33.7 18.3 28.9
z!r ;t * f f
2.05b 3.2 4.Y 3.2# 1.2
a P = phosphorus b Mean zt SE of mean. ’ Difference between stock group d Difference between stock group ’ Difference between stock group
366 372 651 1175 552
3z zt f f zk
69 88 86d
12od SSd
Llnlecithinl
‘$F$fi-
and experimental and experimental and experimental
Diet
-I
PHOSPHOLIPIDS
Phosphorus OIgjlOO mg wet tissue)
group group group
IN
a b c d c
11.4 27.4 18.0 15.7 17.0
zt f zt f *
P = phosphorus. Mean zt SE of mean. Difference between stick Difference between stock Difference between stock
I.+ 2.4c 3.2 5.2 2.2’
group group group
26.0 21.3 37.0 26.4 25.0
i f ;f; i zt
15 1’ 15* 16d 10d
and experimental and experimental and experimental
(‘%)
Lecithin
I ,,“iziin
1
Cephalin
zk f * * zk
6.6 1.6 l.gd 4.4 1.8
37.0 40.0 13.3 27.0 32.0
f * zt * f
significant significant significant
8.3 3.4 z.2d 2..+ 1.5
3.2 6.3 14.3 17.0
0 3~ z!z f f
24.6 22.6 20.4 18.0 25.0
0.4 0.8 0.8 1.1
z!z f f f f
11.5 2.1 1.6 z.ad 0.7
at P < .05. at P < ,001. at P < .Ol.
IV FJGD
IT,
CHOLESTJGROL
Phospholipids
Phospholi ids Gg of P?lOO me of lipids) Lysolecithin
173 408 462 552 482
/
is statistically is statistically is statistically
RABBITS
Distal Stock Cholesterol 3 weeks Cholesterol 7 weeks Cholesterol 12 weeks Stock 12 weeks
CHOLESTEROL
segment
0 10.5 zk 1.4 21.0 A 2.2 19.8 j, 2.4 0
TABLE AORTIC
1%
FED
Phospholipids
Proximal Stock Cholesterol 3 weeks Cholesterol 7 weeks Cholesterol 12 weeks Stock 12 weeks
25
PERMEABILITY
“gpg-
(
(%)
Lecithin
( ,t$&
1
Cephalin
segment
0 6.5 zt 2.8 4.4 It 0.9 1.8 i 1.8 0
group group gulp
27.4 27.5 28.0 22.0 24.4
+ 2.3 f 1.1 * 1.9 f 4.3 f 2.ge
is statistically is statistically is statistically
44.6 39.2 45.0 25.7 41.6
significant significspt significant
* zt zk zt 4~
1.3 2.4 1.8 4.0c 3.0c
5.5 4.0 17.0 11.0
0 f * f f
0.7 0.9 0.9 1.3
20.6 21.3 14.8 18.0 22.5
f f f f zt
2.3 1.3 2.0’ 1.4 2.8
at P < ,001. at P < .Ol. at P < .05.
lysocephalin, even after 3 weeks of diet. A similar situation was observed in the distal segment (Table IV) where the relative increase in phospholipids was observed after 3 weeks of atherogenic diet and continued up to 12 weeks. In both proximal and distal segments of the aorta of rabbits fed for 12 weeks on the stock diet, phospholipids were increased and some amounts of lysocephalin were noted. In regard to lysolecithin, there was always a significantly higher concentration of this phospholipid in the proximal segment then in the distal segment and at 7 and 12 weeks of cholesterol diet this difference in lysolecithin content between the proximal segment and the distal segment was statistically significant (P < .OOl) . This could indicate perhaps a somewhat different metabolism in the distal than in the proximal segment. There was a significant decrease in aortic lecithin in the proximal segment particularly at 7 weeks and also at 12 weeks of the cholesterol diet. In the distal segment a significant decrease in lecithin was observed only after 12 weeks of the cholesterol diet, but not after 7 weeks. This data could suggest that the proximal segment is more susceptible toward at,herosclerosis than
26
STEFANOVICH
Die1
.UIPS acid
AX-D
GORE
as uranic img;‘g)
the distal segment. It also agrcc~ with t,he higher concentration of lysolec~ithin obscrvetl in the proximal scgmcnt at 7 and 12 weeks of the atherogcnic dkt. Total acid mucopolysaccharide contcnnt (determined a:: uranic acid) did not change during atherogriiic lwriods in the prosimal segment (Tahlc VI, but :t significant increase \vas obscrrcd in the distal part for the 3%and 12%wek groups (Tnblc VI). Thcrc VW a constant clecrcnw in the concentration of choutlroitiu sulfat,c B in proximal wgnwntP iu rabbits ftid cholesterol. However. a d~crraw of cliondroitin ,sulfatc B was also noted in tlios:c rabljits fchclstock clkt for 12 w.c~~ki.
It is gvnerally argued that’ the accun~ulation of certain lipiclr in the intima of diseawtl nrteriw is partially due to increased ent,ry of p-lipoprotcin~. Clinical obxcrrations indicat.c the importaucc of thcl plasma lipid rontcnt. It is also possiblc that the altered permc~ahility of arterial endothelium contributes to these changes. Although t#lle molecular weight of human serum albumin used in the present study is lower than that of P-lipoprotein [69 x lo4 for human wrum albumin and 5-20 X 10” for l)rc-P-lipoprot,~iil 6 0.98-1.002 (Putnam, 1965 )] they are both significantly large macromolecules and, it can be presumed for purposed of this st,udy, that similar mechanisms arc involved for traversing the endothelium. It is pertincnt in this respect, moreover, that some lipids (e.g., lyaolccithin and free fatty acids) are bound and transported by albumin 18witzer and Etlcr, 1965; Fredrickson and Gordon, Jr., 1958). Our results indicate that the permeability (ratio of pla.wla/‘nortic tissue+ lwlIalbumin concentration) of the aortic wall increased significantly after only 3 wcck~
BTHEROGENESIS
.4&-D
AORTIC
TABLE Ac~o Diet stock Cholesterol 3 weeks Cholesterol 7 weeks Cholesterol 13 weeks Stock 12 weeks
MUCOPOLYS.~CCHARIDES
AMPS acid
as uranic (w/d
IN
VI SEGMJ
PROSIM.ZL
HS (y;)b
HS (%ja
27
PERMEABILITY
OF R.~~F~ITs CS-B
(s,)<
AORT.~ cs-c(y~y~
3.44 4.31
f l
0.15e 0.21r
13.0 14.9
f f
0.4 1.1
36.4 32.4
+ *
0.7 l.lQ
26.1 25.6
f *
1.1 0.x
24.5 27.0
+ f
1.2 0.x
3.47
f
0.12
12.1
+
0.7
34.4
*
0.8
2Y.8
f
0.9
24.7
+
1.1
3.99
*
0.13Q
13.x
+
1.2
29.5
+
1.3*
29.4
+
1.7
27.3
f
1.5
4.25
f
0.W
15.1
f
1.4
31.3
*
0.5*
20.4
zt
1.08
34.3
f
1.1
n HA = hyaluronic acid. 6 HS = heparitin sulfate. c CS-B = choudroitiu sulfate B. r( CS-C = chondroitin sulfate C. p ,Mean and SE of mean. ’ Difference between stock group P < .Ol. g Difference between stock group I’ < .05. h 1)ifferenre between stock group P < .OOl.
arLd experimental
grollp
is statistically
significant
at
and
experimental
grollp
is statistically
significant
at
and
experimental
group
is statistically
significaut
at,
of cholesterol feeding. By this time only minor observable gross atherosclerotic changes had developed in the aorta. It is known that some minor changes can appear in the arterial wall after 2 weeks of an atherogenic diet, as observed by light and electron microscopy (Parker and Odland, 1966). Still, it is important to note that in our present experiment significantly increased permeability precedes any dramatic damage to the aortic wall in rabbits fed the atherogenic diet. The permeability, rather than increasing progressively with the continuation of the diet for the additional 9 weeks, appears to stay approximately at the same level regardless of the significant increase in involvement of the aorta by atherosclerotic lesions (e.g., 2.3 vs. 59.3% in the proximal segment at 3 and 12 weeks of cholesterol diet respectively). It should also be noted that in our experiment the atherosclerotic involvement of rabbit aortas was similar for both the proximal and distal parts. The appearance of lysolecithin and lysocephalin seems to be the most significant and outstanding chemical change resulting from only 3 weeks of cholesterol feeding. One can speculate that the appearance of lysolecithin and lysocephalin, which are more water soluble than the corresponding original compounds, increase osmotic pressure within the cell. The experimental results of Kellaway and Saunders (1967) may be pertinent here. They found that the quantity of progesterone solubilized in an ultrasonically irradiated aqueous solution increased when lysolecithin was included. It is possible that the increased concentration of lysolecithin favors both solubilization and dispersion of the other lipids present in the cells of arterial tissue, thus further increasing the osmotic pressure within the cell. This, in
28
STEFANOT’ICH
AND
GORE
turn, may cause water-carrying macromolecules to permea,t,e such cells.* Furthermore, the membrane lecithin may be hydrolyzed to water soluble lysolecithin which may provide abnormal “openings” in the membrane, facilitating the rupture of such membranes. The release of p-glucuronidase attached to the membrane was observed in lysosome granules by Weissman et al. (1964) with concentrations of lysolecithin as low as 2 X low6 M. The origin of lysolecithin and lysocephalin in the aorta tissues of rabbits fed an atherogenic diet remains unresoh-ed. However, another view can be proposed. This explains that at least the appearance of lysolecithin may be due to the action of the lecithin-cholesterol acyhraneferase. Such a mechanism for cholesterol est’erification in rabbit aortic tissue was recently reported (hbdula et al., 1968) ..i An hypothesis for the etiology of dietary atherosclerosis can be proposed from the above discussion: /?-lipoproteins, carrying free cholesterol, enter the intimal layer of the aortic wall. Some quantity of cholesterol becomes esterified at the cell membranes. For this esterification, lecithin (and perhaps cephalini serves as a donor of fatty acids, simultaneously producing water soluble lysolecithin which is then ‘keleased” from the cell membrane. This may cause the disruption of the membrane. Lysolecithin may also produce an increase in osmotic pressure due to its solubility and its ability to solubilize other lipids. The final result may be an increased influx of ,&lipoproteins, carrying the excess cholesterol which is then continually esterified, ultimately causing an abnormal accumulation of cholesteryl esters within the cell. Such an hypothesis is consistent with the pathological manifestations of the early disease process. ACKNOWLEDGMENT We are indebted technical assistance
to Dr. L. C. Fillios for providing several of Mr. D. Shapiro is greatly appreciated.
helpful
comments.
The
excellent
REFERENCES T. transacylation 973.
ABDULLA,
ADAMS,
arterial ADAMS,
H.,
ORTEN,
in human
C. C., and ADAXS, and experimental
C. W. M. atheromatous
(1968). Cholesterol esterificntion by lesions. J. dtheroscler. Res. 8, 96i-
M., BAYLISS, 0. B.. and ORTOS. C. C. (1967). Plasma prokin accumulation in degeneration. J. Atheroscler. Res. 7,473-489. C. W. M.. VIRM. S., MORGAS. R. S.. and OSTOS, C. C. (1968). Dissociation of 3HC. W.
’ It should also be understood that there appears to be a relative increase in the tritiated water influx in atherosclerotic rabbit aortas compared with normal aortas. Two rabbits, one healthy and one atherosclerotic were injected with the same amount of tritiated water. Both rabbits were sacrificed and the aortas were immersed into separate beakers of distilled water. Water was changed three times and radioactivity in the water counted. The norta of the atherosclerotic rabbit “released” 92% more tritiated water than the aorta of the normal rabbit in respect to 1 gm of wet tissue. 5 In our experiment when employing lecithin with 1-l”C-labeled oleic acid in the C-2 position of lecithin, 4% (mean of three experiments) of the labeled acid was recovered as a cholesteryl ester after incubation with an homogenate of rabbit aorta. The homogenate was prepared by homogenizing rabbit aortas (1 gm./lO ml) in Tris buffer (0.1 M, pH 8.0). The supernatant obtained after centrifugation at 1OOOg. for 15 minutes was discarded and the residue in 0.1 M, pH 6.8 phosphate buffer (1 gm./l ml) was incubated for 18 hours at 37” C with labeled lecithin.
ATHEROGENESIS
AND
AORTIC
PERMEABILITY
29
cholesterol and l9-labeled plasma protein influx in normal and atheromatous rabbit aorta. J. Atheroscler. Res. 8, 67S696. BARTLETT, G. R. (1959). Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466471. DCNCAN, L. E. JR.! CORSFIELD, J.: and BUCK. K. (1959). Circulation of labeled albumin through the aortic wall of the dog. Circ. Res. 7,390-397. DUNCAN, L. E. JR., CORNFIELD, J., and Bucrc. K. (1962). The effect of blood pressure on the passage of labeled plasma albumin into canine aortic wall. J. Clin. Invest. 41, 1537-1545. FRIEDMAN, M., and Byers, S. 0. (1963). Endothelial permeability in atherosclerosis. Arch. Pathol. 76,99-105. FREDRICKSON, D. S.. and GORDON, R. S. JR. (1958). Transport of fatty acids. Physiol. Rev. 38,585X30. FOLCH, J., LEES, M., and SLONE-STANLEI., G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226,497-509. GORE, I., and LARKEY. B. J. (1960). Functional activity of aortic mucopolysaccharides. J. Lab. Clin. Med. 56, 839-846. KAO, V. C. Y., and WISSLER, R. W. (1965). A study of the immunohistochemical localization of serum lipoproteins and other plasma proteins in human atherosclerotic lesions. Ez~. Mol. Pathol. 4,465479. KELLAWAY, I. W., and SAUNDERS. L. (1967). The solubilization of some steroids by phosphatidyl choline and lysophosphatidyl choline. Biochim. Biophys. Acta 144, 145-148. MANGOLD, H. K. (1961). Thin-layer chromatography of lipids. J. Amer. Oil Chem. Sot. 38, 708-727. OKISHIO, T. (1961). Study on the transfer of ‘“‘I-labeled serum lipoproteins into the aorta of rabbits with experimental atherosclerosis. Med. J. Osaka Univ. 11, 367-381. PACKHAM, M. A., ROSWELL, H. C., JORGENSEN, L., and MUSTARD. J. F. (1967). Localized protein accumulation in the wall of the aorta. Exp. Mol. Pathol. 7,214-232. PAGE, I. H. (1954). Atherosclerosis: an introduction. Circulation 10, l-27. PARKER, F., and PETERSOS, N. F. (1965). Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin layer chromatograms. J. Lipid Res. 6,455-460. PARKER, F., and ODLAXD, G. F. (1966). A correlative histochemical, biochemical, and electron microscopic study of experimental atherosclerosis in rabbit aorta with specific reference to the myointimal cell. Amer. J. Pathol. 48, 197-239. PUTPI’AM, F. W. (1965). Structure and function of the plasma proteins. In “The Proteins” (H. Neuoth. ed.), 2nd ed., Vol. III. Academic Press, New York: N. Y. STEFANOVICH, V., and GORE, I. (1967). A micromethod for the determination of acid mucopolysaccharides in vascular tissue. J. Chromntog. 31,473-478. SWITZER, S., and EDER, H. A. (1965). Transport of lysolecithin hg albumin in human and rat plasma. J. Lipid Res. 6,506511. WALTON, K. W.. and WILLIAMSON, N. (1968). Histological and immunofluorescent studies on the evolution of the human atheromatous plaque. J. Atheloscler. Res. 8,599-624. WATTS. H. F. (1963). Role of lipoproteins in the formation of atherosclerotic lesions. In “Evolution of the Atherosclerotic Plaque” (R. J. Jones, ed.). Chicago University Press. Chicago, Ill. WEISWAN, G., BECKER. B.. and THOWS, L. (1964). Studies on lysosomes V. The effects of streptolysins and other hemolytic agents on isolated leurocyte granules. J. Cell Biol. 22,
115-126.