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In vivo fluxes of plasma cholesterol, phosphatidylcholine and protein into mini-pig aortic and pulmonary segments
309
Atherosclerosis, 41 (1982) 309-319 @ Elsevier/North-Holland Scientific
Publishers,
Ltd
IN VIVO FLUXES OF PLASMA CHOLESTEROL, PHOSPHATIDYLCHOLINE AND PROTEIN INTO MINI-PIG AORTIC AND PULMONARY SEGMENTS
SBREN CHRISTENSEN BAGGER 1
l**, STEEN
STENDER
of Physiology, University of Aarhus, ’ Department Clinical Chemistry CL, Rigshospitalet, Blegdamsvej (Received (Revised, (Accepted
*, OLE NYVAD
’ and HENNING
DK-8000 Aarhus C and * Department 9, DK-2100 Copenhagen (Denmark)
of
21 April, 1981) received 22 June, 1981) 22 June, 1981)
Summary
Levels of radioactive cholesterol found in the arterial wall after a few hours’ in vivo exposure to plasma containing labelled cholesterol are so small that the contaminating plasma in the tissue may contribute significantly to the radioactivity measured in the wall. By the simultaneous use of [3H]- and [ 14C]cholesterol the contamination was found to be less than 15 nl of plasma/cm* of intimal surface of the thoracic aorta in mini-pigs, and this contamination accounted for less than 5% of the total radioactivity found in the intimamedia tissue 7 h after the injection of serum with bioincorporated labelled cholesterol. The radioactivity in the arterial wall divided by the area below the plasma radioactivity versus the time curve was named the intimal clearance. For a particular mini-pig 2 nearly identical estimates were obtained for the intimal clearance of labelled plasma cholesterol, using an influx period of about 4 h for [3H]cholesterol and 7 h for [ 14C]cholesterol. This indicates a negligible loss of labelled cholesterol from the wall during a 7-h period. The intimal clearances as calculated for labelled plasma cholesterol were about 500, 200, 100 and 50 nl cm-* h-’ for pulmonary trunk, aortic arch, proximal and distal thoracic aorta, respectively. The concomitantly measured intimal clearances of plasma [32P]phosphatidylcholine were about 1.1 times those for plasma cholesterol for the 4 segments. This suggests that the appearance of labelled plasma phosphatidylcholine and of cholesterol in the intimamedia is essentially a consequence of a lipoprotein influx. Similarly, the intimal clearances of plasma [35S]albumin for the same 4 segThis work was supported by a grant from the Danish Heart Foundation. * Requests for reprints should be addressed to S. Christensen.
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310
ments were about 1.5 times those of the labelled lipids, suggesting a nonspecific mechanism for the transfer of plasma lipoproteins into the artery. The intimal clearances of [35S]albumin were within the same order of magnitude as capillary clearance values for albumin reported for mammalian tissues.
Key words:
Arteries albumin
- Atherogenesis - Endothelium -Plasma lipoprotein
-
Mini-pig
-
Permeability
-Plasma
Introduction This paper presents new aspects of methodology for the measurement of influxes of macromolecular components into the arterial intima-media, with particular attention paid to plasma cholesterol [l-12]. Special attention is given to the unavoidable plasma contamination of arterial layers and to one of the basic assumptions often used for influx experiments, namely that the intima-media can be treated for at least several hours as a sink for plasma cholesterol, i.e. that the loss of labelled cholesterol from an intima-media layer, including the avascular layer, is small when compared with the appearance in that layer of labelled plasma cholesterol within the same time period. Cholesterol influx into the arterial wall is compared with the simultaneous influx of phosphatidylcholine (PC) and plasma albumin, all labelled according to biosynthetic principles. Moreover, the permeabilities derived from the present work for aortic endothelium are compared with values reported for non-fenestrated mammalian capillaries. Materials and Methods Animals
Mini-pigs of either sex were obtained from Korselitze, Nykq5bing F., Denmark, and from the Institut fiir Tierzucht und Haustiergenetik, University of Gottingen, F.R.G. Weights ranged between 20 and 37 kg, and ages between 6 and 34 months. Some of the animals were given a diet composed of 7% by weight of dried egg yolk, 0.5% of cholesterol (Merck) and the remainder composed of ordinary sow pellet. No atheromas were observed. Preparation
of dialyzed
donor serum
Four serum-donor animals were used for the bio-incorporation of 1, 2 or 3 radiochemicals. The animals were anesthetized by in$amuscular injection of a mixture of etorfin and acepromazine (Immobilon , Pharmacia, Sweden), 4 ml/25 kg of body weight. An angiographic pigtail catheter, type Fr. (W. Cook), was introduced into the external jugular vein until the tip was located in the right atrium, according to screen observation. The catheter emerged through an incision into the subcutaneous layer of the neck. In a mini-pig 10 mCi of [3sS]methionine (spec. act. 387 mCi/mmol, Amersham, England) were administered intramuscularly at time zero. After 11.5 h, 3.6 mCi of [14C]cholesterol (spec. act. 54 mCi/mmol, NEN, U.S.A.)
311
were administered by means of the catheter as a suspension produced by mixing a small volume of ethanol and 20 ml of saline. Finally, at 18 h, 80 mCi of 32P were injected i.m. as sodium orthophosphate (spec. act. 261 mCi/mg P, Ris$, Denmark) dissolved in 5 ml of saline. Isotope incorporation into plasma components was studied using EDTA-plasma from blood samples drawn at appropriate intervals. Exsanguination was performed at 41.5 h. The left common carotid artery was cannulated and blood collected into sterile bottles without anticoagulants. Serum was dialyzed at ambient temperature against a physiological salt solution (Robinson buffer) in an artificial kidney for 1 h (AHFK Kll Asahi Med. Corp., Japan). A reduction of more than 99.5% in the levels of trichloroacetic acidsoluble 32P and 35S radioactivity was obtained. The dialyzed serum was then injected into recipient mini-pigs. Serum injection experiments In one experiment, used for plasma contamination studies, a volume of the triple-labelled donor serum was infused at about 7 h prior to killing. At about 0.3 h before killing the same animal received a volume of serum from a 26-kg donor mini-pig, labelled exclusively with [3H]cholesterol (50 mCi of [1,2(n)3H]cholestero1, spec. act. 53 Ci/mmol, NEN, U.S.A.). In another mini-pig the two differently labelled donor sera were injected, one at 6.5 h and the other at 3.4 h before killing. That these two differently labelled donor sera were eliminated identically from the plasma subsequent to i.v. injection of mixed serum was verified in a young mini-pig. The animals were killed by an overdose of anaesthetic. Blood was rapidly removed from the arteries by rinsing with large volumes of saline. Loose connective tissue was stripped from the arteries and arterial segments were then sectioned and frozen in liquid nitrogen with subsequent storage at -20°C. Immediately before homogenization a segment was thawed and dissected in the cold into a maximum of 4 layers.
Analytical procedures Plasma and tissue lipids were extracted, purified and subjected to 2 thinlayer chromatographic (TLC) separation steps, yielding free cholesterol (FC), esterified cholesterol (EC) and phosphatidylcholine (PC). These fractions were then processed for chemical and radioactivity analyses, also taking into account the percentages of recovery [13]. Levels of 3H and 14C radioactivities were recorded by means of appropriate settings of the liquid scintillation counter (LSC). The areas of TLC silica gel containing PC were subjected to wet combustion. The inorganic phosphate thus obtained was precipitated as magnesium ammonium phosphate in an amount suitable for collection of the crystals on glass filter circles. For 32P counting the circles were positioned below a Berthold low-level proportional argon flow detector [ 141. Subsequent to lipid extraction, non-protein 35S was removed by trichloroacetic acid washings, leaving a pellet with the protein-incorporated 35S. The pellet was treated with formic acid, and a clear 200 ~1 volume of supernatant was mixed with 10 ml of 4% Cab-0-Sil in Permablend @ for counting of “S by means of LSC [13]. It was verified by experiment that the 35S-labelled proteins fraction measured by
312
this method was recovered virtually 100% when plasma containing labelled proteins was mixed with non-radioactive arterial tissue, and then subjected to the entire separation procedure. Influx
calculation
and terms
Results are presented the formula:
by means of an uptake
coefficient
k, calculated
from
where L is the radioactivity measured at killing in a luminal layer of area A and corrected for contaminating plasma radioactivity. The FL represents the average level of radioactivity per ml plasma during the influx experiment for which T is the duration. Provided that the luminal layer functions as a sink within the time interval T, k can be visualized as an influx parameter, here expressed as an intimal clearance having the designation nl cme2 * h-l. In this study 30% of the cholesterol radioactivity was found in low density and about 70% in high density lipoproteins, Only if it is assumed that the labelled phosphatidylcholine had a similar distribution on these lipoprotein classes would identical intimal clearances for phosphatidylcholine and cholesterol be in accordance with the influx of these lipids into the arterial tissue as components in lipoproteins. The term cholesterol designates the sum of free and esterified cholesterol. Where an influx is proportional to the plasma concentration of the substance in question, the clearance can be conceived of as a permeability coefficient. A clearance is reducible to a velocity, in so far as 1 cm * s-’ equals 3.6 X 10’ nl cmV2 . h-l. In capillary physiology the permeability coefficient has been envisaged as a clearance [ 151. Results and Discussion Contamination
The contaminating plasma volumes were obtained from a 0.3 h experiment by division of 3H-E.C label, measured per cm’, by the terminal value, c&, for 3H-EC per nl of plasma (Table 1, col. 5). Since an influx may contribute some label even during that short period, the values in col. 5 are overestimates. Nevertheless, values as low as 3-14 nl/cm2 were found for the intimal surface of aortic segments. The same animal was injected 7 h before killing with [ i4C]cholesterol-containing serum. The 14C-EC found in layer I (intima-media) exceeded by more than 20-fold the 14C-EC ascribeable to the contamination derived above. On the other hand, 1 cm2 of the pure media layer, M, at 0.3 h manifests the presence of label equivalent to about 50 nl of terminal plasma. The value is equal to about 1 I.tl/g of media tissue, which corresponds to the intravascular volume expected in the microcirculation of average vascularized tissue [ 181. Intima-media
conceived
as a sink
The independence of k from T for aortic influx calculation phoprotein, phosphatidylcholine and phosphatidylethanolamine
of plasma phoswas observed
1
aorta
’ The
The
method
concentrations
tively.
that
in footnotes
implies
concentration
concentration
average
plasma
terminal
a The
b The
Tp,
b for
TC
media.
are 0.53,
were
2.92
X lo-
17 and -2
2.70
3, 0.57
and
text,
dpm/nl
from
5.04
1230
130
67
278
130
241
985
274
393
433
3 330
2 720
2 760
(nl/cm2)
4a
I4C-EC/ck
EXPERIMENTS
3H0.421 dpm/nl,
are added.
was
for
respectively.
and
14C-EC,
arterial
dpm/nl
and
saline-washed
X 10m3
trunk,
in the
X 10m3
dissected
3.32
0.35
0.18
0.75
0.35
0.65
2.66
0.74
1.06
1.17
8.99
7.35
7.45
(dpm/cm2)
3
for pulmonary
described
and
layers
dpm/nl
The
in a method
e.g.
used
was 0.415
M values,
label,
I and
a and
the
of
ck.
experiment.
of label.
of influx
A = adventitia
78
A
T = duration
243.9
39
M = media,
1.5 13.1
38
M
73.9
53
A
I
4.5 23.6
54
95
A
34
44
M2
M
20.8 97.8
50
I
6.0 22.8
54
Ml
109.3
94
A
I
25.2
+ some
(dpmlcm2)
(mg/cm2) 33.0
2
1
(T = 7 h)
h)
(T = 0.3
INFLUX
14C-EC,
27
respectively.
ARTERIAL
esterified
IN
3 H-EC,
cholesterol
Labelled
SURFACE
44
terol,
plasma
weight
INTIMAL
to area ratio
Wet
THE
I
OF
M
I = intima-media,
aorta
descending
thoracic
Distal
thoracic
descending
and arch
aorta
Proximal
thoracic
Ascending
(trunk)
Layer
CONTAMINATION
artery
segment
Pulmonary
Arterial
PLASMA
TABLE
THE
3
)
4.63
5
and
dpm/nl
for
3H-
and
TC = esterified
29
2
54
5
44
46
-2
17
(nllcm2)
6b
14C-EC
3H-EC
from
volume
calculated
as plasma
EC and
X 10-j
respectively.
segments.
580
31
176
56
11
233
49
54
14
260
60
78
@l/cm2
5a
as 3H-EC/c$
calculated
Contamination
9
total
14C-EC,
and
33
8
61
10
56
61
-3
12
@l/cm2)
7c
1 4C-TC
3H-TC
respec-
choles-
and
G
w
314
for at least 5 h in the stilbestrol-treated cockerel [14]. In the same animal model this finding was subsequently extended to plasma cholesterol through use of the double-isotope technique outlined in the section on serum injection experiments [17]. By this technique, 2 nearly identical influx values for 2 different time periods were obtained. A mini-pig experiment of that type is the basis for Fig. 1, where the tissue radioactivity expressed as a plasma volume equivalent (kT, i.e. L/AZP) is plotted versus T. Again, k essentially appears to be independent of T within the time period chosen. The tissues studied included layers I and M, as detailed in the legend. In a similar plot concerned with only I-layer radioactivity, the intercept with the Y-axis was positive in accordance with a loss of label from that layer during the latter part of the experiment. If a similar plot was based upon layer M, the intercept was negative. These findings suggest that the activity from layer I is transferred to layer M within the experimental period. An intercept close to zero for the I + M layer (Fig. 1) is in accordance with a negligable loss of cholesterol or with the presence of a sink for plasma cholesterol within a period of 6-7 h. The role of the vasa vasorum of layer M, relative to the role of the intimal surface as entrances for labelled cholesterol remaining in the wall at any given time, may well have been reversed as time passed, as was the case for cockerel aortic wall [16]. (Consider also col. 2 versus col. 3, Table 1.) The difference in slopes observed for TC and EC in Fig. 1 may be explained by the hydrolysis at a fixed fractional rate of newly entered plasma EC [ 171. In spite of any hydrolysis, labelled FC contributed less than 10% of the TC label in the artery. In plasma the percentage was even less. The expected exchange of
32w
TC0 EC--_
??
24C0..
0
2
4 HOURS
6
8
Fig. 1. Evidence for the transient sink function for the luminal half of arterial wall segments. The wet weights of the luminal layers ranged from 83 to 102 mg/cm*, and they comprised both sublayers I and M as defined in Table 1. 1: Pulmonary trunk; 2: ascending aorta plus arch; 3 and 4: proximal and distal halves of thoracic aorta, respectively. With reference to the Methods section a zero intercept suggests a flux proceeding with a constant rate into a sink. Cl4 CJcholesterol-labelled lipoproteins circulated for 6.5 h and [3H]cholesterol-labelled lipoproteins circulated for 3.4 h before termination of the experiment.
315
FC between the endothelial cell surface and the circulating plasma lipoproteins would further influence the small fraction of label found in FC. Finally, assuming the existence of a transient sink, the label/cm* of the sink
260
4bo
6bO
nl UII-~h-l for PC Fig. 2. Intimal clearances for labelled plasma total cholesterol (TC) (ordinate), and labelled phosphatidylcholine (PC) (abscissa). Results are from influx experiments lasting 6.5 and 7 h. 0: pulmonary trunk, ‘3: .ascending aorta plus the arch: ??and A: proximal and distal halves of thoracic aorta. respectively. The most probable linear regression is y = 0.88 (tO.06) x - 14 (k20).
layer is a sum of the contaminating volume X ck and k X3; X T. Since the sum can be written for 3H as well as for i4C data, 2 equations are thus available for the determination of the contaminating volume and k. For an arterial segment subjected to a high influx (pulmonary trunk), this method appears to give the more reliable contamination value (columns 6 and 7 of Table 1. I and M (M,) values have to be added). Influx
of phosphatidylcholine
The intimal clearance values obtained for PC and their linear relationship to those obtained for TC are presented in Fig. 2. This relationship is in accordance with a coupling between the arterial influx of plasma PC and TC. Provided that a transient sink in the artery for the labelled PC is also present and that a similar distribution of labelled PC and TC among the plasma lipoproteins exists, the fact that the value for the slope 0.88 + 0.06 is very nearly unity favours the concept that the labelled lipids were found in the wall as a result of lipoprotein influx. The stilbestrol-treated, hyperlipaemic cockerel provided similar findings [171. Influx
of plasma albumin
Plasma total [35S]protein appeared to be a useful tracer for albumin because 80438% of donor [35S]protein behaved as albumin in being non-precipitable in ammonium sulphate at half-saturation, and because the permeability of the endothelium to albumin is probably greater than the permeability to other predominant plasma proteins [lo]. For the relatively small albumin molecule it is particularly pertinent to contemplate whether the intima-media tissue functioned as an equilibrating tissue rather than as a sink for period T. Evaluation of the data in terms of equilibrium spaces was enhanced by a nearly constant [35S]protein plasma concentration, which at the time of killing was as high as
316 TABLE
2
PERMEABILITY Animal
COEFFICIENTS
FOR THE ENDOTHELIUM
Arterial segment
OF MINI-PIG
ARTERIAL
lo8 X Intima-media uptake coefficients (“vascular permeabilities”) as calculated for 4 macromolecular
SEGMENTS surface markers a
I14Cl
r3H1
C32Pl
cholesterol
cholesterol
[35Sl
phosphatidylcholine
protein
(cm
.
s-l)
(cm
.
s-1 )
(cm
.
s-l)
b
(albumin) (cm
.
s-1 )
Asc. aorta +
13
arch Prox. dew.
4.9
4.7
6.4
8.1
thor. aorta Dist. desc.
3.4
3.4
4.3
3.7
thor. aorta
1.3
1.3
1.6
3.1
Asc. aorta + arch Prox. desc.
2.9
5.6
5.3
thor. aorta Dist. desc.
1.5
1.0
3.5
thor. aorta 13 9
Pulmonary Pulmonary
trunc trunc
1.0
1.4
4.0
12.0 15.7
15.1 16.7
(18.7) (19.3)
a Influx periods 6.5 and 7 h for animals 13 and 9, respectively. During the final 3.4 h of the 6.5 h experiment, lipoproteins labelled with [ 3 HI cholesterol were circulating. The layer studied was the sink layer described in legends to Figs. 1 and 2. b The figures 3.7 and 4.0 are based upon uptake of label by only one of the two sublayers, I and M, plus an estimated uptake for the other sublayer. The values in parentheses are theoretically invalidated as discussed in the text.
82% of the average value. For the spaces obtained the average values in 2 minipigs were 55,20,14 and 10 pi/g W.W. for intima-media layers from pulmonary trunk, aortic arch and proximal and distal thoracic aorta, respectively. For pure media layers values of 104, 19, 10 and 13 /A/g, were obtained. An albumin equilibrium space of 59 pll/g was reported for dog carotid artery [ 201.
.=
PHOw!ATIOnCHOLINE
0=
CHOLESTEROL
PROTEIN PERMEABILITY Cc.xs&'xlO? Fig. 3. The figure gives a graphic representation of the values in Table 2. The most probable linear regressions are y = 0.88 (r0.07) x - 0.71 (t0.76) for PC, and y = 0.79 (kO.07) x - 1.12 (* 0.71) for TC.
317
These values indicate that equilibrium may have been reached in the case of the pulmonary trunk. The aortic segments, on the other hand, showed only about 25% of the equilibrium value cited. At this value in a well-stirred steady state compartment, equilibrating with plasma of constant specific activity, an influx calculated on the sink assumption would introduce a negative error of 14%. In conclusion, aortic [%]protein data are treated on the assumption of a sink. Since the pulmonary trunk k values for [3sS]protein are open to criticism, they have been bracketed in Table 2. This table, in conjunction with Fig. 3, presents k -the intimal clearance - in units of cm X s-l. It is evident in Fig. 3 that the intimal clearances for albumin are about 1.5 times those for “lipoprotein”, as determined with labelled cholesterol and phosphatidylcholine. This suggests that albumin and plasma lipoproteins enter the arterial wall by a similar mechanism, Regional
patterns
The coefficients for influx of labelled TC, EC, PC and albumin decreased with increasing distance from the heart (Table 2). This feature has been described for fibrinogen and albumin in pig experiments [lo-121 and for albumin, low density lipoprotein and cholesterol in dogs [7,8]. A new feature is the influx into the pulmonary trunk. The intimal clearances were 3-5 times greater in the pulmonary trunk than in the aortic arch, irrespective of the macromolecular label (Table 2). * Capillary and arterial intimal permeabilities
to plasma macromolecules
In order to characterize the permeability of capillary and intimal endothelium the same parameters have to be evaluated. These parameters include the area of endothelium, concentrations on both sides of the endothelium, and transendothelial fluxes. Strictly speaking the use of the permeability concept requires a proportionality between net influx of moles and molar concentration differences created or found across the barrier studied. In the case of the intima-media tissue the trans-endothelial flux is not obtainable from an accessible flow of lymph, but the influx at the prevailing plasma concentration can be obtained with tracer methods from the ratio between arterial radioactivity and plasma specific activity. It is important to keep in mind that the ratio between labelled and unlabelled molecules is much lower in the tissue than in the plasma (the sink condition). The presence of such conditions for labelled cholesterol is supported by the kinetic findings, particularly those given in Fig. 1. Vasavasorum are probably not present in our intimamedia (I) but in part of our media (M) layers [21]. If it is assumed that sink conditions apply also to the labelled albumin, the intimal clearance of albumin can be conceived of as a permeability coefficient, in the same way that the intimal clearance of phosphatidylcholine and cholesterol is visualized as a permeability coefficient for plasma lipoproteins. The permeabilities of “lipoproteins” calculated in this way are almost the same as those found by Bratzler et al. for rabbit aortic endothelium using plasma low density lipoprotein, labelled in the protein moiety with 1311 [ 91. The albumin permeability values given in Table 2 are near to those published for various mammalian non-
318
fenestrated capillaries as summarized in Table 4 of Ref. 15. In conclusion, the similarity of the simultaneously measured intimal clearances of albumin, cholesterol and phosphatidylcholine over a wide range of values (Fig. 3) suggests that the in vivo transfer of plasma lipoproteins into the arterial wall of mini-pigs occurs mainly by a nonspecific mechanism. Diffusion, filtration or pinocytosis [ 22,231 may be involved. Acknowledgements Research Establishment Risq5, Denmark, through the cooperation of Dr. H. Hansen.
was helpful
in various
respects
References 1 Walton, K.W., Pathogenetic mechanisms in atherosclerosis, Amer. J. Cardiol., 35 (1975) 542. 2 Bradby, G.H.V.. Walton, K.W. and Watts, R., The binding of total low density lipoproteins in human arterial intima affected and unaffected by atherosclerosis, Atherosclerosis, 32 (1979) 403. 3 Stein, O., Stein, Y. and Eisenberg, S., A radioautographic study of the transport of 125-I-labelled serum lipoproteins in rat aorta. Z. Zellforsch., 138 (1973) 223. 4 Hoff, H.F., Karagas. M., Heideman, C.L.. Gaubatz, J.W. and Gotto. Jr., A.M., Correlation in the human aorta of APO B fractions with tissue cholesterol and collagen content, Atherosclerosis. 32 (1979) 259. 5 Hoff, H.F., Bradley, W.A.. Heideman, C.L., Gaubatz. J.W., Karagas. M.D. and Gotto, Jr., A.M.. Characterization of low density lipoprotein-like particle in the human aorta from grossly normal and atherosclerotic regions, Biochim. Biophys. Acta, 573 (1979) 361. 6 Smith. E.B. and Slater. R.S., Lipids and low density lipoproteins in intima in relation to its morphological characteristics. In: R. Porter and J. Knight (Ed%), Initiating Factors (Ciba Symposium No. 12, New Series), Elsevier. Amsterdam, 1973, p. 39. 7 Duncan, L.E.. Cornfield, J. and Buck, K., Circulation of labelled albumin through the aortic walI of the dog, Circ. Res., 7 (1959) 390. 8 Duncan, L.E., Buck, K. and Lynch, A., Lipoprotein movement through canine aortic wall. Science, 142 (1963) 972. 9 Bratzler. R.L., Chisohn, G.M., Colton, C.K., Smith, K.A. and Lees, R.S., The distribution of labelled low-density lipoproteins across the rabbit thoracic aorta in Volvo, Atherosclerosis, 28 (1977) 289. 10 Bell, F.P., Gallus, A.S. and Schwartz. C.J., Focal and regional patterns of uptake and the transmural distribution of 131-I-fibrinogen in the pig aorta in viva, Exp. Mol. Path., 20 (1974) 281. 11 Bell, F.P.. Adamson; I.L. and Schwartz, C.J., Aortic endothelial permeability to albumin - Focal and regional patterns of uptake and transmural distribution of 131-I-albumin in the young pig. Exp. Mol. Path.. 20 (1974) 57. 12 Somer. J.B., Evans, G. and Schwartz. C.J.. Influence of experimental aortic coarctation on the pattern of aortic Evans Blue uptake in viva, Atherosclerosis, 16 (1972) 127. 13 Stender, S., Christensen, S. and Nyvad, 0.. Uptake of labelled free and esterified cholesterol from plasma by the aortic intima-media tissue measured in viva in three animal species. Atherosclerosis, 31 (1978) 279. 14 Christensen, S. and Nielsen, H., Permeability of arterial end&helium to plasma macromolecules, Atherosclerosis, 27 (1977) 447. 15 Renkin, E.M., Multiple pathways of capillary permeability. Circ. Res., 41 (1977) 735. 16 Christensen, S., Transfer of labelled cholesterol acrnss the aortic intiial surface of normal and cholesterol-fed cockerels, Atherosclerosis. 4 (1964) 151. 17 Stender, S. and Christensen, S.. The concomitantly measured transfer of free cholesterol, esterified cholesterol, phospholipids and phosphoprotein from plasma into the aortic wall of stilboestroltreated cockerels. Atherosclerosis, 28 (1977) 15. 18 Ganong, W.F., Review of Medical Physiology, 8th edition, Lange Medical Publications, LOS Altos. CA, 1977 (Lot. cit. Table 30.1.) 19 Adams, C.W.M. and Morgan, R.S.. Autoradiographic demonstration of cholesterol filtration and accumulation in atheromatous rabbit aorta, Nature (Land.), 210 (1966) 175. 20 Villamil. M.F., Rettori, V., Barajas, L. and Kleeman, C.R., Extracellular space and the ionic distribution in the isolated arterial wall, Amer. J. Physiol., 214 (1968) 1104.
319 21 22
Wolinsky, H. and Glagov, S.. Nature of species vascmm in mammals, Circ. Res., 20 (1967) 409.
differences
in the medial
distribution
of aortic
vasa
Jensen, J., On the relationship between metabolic activity and cholesterol uptake by intima-media of the rabbit aorta. Biochim. Biophys. Acta, 183 (1969) 204. 23 Stender, S. and Zilversmit, D.B., Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits - Molecular size as a determinant of plasma lipoprotein influx, Arteriosclerosis, 1 (1981) 38.
Atherosclerosis, 41 (1982) 309-319 @ Elsevier/North-Holland Scientific
Publishers,
Ltd
IN VIVO FLUXES OF PLASMA CHOLESTEROL, PHOSPHATIDYLCHOLINE AND PROTEIN INTO MINI-PIG AORTIC AND PULMONARY SEGMENTS
SBREN CHRISTENSEN BAGGER 1
l**, STEEN
STENDER
of Physiology, University of Aarhus, ’ Department Clinical Chemistry CL, Rigshospitalet, Blegdamsvej (Received (Revised, (Accepted
*, OLE NYVAD
’ and HENNING
DK-8000 Aarhus C and * Department 9, DK-2100 Copenhagen (Denmark)
of
21 April, 1981) received 22 June, 1981) 22 June, 1981)
Summary
Levels of radioactive cholesterol found in the arterial wall after a few hours’ in vivo exposure to plasma containing labelled cholesterol are so small that the contaminating plasma in the tissue may contribute significantly to the radioactivity measured in the wall. By the simultaneous use of [3H]- and [ 14C]cholesterol the contamination was found to be less than 15 nl of plasma/cm* of intimal surface of the thoracic aorta in mini-pigs, and this contamination accounted for less than 5% of the total radioactivity found in the intimamedia tissue 7 h after the injection of serum with bioincorporated labelled cholesterol. The radioactivity in the arterial wall divided by the area below the plasma radioactivity versus the time curve was named the intimal clearance. For a particular mini-pig 2 nearly identical estimates were obtained for the intimal clearance of labelled plasma cholesterol, using an influx period of about 4 h for [3H]cholesterol and 7 h for [ 14C]cholesterol. This indicates a negligible loss of labelled cholesterol from the wall during a 7-h period. The intimal clearances as calculated for labelled plasma cholesterol were about 500, 200, 100 and 50 nl cm-* h-’ for pulmonary trunk, aortic arch, proximal and distal thoracic aorta, respectively. The concomitantly measured intimal clearances of plasma [32P]phosphatidylcholine were about 1.1 times those for plasma cholesterol for the 4 segments. This suggests that the appearance of labelled plasma phosphatidylcholine and of cholesterol in the intimamedia is essentially a consequence of a lipoprotein influx. Similarly, the intimal clearances of plasma [35S]albumin for the same 4 segThis work was supported by a grant from the Danish Heart Foundation. * Requests for reprints should be addressed to S. Christensen.
0021-9150/82/0000--0000!$02.75
0 1982
Elsevier/North-Holland
Scientific
Publishers,
Ltd.
310
ments were about 1.5 times those of the labelled lipids, suggesting a nonspecific mechanism for the transfer of plasma lipoproteins into the artery. The intimal clearances of [35S]albumin were within the same order of magnitude as capillary clearance values for albumin reported for mammalian tissues.
Key words:
Arteries albumin
- Atherogenesis - Endothelium -Plasma lipoprotein
-
Mini-pig
-
Permeability
-Plasma
Introduction This paper presents new aspects of methodology for the measurement of influxes of macromolecular components into the arterial intima-media, with particular attention paid to plasma cholesterol [l-12]. Special attention is given to the unavoidable plasma contamination of arterial layers and to one of the basic assumptions often used for influx experiments, namely that the intima-media can be treated for at least several hours as a sink for plasma cholesterol, i.e. that the loss of labelled cholesterol from an intima-media layer, including the avascular layer, is small when compared with the appearance in that layer of labelled plasma cholesterol within the same time period. Cholesterol influx into the arterial wall is compared with the simultaneous influx of phosphatidylcholine (PC) and plasma albumin, all labelled according to biosynthetic principles. Moreover, the permeabilities derived from the present work for aortic endothelium are compared with values reported for non-fenestrated mammalian capillaries. Materials and Methods Animals
Mini-pigs of either sex were obtained from Korselitze, Nykq5bing F., Denmark, and from the Institut fiir Tierzucht und Haustiergenetik, University of Gottingen, F.R.G. Weights ranged between 20 and 37 kg, and ages between 6 and 34 months. Some of the animals were given a diet composed of 7% by weight of dried egg yolk, 0.5% of cholesterol (Merck) and the remainder composed of ordinary sow pellet. No atheromas were observed. Preparation
of dialyzed
donor serum
Four serum-donor animals were used for the bio-incorporation of 1, 2 or 3 radiochemicals. The animals were anesthetized by in$amuscular injection of a mixture of etorfin and acepromazine (Immobilon , Pharmacia, Sweden), 4 ml/25 kg of body weight. An angiographic pigtail catheter, type Fr. (W. Cook), was introduced into the external jugular vein until the tip was located in the right atrium, according to screen observation. The catheter emerged through an incision into the subcutaneous layer of the neck. In a mini-pig 10 mCi of [3sS]methionine (spec. act. 387 mCi/mmol, Amersham, England) were administered intramuscularly at time zero. After 11.5 h, 3.6 mCi of [14C]cholesterol (spec. act. 54 mCi/mmol, NEN, U.S.A.)
311
were administered by means of the catheter as a suspension produced by mixing a small volume of ethanol and 20 ml of saline. Finally, at 18 h, 80 mCi of 32P were injected i.m. as sodium orthophosphate (spec. act. 261 mCi/mg P, Ris$, Denmark) dissolved in 5 ml of saline. Isotope incorporation into plasma components was studied using EDTA-plasma from blood samples drawn at appropriate intervals. Exsanguination was performed at 41.5 h. The left common carotid artery was cannulated and blood collected into sterile bottles without anticoagulants. Serum was dialyzed at ambient temperature against a physiological salt solution (Robinson buffer) in an artificial kidney for 1 h (AHFK Kll Asahi Med. Corp., Japan). A reduction of more than 99.5% in the levels of trichloroacetic acidsoluble 32P and 35S radioactivity was obtained. The dialyzed serum was then injected into recipient mini-pigs. Serum injection experiments In one experiment, used for plasma contamination studies, a volume of the triple-labelled donor serum was infused at about 7 h prior to killing. At about 0.3 h before killing the same animal received a volume of serum from a 26-kg donor mini-pig, labelled exclusively with [3H]cholesterol (50 mCi of [1,2(n)3H]cholestero1, spec. act. 53 Ci/mmol, NEN, U.S.A.). In another mini-pig the two differently labelled donor sera were injected, one at 6.5 h and the other at 3.4 h before killing. That these two differently labelled donor sera were eliminated identically from the plasma subsequent to i.v. injection of mixed serum was verified in a young mini-pig. The animals were killed by an overdose of anaesthetic. Blood was rapidly removed from the arteries by rinsing with large volumes of saline. Loose connective tissue was stripped from the arteries and arterial segments were then sectioned and frozen in liquid nitrogen with subsequent storage at -20°C. Immediately before homogenization a segment was thawed and dissected in the cold into a maximum of 4 layers.
Analytical procedures Plasma and tissue lipids were extracted, purified and subjected to 2 thinlayer chromatographic (TLC) separation steps, yielding free cholesterol (FC), esterified cholesterol (EC) and phosphatidylcholine (PC). These fractions were then processed for chemical and radioactivity analyses, also taking into account the percentages of recovery [13]. Levels of 3H and 14C radioactivities were recorded by means of appropriate settings of the liquid scintillation counter (LSC). The areas of TLC silica gel containing PC were subjected to wet combustion. The inorganic phosphate thus obtained was precipitated as magnesium ammonium phosphate in an amount suitable for collection of the crystals on glass filter circles. For 32P counting the circles were positioned below a Berthold low-level proportional argon flow detector [ 141. Subsequent to lipid extraction, non-protein 35S was removed by trichloroacetic acid washings, leaving a pellet with the protein-incorporated 35S. The pellet was treated with formic acid, and a clear 200 ~1 volume of supernatant was mixed with 10 ml of 4% Cab-0-Sil in Permablend @ for counting of “S by means of LSC [13]. It was verified by experiment that the 35S-labelled proteins fraction measured by
312
this method was recovered virtually 100% when plasma containing labelled proteins was mixed with non-radioactive arterial tissue, and then subjected to the entire separation procedure. Influx
calculation
and terms
Results are presented the formula:
by means of an uptake
coefficient
k, calculated
from
where L is the radioactivity measured at killing in a luminal layer of area A and corrected for contaminating plasma radioactivity. The FL represents the average level of radioactivity per ml plasma during the influx experiment for which T is the duration. Provided that the luminal layer functions as a sink within the time interval T, k can be visualized as an influx parameter, here expressed as an intimal clearance having the designation nl cme2 * h-l. In this study 30% of the cholesterol radioactivity was found in low density and about 70% in high density lipoproteins, Only if it is assumed that the labelled phosphatidylcholine had a similar distribution on these lipoprotein classes would identical intimal clearances for phosphatidylcholine and cholesterol be in accordance with the influx of these lipids into the arterial tissue as components in lipoproteins. The term cholesterol designates the sum of free and esterified cholesterol. Where an influx is proportional to the plasma concentration of the substance in question, the clearance can be conceived of as a permeability coefficient. A clearance is reducible to a velocity, in so far as 1 cm * s-’ equals 3.6 X 10’ nl cmV2 . h-l. In capillary physiology the permeability coefficient has been envisaged as a clearance [ 151. Results and Discussion Contamination
The contaminating plasma volumes were obtained from a 0.3 h experiment by division of 3H-E.C label, measured per cm’, by the terminal value, c&, for 3H-EC per nl of plasma (Table 1, col. 5). Since an influx may contribute some label even during that short period, the values in col. 5 are overestimates. Nevertheless, values as low as 3-14 nl/cm2 were found for the intimal surface of aortic segments. The same animal was injected 7 h before killing with [ i4C]cholesterol-containing serum. The 14C-EC found in layer I (intima-media) exceeded by more than 20-fold the 14C-EC ascribeable to the contamination derived above. On the other hand, 1 cm2 of the pure media layer, M, at 0.3 h manifests the presence of label equivalent to about 50 nl of terminal plasma. The value is equal to about 1 I.tl/g of media tissue, which corresponds to the intravascular volume expected in the microcirculation of average vascularized tissue [ 181. Intima-media
conceived
as a sink
The independence of k from T for aortic influx calculation phoprotein, phosphatidylcholine and phosphatidylethanolamine
of plasma phoswas observed
1
aorta
’ The
The
method
concentrations
tively.
that
in footnotes
implies
concentration
concentration
average
plasma
terminal
a The
b The
Tp,
b for
TC
media.
are 0.53,
were
2.92
X lo-
17 and -2
2.70
3, 0.57
and
text,
dpm/nl
from
5.04
1230
130
67
278
130
241
985
274
393
433
3 330
2 720
2 760
(nl/cm2)
4a
I4C-EC/ck
EXPERIMENTS
3H0.421 dpm/nl,
are added.
was
for
respectively.
and
14C-EC,
arterial
dpm/nl
and
saline-washed
X 10m3
trunk,
in the
X 10m3
dissected
3.32
0.35
0.18
0.75
0.35
0.65
2.66
0.74
1.06
1.17
8.99
7.35
7.45
(dpm/cm2)
3
for pulmonary
described
and
layers
dpm/nl
The
in a method
e.g.
used
was 0.415
M values,
label,
I and
a and
the
of
ck.
experiment.
of label.
of influx
A = adventitia
78
A
T = duration
243.9
39
M = media,
1.5 13.1
38
M
73.9
53
A
I
4.5 23.6
54
95
A
34
44
M2
M
20.8 97.8
50
I
6.0 22.8
54
Ml
109.3
94
A
I
25.2
+ some
(dpmlcm2)
(mg/cm2) 33.0
2
1
(T = 7 h)
h)
(T = 0.3
INFLUX
14C-EC,
27
respectively.
ARTERIAL
esterified
IN
3 H-EC,
cholesterol
Labelled
SURFACE
44
terol,
plasma
weight
INTIMAL
to area ratio
Wet
THE
I
OF
M
I = intima-media,
aorta
descending
thoracic
Distal
thoracic
descending
and arch
aorta
Proximal
thoracic
Ascending
(trunk)
Layer
CONTAMINATION
artery
segment
Pulmonary
Arterial
PLASMA
TABLE
THE
3
)
4.63
5
and
dpm/nl
for
3H-
and
TC = esterified
29
2
54
5
44
46
-2
17
(nllcm2)
6b
14C-EC
3H-EC
from
volume
calculated
as plasma
EC and
X 10-j
respectively.
segments.
580
31
176
56
11
233
49
54
14
260
60
78
@l/cm2
5a
as 3H-EC/c$
calculated
Contamination
9
total
14C-EC,
and
33
8
61
10
56
61
-3
12
@l/cm2)
7c
1 4C-TC
3H-TC
respec-
choles-
and
G
w
314
for at least 5 h in the stilbestrol-treated cockerel [14]. In the same animal model this finding was subsequently extended to plasma cholesterol through use of the double-isotope technique outlined in the section on serum injection experiments [17]. By this technique, 2 nearly identical influx values for 2 different time periods were obtained. A mini-pig experiment of that type is the basis for Fig. 1, where the tissue radioactivity expressed as a plasma volume equivalent (kT, i.e. L/AZP) is plotted versus T. Again, k essentially appears to be independent of T within the time period chosen. The tissues studied included layers I and M, as detailed in the legend. In a similar plot concerned with only I-layer radioactivity, the intercept with the Y-axis was positive in accordance with a loss of label from that layer during the latter part of the experiment. If a similar plot was based upon layer M, the intercept was negative. These findings suggest that the activity from layer I is transferred to layer M within the experimental period. An intercept close to zero for the I + M layer (Fig. 1) is in accordance with a negligable loss of cholesterol or with the presence of a sink for plasma cholesterol within a period of 6-7 h. The role of the vasa vasorum of layer M, relative to the role of the intimal surface as entrances for labelled cholesterol remaining in the wall at any given time, may well have been reversed as time passed, as was the case for cockerel aortic wall [16]. (Consider also col. 2 versus col. 3, Table 1.) The difference in slopes observed for TC and EC in Fig. 1 may be explained by the hydrolysis at a fixed fractional rate of newly entered plasma EC [ 171. In spite of any hydrolysis, labelled FC contributed less than 10% of the TC label in the artery. In plasma the percentage was even less. The expected exchange of
32w
TC0 EC--_
??
24C0..
0
2
4 HOURS
6
8
Fig. 1. Evidence for the transient sink function for the luminal half of arterial wall segments. The wet weights of the luminal layers ranged from 83 to 102 mg/cm*, and they comprised both sublayers I and M as defined in Table 1. 1: Pulmonary trunk; 2: ascending aorta plus arch; 3 and 4: proximal and distal halves of thoracic aorta, respectively. With reference to the Methods section a zero intercept suggests a flux proceeding with a constant rate into a sink. Cl4 CJcholesterol-labelled lipoproteins circulated for 6.5 h and [3H]cholesterol-labelled lipoproteins circulated for 3.4 h before termination of the experiment.
315
FC between the endothelial cell surface and the circulating plasma lipoproteins would further influence the small fraction of label found in FC. Finally, assuming the existence of a transient sink, the label/cm* of the sink
260
4bo
6bO
nl UII-~h-l for PC Fig. 2. Intimal clearances for labelled plasma total cholesterol (TC) (ordinate), and labelled phosphatidylcholine (PC) (abscissa). Results are from influx experiments lasting 6.5 and 7 h. 0: pulmonary trunk, ‘3: .ascending aorta plus the arch: ??and A: proximal and distal halves of thoracic aorta. respectively. The most probable linear regression is y = 0.88 (tO.06) x - 14 (k20).
layer is a sum of the contaminating volume X ck and k X3; X T. Since the sum can be written for 3H as well as for i4C data, 2 equations are thus available for the determination of the contaminating volume and k. For an arterial segment subjected to a high influx (pulmonary trunk), this method appears to give the more reliable contamination value (columns 6 and 7 of Table 1. I and M (M,) values have to be added). Influx
of phosphatidylcholine
The intimal clearance values obtained for PC and their linear relationship to those obtained for TC are presented in Fig. 2. This relationship is in accordance with a coupling between the arterial influx of plasma PC and TC. Provided that a transient sink in the artery for the labelled PC is also present and that a similar distribution of labelled PC and TC among the plasma lipoproteins exists, the fact that the value for the slope 0.88 + 0.06 is very nearly unity favours the concept that the labelled lipids were found in the wall as a result of lipoprotein influx. The stilbestrol-treated, hyperlipaemic cockerel provided similar findings [171. Influx
of plasma albumin
Plasma total [35S]protein appeared to be a useful tracer for albumin because 80438% of donor [35S]protein behaved as albumin in being non-precipitable in ammonium sulphate at half-saturation, and because the permeability of the endothelium to albumin is probably greater than the permeability to other predominant plasma proteins [lo]. For the relatively small albumin molecule it is particularly pertinent to contemplate whether the intima-media tissue functioned as an equilibrating tissue rather than as a sink for period T. Evaluation of the data in terms of equilibrium spaces was enhanced by a nearly constant [35S]protein plasma concentration, which at the time of killing was as high as
316 TABLE
2
PERMEABILITY Animal
COEFFICIENTS
FOR THE ENDOTHELIUM
Arterial segment
OF MINI-PIG
ARTERIAL
lo8 X Intima-media uptake coefficients (“vascular permeabilities”) as calculated for 4 macromolecular
SEGMENTS surface markers a
I14Cl
r3H1
C32Pl
cholesterol
cholesterol
[35Sl
phosphatidylcholine
protein
(cm
.
s-l)
(cm
.
s-1 )
(cm
.
s-l)
b
(albumin) (cm
.
s-1 )
Asc. aorta +
13
arch Prox. dew.
4.9
4.7
6.4
8.1
thor. aorta Dist. desc.
3.4
3.4
4.3
3.7
thor. aorta
1.3
1.3
1.6
3.1
Asc. aorta + arch Prox. desc.
2.9
5.6
5.3
thor. aorta Dist. desc.
1.5
1.0
3.5
thor. aorta 13 9
Pulmonary Pulmonary
trunc trunc
1.0
1.4
4.0
12.0 15.7
15.1 16.7
(18.7) (19.3)
a Influx periods 6.5 and 7 h for animals 13 and 9, respectively. During the final 3.4 h of the 6.5 h experiment, lipoproteins labelled with [ 3 HI cholesterol were circulating. The layer studied was the sink layer described in legends to Figs. 1 and 2. b The figures 3.7 and 4.0 are based upon uptake of label by only one of the two sublayers, I and M, plus an estimated uptake for the other sublayer. The values in parentheses are theoretically invalidated as discussed in the text.
82% of the average value. For the spaces obtained the average values in 2 minipigs were 55,20,14 and 10 pi/g W.W. for intima-media layers from pulmonary trunk, aortic arch and proximal and distal thoracic aorta, respectively. For pure media layers values of 104, 19, 10 and 13 /A/g, were obtained. An albumin equilibrium space of 59 pll/g was reported for dog carotid artery [ 201.
.=
PHOw!ATIOnCHOLINE
0=
CHOLESTEROL
PROTEIN PERMEABILITY Cc.xs&'xlO? Fig. 3. The figure gives a graphic representation of the values in Table 2. The most probable linear regressions are y = 0.88 (r0.07) x - 0.71 (t0.76) for PC, and y = 0.79 (kO.07) x - 1.12 (* 0.71) for TC.
317
These values indicate that equilibrium may have been reached in the case of the pulmonary trunk. The aortic segments, on the other hand, showed only about 25% of the equilibrium value cited. At this value in a well-stirred steady state compartment, equilibrating with plasma of constant specific activity, an influx calculated on the sink assumption would introduce a negative error of 14%. In conclusion, aortic [%]protein data are treated on the assumption of a sink. Since the pulmonary trunk k values for [3sS]protein are open to criticism, they have been bracketed in Table 2. This table, in conjunction with Fig. 3, presents k -the intimal clearance - in units of cm X s-l. It is evident in Fig. 3 that the intimal clearances for albumin are about 1.5 times those for “lipoprotein”, as determined with labelled cholesterol and phosphatidylcholine. This suggests that albumin and plasma lipoproteins enter the arterial wall by a similar mechanism, Regional
patterns
The coefficients for influx of labelled TC, EC, PC and albumin decreased with increasing distance from the heart (Table 2). This feature has been described for fibrinogen and albumin in pig experiments [lo-121 and for albumin, low density lipoprotein and cholesterol in dogs [7,8]. A new feature is the influx into the pulmonary trunk. The intimal clearances were 3-5 times greater in the pulmonary trunk than in the aortic arch, irrespective of the macromolecular label (Table 2). * Capillary and arterial intimal permeabilities
to plasma macromolecules
In order to characterize the permeability of capillary and intimal endothelium the same parameters have to be evaluated. These parameters include the area of endothelium, concentrations on both sides of the endothelium, and transendothelial fluxes. Strictly speaking the use of the permeability concept requires a proportionality between net influx of moles and molar concentration differences created or found across the barrier studied. In the case of the intima-media tissue the trans-endothelial flux is not obtainable from an accessible flow of lymph, but the influx at the prevailing plasma concentration can be obtained with tracer methods from the ratio between arterial radioactivity and plasma specific activity. It is important to keep in mind that the ratio between labelled and unlabelled molecules is much lower in the tissue than in the plasma (the sink condition). The presence of such conditions for labelled cholesterol is supported by the kinetic findings, particularly those given in Fig. 1. Vasavasorum are probably not present in our intimamedia (I) but in part of our media (M) layers [21]. If it is assumed that sink conditions apply also to the labelled albumin, the intimal clearance of albumin can be conceived of as a permeability coefficient, in the same way that the intimal clearance of phosphatidylcholine and cholesterol is visualized as a permeability coefficient for plasma lipoproteins. The permeabilities of “lipoproteins” calculated in this way are almost the same as those found by Bratzler et al. for rabbit aortic endothelium using plasma low density lipoprotein, labelled in the protein moiety with 1311 [ 91. The albumin permeability values given in Table 2 are near to those published for various mammalian non-
318
fenestrated capillaries as summarized in Table 4 of Ref. 15. In conclusion, the similarity of the simultaneously measured intimal clearances of albumin, cholesterol and phosphatidylcholine over a wide range of values (Fig. 3) suggests that the in vivo transfer of plasma lipoproteins into the arterial wall of mini-pigs occurs mainly by a nonspecific mechanism. Diffusion, filtration or pinocytosis [ 22,231 may be involved. Acknowledgements Research Establishment Risq5, Denmark, through the cooperation of Dr. H. Hansen.
was helpful
in various
respects
References 1 Walton, K.W., Pathogenetic mechanisms in atherosclerosis, Amer. J. Cardiol., 35 (1975) 542. 2 Bradby, G.H.V.. Walton, K.W. and Watts, R., The binding of total low density lipoproteins in human arterial intima affected and unaffected by atherosclerosis, Atherosclerosis, 32 (1979) 403. 3 Stein, O., Stein, Y. and Eisenberg, S., A radioautographic study of the transport of 125-I-labelled serum lipoproteins in rat aorta. Z. Zellforsch., 138 (1973) 223. 4 Hoff, H.F., Karagas. M., Heideman, C.L.. Gaubatz, J.W. and Gotto. Jr., A.M., Correlation in the human aorta of APO B fractions with tissue cholesterol and collagen content, Atherosclerosis. 32 (1979) 259. 5 Hoff, H.F., Bradley, W.A.. Heideman, C.L., Gaubatz. J.W., Karagas. M.D. and Gotto, Jr., A.M.. Characterization of low density lipoprotein-like particle in the human aorta from grossly normal and atherosclerotic regions, Biochim. Biophys. Acta, 573 (1979) 361. 6 Smith. E.B. and Slater. R.S., Lipids and low density lipoproteins in intima in relation to its morphological characteristics. In: R. Porter and J. Knight (Ed%), Initiating Factors (Ciba Symposium No. 12, New Series), Elsevier. Amsterdam, 1973, p. 39. 7 Duncan, L.E.. Cornfield, J. and Buck, K., Circulation of labelled albumin through the aortic walI of the dog, Circ. Res., 7 (1959) 390. 8 Duncan, L.E., Buck, K. and Lynch, A., Lipoprotein movement through canine aortic wall. Science, 142 (1963) 972. 9 Bratzler. R.L., Chisohn, G.M., Colton, C.K., Smith, K.A. and Lees, R.S., The distribution of labelled low-density lipoproteins across the rabbit thoracic aorta in Volvo, Atherosclerosis, 28 (1977) 289. 10 Bell, F.P., Gallus, A.S. and Schwartz. C.J., Focal and regional patterns of uptake and the transmural distribution of 131-I-fibrinogen in the pig aorta in viva, Exp. Mol. Path., 20 (1974) 281. 11 Bell, F.P.. Adamson; I.L. and Schwartz, C.J., Aortic endothelial permeability to albumin - Focal and regional patterns of uptake and transmural distribution of 131-I-albumin in the young pig. Exp. Mol. Path.. 20 (1974) 57. 12 Somer. J.B., Evans, G. and Schwartz. C.J.. Influence of experimental aortic coarctation on the pattern of aortic Evans Blue uptake in viva, Atherosclerosis, 16 (1972) 127. 13 Stender, S., Christensen, S. and Nyvad, 0.. Uptake of labelled free and esterified cholesterol from plasma by the aortic intima-media tissue measured in viva in three animal species. Atherosclerosis, 31 (1978) 279. 14 Christensen, S. and Nielsen, H., Permeability of arterial end&helium to plasma macromolecules, Atherosclerosis, 27 (1977) 447. 15 Renkin, E.M., Multiple pathways of capillary permeability. Circ. Res., 41 (1977) 735. 16 Christensen, S., Transfer of labelled cholesterol acrnss the aortic intiial surface of normal and cholesterol-fed cockerels, Atherosclerosis. 4 (1964) 151. 17 Stender, S. and Christensen, S.. The concomitantly measured transfer of free cholesterol, esterified cholesterol, phospholipids and phosphoprotein from plasma into the aortic wall of stilboestroltreated cockerels. Atherosclerosis, 28 (1977) 15. 18 Ganong, W.F., Review of Medical Physiology, 8th edition, Lange Medical Publications, LOS Altos. CA, 1977 (Lot. cit. Table 30.1.) 19 Adams, C.W.M. and Morgan, R.S.. Autoradiographic demonstration of cholesterol filtration and accumulation in atheromatous rabbit aorta, Nature (Land.), 210 (1966) 175. 20 Villamil. M.F., Rettori, V., Barajas, L. and Kleeman, C.R., Extracellular space and the ionic distribution in the isolated arterial wall, Amer. J. Physiol., 214 (1968) 1104.
319 21 22
Wolinsky, H. and Glagov, S.. Nature of species vascmm in mammals, Circ. Res., 20 (1967) 409.
differences
in the medial
distribution
of aortic
vasa
Jensen, J., On the relationship between metabolic activity and cholesterol uptake by intima-media of the rabbit aorta. Biochim. Biophys. Acta, 183 (1969) 204. 23 Stender, S. and Zilversmit, D.B., Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits - Molecular size as a determinant of plasma lipoprotein influx, Arteriosclerosis, 1 (1981) 38.