© 1999 American Heart Association, Inc.
Original Contributions |
Evidence of Hypoxic Areas Within the Arterial Wall In Vivo
From the Wallenberg Laboratory for Cardiovascular Research, University of Göteborg, Göteborg, Sweden.
Correspondence and reprint requests to Dr Tom Björnheden, Wallenberg Laboratory, Sahlgrenska University Hospital/S, 413 45 Göteborg, Sweden. E mail tom.bjornheden@wlab.wall.gu.se
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Abstract—The anoxemia theory of atherosclerosis states that an imbalance between the demand and supply of oxygen in the arterial wall is a key factor for the development of atherosclerotic lesions. Direct in vitro and in situ measurements have shown that PO2 is decreased in the more deeply situated parts of the media, but the degree of hypoxia in vivo or the distribution of hypoxia along the arterial tree is not known. For this reason, we have developed a method for the detection of hypoxia in the arterial wall in vivo by using a hypoxia marker, 7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline, that may be visualized by immunofluorescence. In the present study, we have used this method in rabbits with experimentally induced atherosclerosis. Our results indicate that zones of hypoxia occur at depth in the atherosclerotic plaque. The mechanism was probably an impaired oxygen diffusion capacity due to the thickness of the lesion, together with high oxygen consumption by the foam cells. Thus, we have for the first time demonstrated that hypoxia actually does exist in the arterial wall in vivo, lending support to the anoxemia theory of atherosclerosis.
Key Words: atherosclerosis • artery • hypoxia • hypoxia marker
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Introduction |
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The anoxemia theory of atherosclerosis1 highlights the fact that the more deeply situated parts of the arterial wall depend on diffusion to satisfy their need for oxygen and nutrients. When atherosclerotic lesions develop, the arterial wall thickness increases and diffusion capacity is impaired. At the same time, oxygen consumption is augmented,2 3 and an energy imbalance may occur. Local metabolic disturbances may be envisioned that may endanger regression or even result in progression of the atherosclerotic process, with the formation of a necrotic core.
In several studies in animals in vitro and in situ, a decreased oxygen concentration has been demonstrated in the arterial media, with a minimum PO2 of 0.3 to 0.7 kPa (20 to 50 mm Hg).4 5 6 7 So far, however, no data have been presented to verify that hypoxia is present in arterial tissue in the intact animal. At the same time, it is obvious that such data are crucial to validate the presented hypothesis.
In a recent article, we introduced a method for the assessment of hypoxia in arterial tissue in vitro.8 This method is based on the demonstration of a tissue-bound hypoxia marker, 7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline (NITP), which was originally developed by Hodgkiss et al9 to detect hypoxia in tumors. The use of nitroimidazole derivatives (such as NITP) as hypoxia markers was introduced in the early 1980s,10 and applications in tumor research abound (eg, see References 11 through 1411 12 13 14 ). The marker undergoes nitroreduction intracellularly (mainly by cytochrome P450 reductase)15 and reactive radicals are formed, which bind to cellular constituents in the absence of oxygen, hence, a marker of hypoxia. The main advantage with this method is that NITP may be administered in vivo, which makes it possible to assess hypoxia in arterial tissue in the intact animal.
In the present research, we have used this method to study arterial tissue in rabbits in vivo. Our results indicate that hypoxic zones do occur at certain locations in the arterial tree in the living animal. We believe that these results constitute a crucial piece of evidence in support of the anoxemia theory of atherosclerosis.
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Methods |
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Medium, Chemicals, and Antibodies
Eagle's minimum essential medium with Earle's salts and 10 mmol/L NaHCO3 was used perioperatively, supplemented with 1% nonessential amino acids, 100 µg/mL streptomycin, 100 IU/mL penicillin, and 60 mg/mL BSA (Serva Feinbiochimica). NITP was obtained from Lancaster Synthesis Ltd. This drug is poorly soluble in water, and the technique developed by Hodgkiss et al9 was used to prepare the solutions for intraperitoneal administration. Thus, NITP was first dissolved in dimethyl sulfoxide (Sigma Chemical Co) and then added to peanut oil (aflatoxin content <0.5 ppb; Apoteksbolaget) at 10% vol/vol. NITP was added at a final concentration of 50 (n=2) or 100 (n=4) µmol/mL to a 30-mL peanut oil mixture. All NITP solutions were prepared fresh before use.
Sheep anti-theophylline antibody (theophylline-8-BSA) was purchased from Biogenesis Inc, Bournemouth, England. Biotinylated rabbit anti-sheep IgG antibody, fluorescein avidin D, biotinylated goat anti-avidin D, and normal rabbit serum were all obtained from Vector Laboratories, Burlingame, Calif. Nonimmune sheep IgG to be used as the nonspecific control antibody was bought from Cedarlane Ltd, Hornby, Canada. Dry milk powder was obtained from Semper.
Chemicals for the modified Russel-Movat pentachrome staining were obtained as follows: alcian blue from Polysciences Inc; crocein scarlet, phosphotungstic acid, and safranin O from Sigma Chemical Co; and acid fuchsin from Histolab Products AB. Sections were mounted on glass slides (SuperFrost/Plus, Menzel-Gläser) with Vectashield as the mounting medium (Vector Laboratories).
Animals
At 2 to 3 months of age, experimental
atherosclerosis was induced in 8 male New Zealand White
rabbits (Lidköpings Kaniufarm, Masslösa, Sweden)
through a combination of a cholesterol-enriched diet
(1% cholesterol in standard rabbit chow fed ad libitum)
and mechanical injury induced by use of an embolectomy
catheter.16 17 Experiments were carried out after an
induction period of 6 to 8 weeks. Extensive lesions developed in 5
animals, whereas the lesions were less prominent in 3.
To 6 rabbits (3 with extensive lesions and 3 with less prominent
lesions), 30 mL NITP solution was given
intraperitoneally at a dose of 0.43 (n=2) and 0.85
(n=4) mmol/kg (body weight, 3.5 kg) at 6.5 (n=1) and 4.7±0.2
(n=5) hours before the animals were killed. During the NITP injection,
the rabbits were sedated with ketamine 7.5 mg/kg and xylazine 3
mg/kg IM. The animals tolerated the injections well and showed no
adverse reactions during the experiment. Blood samples were taken at
intervals for the determination of NITP concentrations in plasma, which
was performed by high-performance liquid
chromatography9 by Dr M. Stratford,
Gray Laboratory of the Cancer Research Campaign, Mount Vernon Hospital,
Northwood, UK. The aortic arch and the thoracic aorta were removed by a
method that has been developed to ensure maximum tissue
integrity.18 After premedication with ketamine 7.5
mg/kg and xylazine 3 mg/kg, anesthesia was induced by
administration of ketamine 8.5 mg/kg IV and xylazine 3.5 mg/kg
IV initially, plus half this amount every 15 minutes thereafter. After
perioperative preparations, the skin was incised and
the peritoneum opened. Before the thoracic cage was opened, (20 to 30
minutes after the induction of anesthesia), an overdose of
pentobarbital sodium (
50 mg/kg) was given, and the rabbit was
exsanguinated. Approximately 3 minutes after the cessation of
spontaneous breathing, continuous rinsing with oxygenated
medium at 37°C was initiated, and the aortic arch and proximal half
of the thoracic aorta were removed. The major part of the adventitia
was dissected away, and the vessel was cut into 6 to 8 segments 4 to
5 mm long and fixed in 4% buffered formaldehyde, pH 7.0, for 1 to
2 days. Specimens were also taken from selected organs, ie,
myocardium, liver, kidney, striated muscle, esophagus, and
spleen. One of the atherosclerotic rabbits with prominent lesions and 1
nonmanipulated rabbit served as controls. These rabbits did not receive
NITP, but arterial tissue was otherwise treated in the same
way.
As another control experiment, atherosclerotic segments were obtained in the same way, but instead of fixation, the tissue was incubated in vitro. The incubation conditions were identical to those that had been used in previously published methodological studies8 (3 hours, 3% O2) but with modified NITP concentrations in the medium; ie, 250, 125, and 63 µmol/L, or 1x, 0.5x, and 0.25x the concentration originally used.
Sections
Three 5-µm paraffin sections were obtained from 8 to 10 evenly
distributed levels along the thoracic aorta and from 3 to 4 levels in
the arch. In 1 rabbit, 1 segment from the aortic arch was sectioned
serially with 300 µm between sections for a length of 8
mm. A total of 200 to 300 sections were examined. Five-micron sections
were also obtained in a random fashion from the myocardium,
liver, kidney, skeletal muscle, esophagus, and spleen.
Immunofluorescence
The paraffin sections of the fixed specimens were
transferred to glass slides. After deparaffinization and rehydration to
buffer in a graded series of alcohol, the sections were prepared
for immunofluorescence.
Blocking of nonspecific binding was performed with normal rabbit serum (1:50, 20 minutes), and the primary antibody was added (theophylline-8-BSA, 1:5000, 60 minutes). After the sections were rinsed (PBS with 5% dry milk powder, 3x for 5 minutes at pH 7.4), the secondary biotinylated antibody was added (rabbit anti-sheep IgG antibody, 1:100, 30 minutes), and the sections were rinsed once again in the same way. To enhance the immunofluorescence signal, consecutive incubations, 30 minutes each, were made with fluorescein–avidin D (1:500), biotinylated goat anti–avidin D antibody (1:100), and fluorescein–avidin D once again (1:500). After being rinsed in tap water, the sections were mounted. Sections incubated in the absence of the primary antibody and sections in incubations where nonimmune IgG substituted for the specific primary antibody served as controls.
Histochemistry
To visualize the structural correlates of the observed
immunofluorescence, parallel sections were stained
with the Russel-Movat pentachrome method in some cases. In our
experience, optimal staining was obtained after some modifications of
the protocol19 by using a higher concentration (2%) of
alcian blue and a considerably lower concentration of the alcoholic
safranin solution (0.1%).
Microscopy
Bright-field or fluorescence microscopy was performed on
an Axiophot or an Axiovert microscope. For fluorescence, filter
set 16 (BP 485/20, FT 510, LP 520; Carl Zeiss) was used.
Photomicrographs of the outlines of the serial sections were obtained
on the same microscope with the use of the differential interference
contrast setup. Images for publication were registered with a color
video camera (Sony 3CCD, DXC-930P, Sony Co) in integrating mode and
KS400 image analysis software (Carl Zeiss).
Statistics
SDs of the mean were used as the measure of dispersion unless
stated otherwise.
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Results |
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NITP in Plasma
To the best of our knowledge, NITP had not been used in rabbits before, which means that no pharmacokinetic data were available on suitable doses and modes of administration. Also, for practical reasons, it was not feasible to perform extensive methodological experiments. Thus, in the present experiments, we were guided by protocols that have been used in tumor-bearing mice in which hypoxia was assessed by flow cytometry.20
The plasma levels of NITP rose to a maximum between 11.3 and 98.1
µmol/L within 11/2 hours and then decreased, with a half-life
of 1 to 3 hours (Figure 1
). The
maximum concentrations tended to be lower and the half-life longer than
those described in the mouse (100 µmol/L and 1/2 hour,
respectively).9 20 The NITP exposure, calculated as area
under the curve (AUC), was 230±157 (range, 29 to 415) µmol
· h-1 · L-1 in the rabbits. In the
tumor-bearing mice, the AUC was 154 µmol ·
h-1 · L-1.
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For comparison, in the in vitro system that we used to test NITP, the concentration was 250 µmol/L during the 3 hours of incubation, which would correspond to an AUC of 750 µmol · h-1 · L-1.8 In the in vitro control experiment that was performed in the present study, the NITP exposure corresponded to an AUC of 190 to 750 µmol · h-1 · L-1.
Immunofluorescence in Arterial Tissue
Rabbits With Experimental Atherosclerosis
Sections (216) from 12 levels in each of 6 NITP-treated rabbits
were studied. In 1/3 of these, only minor lesions or no
lesions were seen. Approximately another 1/3 had prominent
lesions, whereas the remainder were in between. As summarized in the
Table, the overwhelming majority of
lesions exceeding a thickness of 400 to 500 µm exhibited
distinct zones of immunofluorescence. In Figure 2, these zones are shown at low
magnification together with a routine, stained adjacent section. At
higher magnifications (Figures 3 and 4), the fluorescence appears to
be mainly localized intracellularly in groups of foam cells situated in
the interior parts of the lesions. The zones of
immunofluorescence were 200 to 300 µm wide
and situated at a distance of 200 to 300 µm from the
endothelial surface. In Figure 5, a diagrammatic 3-dimensional
reconstruction of 1 serially sectioned segment is depicted. The
fluorescence occupies the center of the lesion like a
wedge.
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In many sections, fluorescence was also noted in the luminal
endothelium (Figure 6)
and in the endothelium of the vasa vasorum. In control
incubations with nonspecific antibodies, this reaction was reproduced
in the endothelium, whereas no fluorescence was
observed in the foam cells (Figure 7).
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It appeared that this nonspecific
immunofluorescence was a suitable marker for the
vasa vasorum. Although not specifically studied, vasa vasorum were
regularly observed in the adventitia and in the outermost portion of
the media. It seemed that the distance from the zones of specific
immunofluorescence to the vasa vasorum was similar
to the distance to the main lumen (Figure 7
).
Fluorescence was sometimes noted in the adventitia (Figure 3); this reaction was also reproduced by staining with
nonspecific primary antibodies (Figure 7).
Controls
No immunofluorescence was seen in aortic
sections obtained from animals that had not received NITP, 1 rabbit
with experimental atherosclerosis and 1 nonmanipulated
rabbit (Figure 8). Furthermore, no
immunofluorescence was seen in control sections
when the primary antibody was omitted (Figure 8).
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In Vitro Incubations
NITP exposure was lower during the in vivo experiments than during
the incubations performed to test the hypoxia marker in
vitro.8 For this reason, aortic segments were incubated at
reduced NITP concentrations under hypoxic conditions that had been
previously shown to induce binding. As shown in Figure 9, the intensity of the
immunofluorescence was reduced, but clearly
detectable, at an NITP exposure (190 µmol ·
h-1 · L-1) within the same range as the one
used in vivo (230±157 µmol · h-1 ·
L-1).
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Immunofluorescence in Other Tissues
Strong immunofluorescence was noted in the
mucosa of the esophagus (Figure 10).
Immunofluorescence was also noted in the
pericentral regions of the liver lobules (Figure 10). Weak
fluorescence was observed in some of the collecting tubuli of
the kidney. No immunofluorescence was noted in the
myocardium, spleen, or skeletal muscle.
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Discussion |
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A method for the demonstration of arterial wall hypoxia by using the hypoxia marker NITP was recently described from our laboratory.8 The testing was carried out in vitro, and it was proposed that the method could also be used in vivo. In the present study, we have applied this technique in rabbits with experimental atherosclerosis.
In the rabbit aorta, distinct NITP-associated
immunofluorescence was observed in atherosclerotic
lesions that were >400 to 500 µm thick (Figures 2, 3, and 7), and the fluorescence was reproduced in
consecutive serial sections (Figure 5). The fluorescence
was localized intracellularly to foam cells (Figure 4), which
occupied a 200- to 300-µm-wide zone at a distance of 200 to 300
µm from the luminal surface and from the adventitial vasa
vasorum.
When a rabbit aorta with experimental atherosclerosis
was incubated under hypoxic conditions in vitro, foam cells in the
atherosclerotic lesions accumulated NITP.8 In those
experiments, smooth muscle cells bound NITP at a
PO2 <2 to 3 mm Hg, whereas
foam cells seemed to bind the hypoxia marker at a somewhat
higher PO2 but still in a clearly
hypoxia-dependent manner. Thus, the most obvious interpretation
of our present findings is that the arterial tissue in
the zones of NITP immunofluorescence had suffered
from local hypoxia in vivo. From the in vitro data, the
PO2 had most likely been 30 to 40
Pa (2 to 3 mm Hg) in the periphery of these zones. It should also
be pointed out that the NITP exposure in vivo was considerably lower
than that in the in vitro "calibration" experiments (AUCs of
230±157 and 750 µmol · h-1 ·
L-1, respectively). Still, control incubations indicated
that hypoxia-dependent immunofluorescence
was already clearly detectable at an NITP exposure of 190
µmol · h-1 · L-1, similar to that
used in vivo (Figure 9). It has been suggested that the binding
of NITP is proportional to the square root of the
concentration,21 which is also in agreement with
estimations from published in vitro data, indicating that a reduction
of the NITP concentration by a factor 5 could lead to a decrease in
hypoxic fluorescence by 50%.9 Thus, our
present data might have underestimated rather than exaggerated the
true zones of hypoxia in vivo. Furthermore, it is interesting
that classic data by Jurrus and Weiss,5 based on
polarographic measurements in vitro, agree well with our observations.
They found a PO2 of 0 Pa in the
atherosclerotic aortic arch in rabbits at a depth of 300 µm from
the lumen when the total arterial wall thickness exceeded
880 µm, which is similar to the observations in the present
study.
Alternative Mechanisms of NITP Binding
Although hypoxia markers have been shown to bind to
cellular constituents in various tissues24 25 in a
clearly hypoxia-dependent manner, binding to apparently
normoxic tissue has also been described. This points to the possibility
that other, oxygen-independent mechanisms may exist and confound the
interpretation.10 26 27 Thus, nitroreduction might be
channeled by the 2-electron transfer enzyme detoxicating
diaphorase [(NAD(P)H:(quinone-acceptor) oxidoreductase, EC
0.1.6.99.2], thus bypassing the oxygen-dependent neutralization of the
reactive radicals that cause binding.27 This mechanism has
been proposed to explain the apparently oxygen-independent NITP binding
to the esophageal mucosa in mice,27 and in fact, the
presence of this enzyme has been demonstrated there.28
However, others failed to show an effect on NITP binding through
inhibition of the enzyme.29 In addition, in the same
article, binding in the esophagus seemed, in fact, to be oxygen
dependent, at least in vitro. Furthermore, it has been argued that the
detoxicating diaphorase reaction is a very poor producer of
reactive radicals in comparison with the reaction mediated by
cytochrome P450.15 Hypoxia markers have also been
used to demonstrate hypoxic areas in the liver,30 31 but
the results have been suspected to merely reflect local differences in
nitroreductase activity. However, by using retrograde perfusion of
murine liver, Arteel et al32 have demonstrated that NITP
binding in the liver was clearly hypoxia dependent. Thus,
whereas some controversy remains, most data favor the interpretation of
NITP binding as a true marker of tissue hypoxia. That our own
findings do reflect tissue hypoxia is supported by the fact
that the NITP binding in vivo reproduced the clearly
hypoxia-dependent binding in vitro within the same type of
cells and tissue. Also, if
PO2-independent mechanisms were
important, one would also have expected some immunoreaction in cells
closer to the vessel lumen in presumably better-oxygenated
areas.
As discussed in our previous article,8 binding to
foam cells might reflect still another alternative mechanism: ie, local
hypoxia could upregulate detoxicating diaphorase
synthesis,33 leading to an increased nitroreductase
activity and the production of reactive intermediates, which
could bind to NITP. However, others have shown that
overexpression of detoxicating diaphorase by 3 orders of
magnitude had very little influence on NITP binding.15 The
observed immunofluorescence in apparently normoxic
endothelial cells may seem paradoxical and in fact, has
been described before.8 Occasionally, diffuse
fluorescence was also noted in the adventitia (Figure 3). Most likely, however, these reactions do not reflect NITP
binding, because they were reproduced in incubations with nonimmune
primary antibodies (Figure 7). We also confirmed hypoxia
marker binding to the esophagus epithelium and pericentral areas in the
liver (Figure 10).
Levels of Hypoxia
Regarding the degree of hypoxia to which the
immunodetectable NITP corresponded in our experiments, the estimates
were derived from comparisons with our previous in vitro
incubations,8 in which the same type of tissue had been
used. Our conclusion was that bound marker corresponded to a
PO2 of 30 to 40 Pa (2 to 3
mm Hg). In cell lines, half-maximal binding of NITP derivatives is
said to occur mostly at oxygen concentrations of 0.1% to
0.5%,9 15 29 32 35 36 probably corresponding to a
detection level of 0.5% to 1% O2. This is
somewhat lower than our estimate. However, in the only true tissue
"calibration" of NITP (in this case, misonidazole), binding at 140
Pa (10 mm Hg) has been mentioned as the practical limit of
detection.37 In that study,
PO2 was measured directly with
microelectrodes, and tissue binding was assessed by
autoradiography. At 140 Pa (corresponding to 10
mm Hg), PO2 binding was 5 times the
background level, whereas at 70 Pa (5 mm Hg), binding was as high
as 9 times the background level. Although these data were obtained in
tumor spheroids, it is possible that tissue binding of the
hypoxia marker may in fact be more sensitive than previously
suggested. One basic fact that it is also highlighted in the quoted
study is that binding requires living cells. This means that, though
hypoxic, most cells within the foam cell accumulations did not seem to
have undergone necrosis.
"The anoxemia theory of atherosclerosis" states that hypoxia is a key factor for the development of atherosclerotic lesions. However, so far no data have been provided on the actual presence of hypoxia in the arterial wall in vivo. In the present study, we have demonstrated that hypoxic zones do occur within atherosclerotic plaques in rabbits when the lesions exceed certain dimensions and at a depth that is readily reached in humans. It seems likely that the hypoxia in these areas reflects an impaired diffusion capacity due to the thickness of the plaque, together with an increased demand in the metabolically active foam cells.
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Acknowledgments |
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This study was supported by Loo and Hans Osterman's Foundation; the Swedish Heart Lung Foundation Project No. 51009, 61007, and 61510; and the Swedish Medical Research Council (to G.B.).
Received July 1, 1997; accepted August 7, 1998.
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