Articles |
From the Institute of Pharmacological Sciences and Center E. Grossi Paoletti, University of Milano (Italy).
Correspondence to Dr Maurizio Soma, Institute of Pharmacological Sciences, via Balzaretti 9, 20133 Milano, Italy.
| Abstract |
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30% and 75% in the intimal
and medial tissues of
rA-IM/A-IMpretreated rabbits. Thus, a
short-term treatment with rA-IM/A-IM
inhibits smooth muscle cell proliferation and intimal thickening in
rabbits, providing evidence for the potential use of
rA-IM/A-IM in the treatment of
atherosclerosis.
Key Words: recombinant apolipoproteins smooth muscle cell proliferation atherosclerosis restenosis
| Introduction |
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Apolipoprotein A-I (apoA-I), the primary protein component of HDL, seems to be responsible for the antiatherogenic activity of HDL. Indeed, apoA-I participates in reverse cholesterol transport, being a cofactor in lecithin/cholesterol acyltransferase activation4 ; it is also a ligand for the putative HDL receptor5 and stimulates cholesterol efflux from lipid-loaded cells.6 It also displays peculiar properties not directly related to its major activities in lipoprotein metabolism, ie, stimulation of prostacyclin release from the arterial wall and its stabilization,7 8 activation of fibrinolysis,9 and modulation of complement function.10 Because of all of these activities, apoA-I overexpression in atherosclerosis-susceptible mice converts them into atherosclerosis-resistant animals,11 whereas apoA-I infusion in cholesterol-fed rabbits exerts an inhibitory effect on the progression of atherosclerotic lesions.12 Taken together, these findings argue for a potential therapeutic use of apoA-I in the treatment of human atherosclerosis.13
Apolipoprotein A-IMilano (apoA-IM), the first described molecular variant of human apolipoproteins,14 15 seems to confer to the carriers a significant protection against vascular disease.16 ApoA-IM is characterized by a cysteine for arginine substitution at position 173 in the primary sequence,17 allowing the formation of disulfide-linked homodimers (A-IM/A-IM) and heterodimers with apoA-II. The presence of these covalently-linked apoA-I molecules is likely to be responsible for the increased metabolic stability of the carriers' HDL. Indeed, in an in vitro system, the interconversion of A-IM HDL is impaired,18 whereas in vivo the A-IM dimers are removed from plasma at a slower rate than monomeric A-IM or normal A-I.19 The A-IM/A-IM has been produced in Escherichia coli in sufficient amounts for in vitro and in vivo studies; the recombinant protein (rA-IM/A-IM) proved to be structurally and functionally identical to the natural protein.20 In the search of innovative approaches for the treatment of neointimal formation,21 we evaluated the effects of a short-term treatment with rA-IM/A-IM in cholesterol-fed rabbits, in which intimal thickening was induced by inserting a flexible extra-arterial collar around the common carotid artery.22
| Materials and Methods |
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Surgical Procedures
Male New Zealand White rabbits (1.8 to 2 kg) (Charles River,
Italy) were used for the study. Insertion of the flexible
extra-arterial collar was performed essentially as described
previously.22 26 Rabbits were anesthetized by an
intramuscular injection of xylazine (5 mg/kg) and ketamine (35 mg/kg).
Animals were then placed in dorsal recumbency, a midline neck incision
was made, and both carotid arteries were surgically exposed. A
nonocclusive, biologically inert, soft, hollow Silastic collar was
positioned around both carotid arteries. The collar was 1.5 cm long and
touched the arterial circumference at two points, 1.0 cm apart. In each
animal, the collar was removed from one carotid (sham-operated artery),
and the wounds were sutured.
Study Protocol
A short-term protocol treatment (10 days) was chosen to avoid
protein-antibody induction: rabbits were intravenously injected with
either reconstituted HDL
(EPC+rA-IM/A-IM) or EPC liposomes.
Twenty-five rabbits were fed a cholesterol-rich (1%) diet at daily
amounts of 100 g for 31 days. Collar placement was performed 21 days
after the beginning of the diet regimen, and all animals were killed 10
days later. Animals were randomly assigned to the following five
treatment groups: group 1, control (hypercholesterolemic diet only);
groups 2 and 4, rabbits receiving EPC liposomes or reconstituted HDL,
respectively, at days 16, 18, 20, 22, and 24; and groups 3 and 5,
rabbits receiving EPC liposomes or reconstituted HDL, respectively, at
days 21, 23, 25, 27, and 29.
Histological and Immunofluorescence Analysis
Histological Analysis
Histological analysis to assess neointimal
formation was performed on carotid arteries from all animals
(sham-operated and collar-applied carotid arteries). Two rabbits from
each group were injected into the marginal ear vein with
5-bromo-2'-deoxyuridine (BrdU, 40 mg/kg) 3 hours before death. All
animals were given a lethal dose of sodium pentobarbital, and the
vasculature was perfused with 0.1 mol/L phosphate-buffered saline (PBS)
for 10 minutes. The three animals from each group not injected with
BrdU were perfusion-fixed for another 20 minutes with 3%
glutaraldehyde buffered with 0.1 mol/L PBS at a pressure of 100 mm Hg.
The carotids from BrdU-injected animals (20 carotids, two animals from
each group) were fixed by immersion in absolute ethanol overnight.
Tissues were paraffin-embedded and stained with hematoxylin and eosin.26 At least 600 cross sections (5 µm thick) were cut for each artery. Neointimal formation was measured on carotids from all animals (50 carotids from 25 animals) by an image-analysis system (see below).
Immunofluorescence Analysis
Immunofluorescence analysis was performed on ethanol-fixed
carotid arteries. Serial sections (n=10) were used to detect BrdU,
-smooth muscle (
-SM) actin, or macrophages by indirect
immunofluorescence with anti-BrdU (Becton Dickinson), anti
-SM1
actin (Sigma Chemical Co), or RAM11 macrophage-specific (DAKO A/S)
monoclonal antibodies, respectively. Staining with these antibodies was
performed according to standard procedures using a fluorescein
isothiocyanateconjugated (FITC) anti-mouse IgG antibody for
visualization (Sigma).26 For the proliferation
analysis, after BrdU-positive cell counting, all nuclei were
stained with propidium iodide, and the labeling index was calculated as
the percentage of BrdU-positive versus total nuclei.
The discrimination between proliferative smooth muscle cells or
macrophages was obtained with a double-labeling procedure.
Proliferative smooth muscle cells were identified by the double
reaction with tetramethylrhodamine isothiocyanate (TRITC)-labeled
anti-BrdU and FITC-labeled anti
-SM1 actin. Proliferative
macrophages were identified by the double reaction with TRITC-labeled
anti-BrdU and FITC-labeled anti-RAM 11.
Image-Analysis System
Total and BrdU-positive cells and the cross-sectional thickness
of the intima and underlying media were analyzed by an
image-analysis system interfaced with a Zeiss Axioscope microscope.
Neointimal formation was quantified as the ratio of the cross-sectional
thickness of intimal and medial tissue. Intima-to-media (I/M) ratios
were calculated to normalize the data. The I/M value obtained from each
animal is the mean of at least 1000 measurements, performed on at least
10 different sections. Thickness, as well as labeling indexes, were
determined for each wall compartment (media and intima) for each
carotid artery.26 Incorporation of BrdU in replicating
cells was monitored by evaluating the femoral bone marrow
labeling26 in each animal. The image-analysis system
consisted of a Macintosh IIx computer (Apple) equipped with a Frame
Grabber Card (QUICKCAPTURE, Data Translation), a Sony
high-resolution video camera, and a Trinitron SuperMac 21-inch color
monitor. All measurements were performed by using National Institutes
of Health (NIH) software (IMAGE, version 1.44; Dr Wayne
Rasband, NIH, Bethesda, Md).
Statistics
Statistical significance of differences between groups of
animals was determined by unpaired Student's t test. A
value of P<.05 was considered to be significant.
| Results |
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Plasma cholesterol levels in control rabbits (group 1) increased progressively from 40 to 60 to 930±245 mg/dL at the day of collar insertion and to 1741±144 mg/dL at the end of the study. These values did not differ from those of animals receiving EPC liposomes (groups 2 and 3) (909±175 mg/dL at surgery and 1875±320 mg/dL at the time of death) or rA-IM/A-IM (groups 4 and 5) (941±225 mg/dL at surgery and 1923±484 mg/dL at the time of death). Agarose gel electrophoresis of serum samples demonstrated that the hypercholesterolemia in all rabbits was accompanied by the accumulation of atherogenic ß-very-low-density lipoprotein particles.
The contralateral carotid arteries showed normal histology in all
treated groups (Fig 2
, top left), with no thickening of
the intimal or medial tissue (Table
), as previously
described.26 This applied to all rabbits independently of
the treatment. Periarterial insertion of the flexible collar in control
animals (group 1) resulted in a marked increase in intimal thickness
(mostly cellular) in the carotid arteries with the collar (Fig 2
, top
right); medial thickness did not change significantly. The extent of
intimal thickening induced by the collar in control animals (I/M ratio
of 0.63±0.11) was within the range observed in a larger series of
cholesterol-fed rabbits (0.63±0.07, n=10). Intimal carotid thickening
in EPC-treated rabbits (groups 2 and 3) (Fig 2
, bottom left) was
slightly lower than in control rabbits (Table
), possibly confirming
previous reports describing modest antiatherogenic activity of
phospholipid infusions in experimental animals.27
Treatment with rA-IM/A-IM significantly
inhibited the formation of intimal lesions after perivascular
manipulation only when it was started before collar insertion (group 4)
(Fig 2
, bottom right). The mean intimal thickness of the
rA-IM/A-IMpretreated rabbits was
40% and
50% of that in control and EPC-treated rabbits,
respectively (Table
). The intima of
rA-IM/A-IMpretreated rabbits contained
significantly fewer cell layers than the intima of control and
EPC-treated rabbits. By contrast, when
rA-IM/A-IM was administered at the day
of collar insertion and thereafter, no significant reductions in
intimal thickening were observed (Table
).
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Cell-specific antibodies were used to evaluate the composition of
intimal and medial cell populations by immunostaining techniques. The
medial tissues of sham-operated arteries and collar-applied arteries of
all animals showed a strong reactivity for
-SM actin. The
neointima induced by collar positioning contained
predominantly
-SM actinpositive cells (>90% of the whole cell
population) (Fig 3
, top). The neointima but
not the media also contained a few macrophages, identified by a
positive reaction with a specific antibody, RAM11 (Fig 3
, bottom).
Thus, the lesions induced by collar insertion consisted mainly of
smooth muscle cells.
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Direct in vivo evaluation of cell proliferation was performed by
measuring the incorporation of BrdU, a thymidine analogue, into
replicating DNA. An average of 6% of total cells in control animals
were BrdU-positive (Fig 4
, top; Table
); by
double-immunofluorescence labeling techniques all BrdU-positive cells
were also recognized by a specific
-SM actin antibody, further
demonstrating the significant contribution of smooth muscle cell
proliferation to the intimal thickening induced by perivascular
manipulation.
|
EPC administration (groups 2 and 3) did not affect myocyte
proliferation (Fig 4
, middle; Table
). By contrast, pretreatment with
rA-IM/A-IM resulted in a significant
inhibition of smooth muscle cell proliferation (Fig 4
, bottom; Table
);
the number of BrdU-positive cells was significantly reduced compared
with control and EPC-treated rabbits in the media, whereas the
difference in the intima was of borderline significance
(P=.065). Again, the inhibition of smooth muscle cell
proliferation was observed only in animals treated with
rA-IM/A-IM before collar insertion
(group 4 versus group 5).
| Discussion |
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The mechanism responsible for the inhibitory effect of
rA-IM/A-IM on the development of intimal
lesions demonstrated here remains to be clarified. In vivo and
epidemiological evidence strongly suggests that HDLs exert a protective
effect against atherosclerosis by promoting reverse cholesterol
transport.1 Indeed, infusions of HDL into cholesterol-fed
rabbits prevented and/or removed cholesterol deposits within the
arterial wall.3 More recently, the intravenous delivery of
plasma-derived human apoA-I (25 mg/wk for 6 weeks) reduced the I/M
ratios in balloon catheterinjured arteries of cholesterol-fed rabbits
by
40%.12 In a different animal model, ie, transgenic
rats overexpressing human apoA-I, modest elevations of plasma apoA-I
levels similarly resulted in a significant reduction (35%) of intimal
smooth muscle cell proliferation after balloon
deendothelialization.31 In spite of the obvious
differences in the injury models, all these data support similar
activities of apoA-I and rA-IM/A-IM in
inhibiting intimal thickening.
The observation of a reduced aortic cholesterol content in the apoA-Itreated animals12 supports the concept that apoA-I, and possibly rA-IM/A-IM also, inhibits intimal thickening through a stimulation of reverse cholesterol transport.1 However, the cellular composition of the arterial lesions in the present study, as well as the short-term treatment protocol, also argues for other potential mechanisms. Since rA-IM/A-IM is effective in preventing intimal thickening only when given before lesion induction, a preventive effect of rA-IM/A-IM on the early events in lesion formation can be hypothesized. Indeed, apoA-I inhibits the deposition of the membrane attack complex of complement on endothelial cells,10 an early finding preceding the appearance of overt lesions in the hypercholesterolemic rabbit model of atherosclerosis.32 ApoA-I and rA-IM/A-IM may also exert a direct effect on smooth muscle cell proliferation, resulting in a reduced incorporation of a thymidine analogue into replicating DNA. Several genes become transiently activated during cell proliferation,33 and proto-oncogene expression is induced early in vessels after balloon denudation.34 ApoA-I and rA-IM/A-IM may affect this process, as suggested by in vitro observations of an inhibitory effect of human HDL on low-density lipoproteinmediated stimulation of proto-oncogene expression in cultured vascular smooth muscle cells.35
Proliferation of vascular smooth muscle cells is one of the major
mechanisms involved in the process of restenosis after successful
endarterectomy or angioplasty.36 37 The problem of
restenosis after angioplasty has become critical in view of the
steadily growing number of procedures and the still high rate of
restenosis.38 Many inhibitors of smooth muscle cell
proliferation have been identified; some, including heparin,
angiotensin-converting enzyme inhibitors,
3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors, factor Xa
inhibitors, and
-interferon have also been found to inhibit
neointimal formation in animal models of arterial
restenosis.26 39 40 41 42 However, a successful inhibitor of
angioplasty restenosis in human trials has yet to be
found.43 Great effort is thus devoted to the search of
innovative approaches to restenosis prevention: blockade of growth
factors by monoclonal antibodies,44 local drug
delivery,45 gene transfer,46 and local
alteration of gene expression by antisense nucleotides47
have proven effective in animal models.
In the present study, another approach to the issue of restenosis is proposed: the use of recombinant apolipoproteins appears to drastically improve the balance between antiatherogenic and proatherogenic lipoprotein concentrations in plasma. ApoA-I seemed to be a good candidate because of its multifaceted activities on atherosclerosis-related systems1 5 6 7 8 9 10 and because of the strong inverse correlation between plasma HDL levels and the rate of restenosis after endarterectomy48 or angioplasty.49 ApoA-I already proved to be effective in reducing the extent of atherosclerosis in balloon-catheterized cholesterol-fed rabbits.12 ApoA-IM/A-IM, because of its improved physicochemical20 and kinetic19 properties versus apoA-I, might exert a more sustained effect than the normal protein, possibly reflecting the increased protection against cardiovascular disease in the A-IM carriers compared with control individuals.16 A relative dose-response study is warranted to solve this issue, but such a study would require very large amounts of the wild-type and mutated proteins, which are unavailable at present. The present study was limited by the small number of animals treated in each group; therefore, the present findings should be implemented once larger amounts of protein become available. However, the very high reproducibility of intimal thickening induced by arterial manipulation and of the effects of treatment, together with the similar results obtained with apoA-I in a different animal model,12 argue for a direct effect of apoA-I and rA-IM/A-IM in preventing neointimal formation, thus supporting the potential use of recombinant apolipoproteins in the treatment of vascular disease.13
Note added in proof. After the submission of this manuscript, we became aware of very similar results demonstrating that rA-IM/A-IM reduces intimal thickening after balloon catheterization in hypercholesterolemic rabbits.50
| Acknowledgments |
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Received August 1, 1994; accepted November 28, 1994.
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