© 2000 American Heart Association, Inc.
Vascular Biology |
Expression of Allograft Inflammatory Factor-1 Is a Marker of Activated Human Vascular Smooth Muscle Cells and Arterial Injury
From the Departments of Physiology and Cardiology, Heart Failure Research Group, Temple University School of Medicine, Philadelphia, Pa.
Correspondence to Michael Autieri, PhD, Departments of Cardiology and Physiology, Temple University School of Medicine, Room 809, MRB, 3420 N Broad St, Philadelphia PA 19140. E-mail mautieri{at}unix.temple.edu
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Abstract |
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Abstract—The cytokine-induced activation and proliferation of medial vascular smooth muscle cells (VSMCs) leading to intimal hyperplasia is one of the most critical cellular events in the formation of transplant arteriopathy and balloon angioplasty-induced restenosis. Allograft inflammatory factor-1 (AIF-1) is a calcium-binding protein that we have previously shown to be expressed in balloon angioplasty–injured rat carotid arteries. We hypothesized that AIF-1 expression may be associated with the VSMC response to injury. In this study, we examined AIF-1 expression in immunologic and mechanical models of arterial injury. Reverse transcription–polymerase chain reaction and Western analysis demonstrated that AIF-1 is acutely and transiently expressed in aortic medial smooth muscle cells of rat cardiac allografts, with mRNA and protein peaking at 3 to 7 days after transplant and declining by 10 days after transplant. Immunohistochemical analysis identified abundant AIF-1 in the medial VSMCs of these vessels. Immunohistochemical analysis of balloon angioplasty–injured swine coronary arteries also demonstrates an acute AIF-1 expression detectable by 24 hours and continuing up to 14 days after the procedure. AIF-1 in these vessels also localizes to the medial VSMCs and cells of the developing neointima. AIF-1 protein is not expressed in quiescent cultured human VSMCs but is induced in cells challenged with various inflammatory cytokines, primarily by interferon-
, interleukin-1ß, and T-cell–conditioned media.
Transfection and overexpression of AIF-1 in human VSMCs result in
enhanced growth of these cells. Taken together, these data indicate
that AIF-1 expression is associated with vascular trauma and suggest
that this protein may play a role in VSMC activation subsequent to
arterial injury.
Key Words: allograft inflammatory factor-1 • vascular smooth muscle cells • cytokines • transplant arteriopathy • balloon angioplasty
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Introduction |
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Vascular narrowing produced by graft vascular disease is a major complication that limits long-term survival of cardiac transplantation.1 2 A primary cause of cardiac allograft vasculopathy (CAV) subsequent to cardiac transplantation is the intimal proliferation of vascular smooth muscle cells (VSMCs) in large- and medium-sized arteries and veins. The initiation of CAV is believed to involve a chronic immune response of the recipient to the donor vasculature in which activated immune cells damage the endothelium, resulting in the production of cytokines.3 These cytokines in turn elicit the activation of medial VSMCs, which migrate into the intimal layer and proliferate. Similarly, the long-term efficacy of percutaneous transluminal coronary angioplasty as a treatment for advanced multivessel coronary artery disease is significantly limited by the high incidence of vascular restenosis observed in as many as 40% of the patients undergoing this procedure.4 Analogous to CAV, restenosis subsequent to balloon angioplasty injury is also a process mediated by activated VSMCs.5 It has been suggested that cytokine-induced activation and proliferation of medial VSMCs, leading to intimal hyperplasia, is the most critical cellular event in the formation of CAV and balloon angioplasty–induced restenosis.6 7 8 9 Accordingly, the activation of several selected genes may reflect the status of smooth muscle cell (SMC) activation and, thus, the development of arteriopathy indicative of the progression of neointimal hyperplasia. Identification and functional characterization of these gene products is a promising approach for the development of antirestenotic therapeutics.10 11 12
Allograft inflammatory factor-1 (AIF-1) is a 143–amino acid
calcium-binding protein.13 14 Initial investigations
indicated that AIF-1 expression was limited to interferon-
(IFN-)–stimulated macrophages and neutrophils, which
infiltrated rat cardiac allografts.13 Our initial
observations came from balloon angioplasty–damaged rat carotid
arteries, in which we observed a rapid transient expression pattern in
AIF-1 mRNA.14 The intimal proliferative response of
balloon angioplasty–induced vascular injury in rats is similar to that
observed in the rat cardiac transplant model with respect to
endothelial cell loss, activation of medial VSMCs, and
elaboration of neointimal
hyperplasia.1 15 Considering the similarities
between mechanical and immunologic injury, we hypothesized that AIF-1
would also be expressed in the VSMCs of allografted tissue and that its
expression in CAV might be associated with the progression of vascular
hypertrophy characteristic of balloon angioplasty–induced
injury. It was our intention to focus our examination of AIF-1
expression to the SMC response to vascular injury, because VSMCs are
the major cell type in the media and in the developing
neointima in this tissue. In the present study, we
examine AIF-1 expression in 2 in vivo models of arterial
injury: immunologic (in rat aortic allografts) and mechanical (in
porcine coronary artery overstretch). We also explore a more
direct role for AIF-1 in human VSMC activation by examining its ability
to influence VSMC growth. The goals of the present study are (1) to
associate AIF-1 expression with arterial trauma in
different models of arterial injury, (2) to compare this
expression with progression of the restenotic lesion over time,
(3) to localize AIF-1 expression to activated medial and
intimal VSMCs, and (4) to characterize a role for AIF-1 in VSMC
activation.
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Methods |
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Heterotropic Rat Heart Transplantation
Lewis rats providing donor hearts were anesthetized with buprenorphine administered subcutaneously and 1% to 2% isoflurane gas inhalation. The hearts were removed and gently perfused with sodium heparin dissolved in saline and placed in a cold saline solution containing sodium heparin until transplantation. Recipient Wistar-Furth rats were similarly anesthetized, and the donor heart was transplanted into the recipient right abdominal cavity with the donor aorta connected to the recipient abdominal aorta and the donor pulmonary artery connected to the recipient inferior vena cava. Once completed, the wound was closed with sterile nonabsorbable sutures and swabbed with Betadine surgical scrub. At various times after transplant, the donor hearts were removed, and the aortas were isolated and freed of any adhesions and external material. Some samples were processed for RNA and protein isolation; others were fixed and processed for immunohistochemistry. All procedures were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC).
Swine Coronary Artery Balloon Angioplasty
Domestic swine weighing 20 to 30 kg were sedated, and general
anesthesia was induced. After an 8F introducer sheath with
a hockey stick curve was placed in the right femoral artery of the
anesthetized animals, they received a bolus of heparin (200
U/kg) and bretylium (2.5 mg/kg). A guiding catheter was advanced to the
aortic root, intracoronary nitroglycerin (200
µg) was injected, and baseline angiograms of the left and right
coronaries were obtained. Coronary injury was achieved by
deliberate stretch of the vessel wall with an oversized angioplasty
balloon inflated to a pressure of 8 to 10 atm for 30 seconds, with a
1-minute rest period, for a total of 3 times. After withdrawal of the
catheter, the femoral sheath was removed, and the cutdown site was
repaired. At various times after angioplasty, the animals were
euthanized according to the standard protocol of massive pentobarbital
overdose (100 mg/kg IV). The left main coronary artery was
cannulated, and injured segments were located by examination, dissected
in block, and processed for immunohistochemistry. All procedures were
performed in accordance with IACUC-approved institutional
guidelines.
Tissue Processing and Immunohistochemistry
Hearts excised from pigs were perfusion-fixed with 10% buffered
formalin at physiological pressures (100
mm Hg), and injured segments were fixed in 10% formalin for 4 hours
and then in 70% ethanol for 18 hours and paraffin-embedded. Rat aortas
were dissected from the heart, cleared of adherent tissue, fixed, and
processed as described for porcine tissue. Tissue sections were
deparaffinized, rehydrated in PBS and ethanol, quenched with
H2O2, and blocked with goat
serum. For primary AIF-1 antibody, rabbits were immunized with a
peptide corresponding to amino acids 17 to 33 (KAQQEERLDEINKQFLH)
present in the AIF-1 protein. Antisera were affinity-purified with
this peptide, and Western analysis of recombinant AIF-1 protein
indicates that this antiserum recognizes a protein of the predicted 17
kDa. SMC 
-actin antibody (Sigma Chemical Co) was used at a 1:2500
dilution to identify VSMCs. AIF antibody was applied at a 1:1000
dilution in PBS/1% BSA, washed, and incubated with biotinylated goat
anti-rabbit secondary antibody (Transduction Laboratories) at 1:2000
dilution. This was washed and incubated with an avidin-biotin enzyme
complex and chromogenic substrate as described by the
manufacturer (Vectastain Elite ABC peroxidase), which develops a
reddish-brown stain. Sections were counterstained with hematoxylin.
Sections treated with secondary antibodies only or nonimmune IgG did
not show any staining. Coronary vessels from at least 2 swine
and 3 sections per vessel were evaluated.
Cells and Culture
Human coronary VSMCs were obtained as cryopreserved
secondary culture from Clonetics Corp and subcultured in growth medium
as described previously.14 Cells from passages 3 to 6 were
used in the described studies. The growth medium was changed every
other day until cells approached confluence. Preconfluent VSMCs were
serum-starved (0.25% FCS) for 48 hours and then exposed to 10% FCS,
10 ng/mL basic fibroblast growth factor (bFGF), 100 U/mL IFN-, 20
ng/mL interleukin (IL)-1ß, 20 ng/mL platelet-derived growth
factor (PDGF)-AB, 2 ng/mL transforming growth factor (TGF)-ß, or
T-cell–conditioned medium for 48 hours, at which times samples were
processed for protein isolation. Some samples remained untreated and
were used as controls. PDGF, bFGF, IFN-, and TGF-ß were purchased
from GIBCO-BRL; IL-1ß was purchased from Boehringer-Mannheim;
and T-cell–conditioned medium was purchased from Fisher Biotech.
Transfection and Proliferation Assay
The protein coding region of the AIF-1 cDNA was cloned by
polymerase chain reaction (PCR) with the use of AIF-1 gene-specific
primers. The 5' PCR primer also contained a Kozak consensus sequence
(GCCGCCGCCATGG) to enhance translation. This was inserted into the
expression vector pBK-CMV (Stratagene), and this purified DNA construct
was termed pBK-CMV-AIF-1. Transfection of human VSMCs has been
described.15 Briefly, human coronary artery SMCs
grown in T-75 flasks were transfected with either no plasmid (mock
control), pBK-CMV plasmid alone, or pBK-CMV-AIF-1 along with 3 µL/mL
LipofectAMINE reagent (Life Technologies) and mixed with 1 µg/mL of
either plasmid. Two days after transfection, the cells were trypsinized
and split 1:2, with one half left to grow in the presence of growth
medium+G418 (Geneticin) for 15 days. The other half was saved for RNA
isolation. After selection for 15 days, the cells were then trypsinized
and counted by use of a standard hemocytometer.
Western Blotting
To prepare cell extracts, human VSMCs grown in T-75 flasks were
cultured and treated as described above, washed with PBS, and treated
with 0.3 mL of ice-cold lysis buffer (50 mmol/L HEPES-KOH, pH 7.5,
150 mmol/L NaCl, and 0.1% Triton X-100) containing protease
inhibitors, as described.16 Lysates were
incubated on ice 20 minutes, withdrawn through a 21-gauge needle 3
times, then centrifuged at 3000 rpm for 15 minutes at 4°C,
and stored at -20°C. Proteins were extracted from rat aorta by
Tri-Reagent (Molecular Research Center). Equal protein concentrations
of cell extracts were electrophoresed through an 18%
polyacrylamide gel under reducing conditions and transferred to
a nitrocellulose membrane. Membranes were blocked with 5% nonfat
powdered milk in TBST buffer (0.1 mol/L Tris-HCl, pH 8.0, containing
1.5 mol/L NaCl and 0.5% Triton X-100). Membranes were incubated with a
1:2500 dilution of primary antibody and a 1:2000 dilution of goat
anti-rabbit secondary antibody. Proliferating cell nuclear antigen and
cyclooxygenase-2 antibody were from Santa Cruz,
Inc. The membrane was washed with TBST, and reactive proteins were
visualized by using the enhanced chemiluminescence method (Amersham)
according to the manufacturer’s instructions.
RNA Isolation and Semiquantitative Reverse
Transcription–PCR
For each time point studied, aorta from allografted rat hearts
were isolated and cleared of adherent tissue. Total RNA was extracted
by Tri-Reagent, which allowed simultaneous extraction of
RNA and protein, and 4 µg of total RNA was reverse-transcribed by
using random hexamers as described previously.14 One fifth
of the cDNA was PCR-amplified by using the following primers: 5' TAT
CAT GTC CTT GAA ACG AAT GCT GG AGA A 3' and 5' TTT GTC TTC TGT TTT AGC
ATT CGG TCT CAG 3', which define a 330-bp region of the human AIF cDNA,
for 32 cycles. This is in the linear assay range with respect to cycle
number, template concentration, and dilution of cDNA. The
glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
amplimers were purchased from Clonetech and define an amplicon of 450
bp. One fifth of the reaction was run on a 2.5% agarose gel, ethidium
bromide–stained, and photographed. PCR products were
Southern-transferred to the hybridization membrane and hybridized with
an end-labeled 45-mer oligonucleotide probe
complementary for a sequence internal to the PCR amplicons. For
verification of transfection efficiency, total RNA was extracted from
transfected cells, and 4 µg was reverse-transcribed. One fifth of the
cDNA was PCR-amplified by using neomycin-specific amplimers, which
define a 290-bp region of the neomycin cDNA, for 32 cycles.
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Results |
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AIF-1 mRNA and Protein Are Expressed in Medial VSMCs in Allografted Rat Aorta
We initially examined the expression of AIF-1 mRNA in VSMCs subject to immunologic insult by using aortas isolated from rat cardiac allografts. Donor hearts from Lewis rats were heterotopically transplanted into the abdomen of Wistar-Furth recipient rats, and semiquantitative reverse transcription–PCR (RT-PCR) was performed on RNA isolated from aortas dissected from Lewis donor hearts at various times after transplant (Figure 1
). Figure 1 indicates an acute expression pattern of AIF-1 mRNA
expression, with mRNA levels increasing within 24 hours of
transplantation, peaking at 3 to 7 days, and declining by 10 days after
surgery. Seven-day syngeneic (Lewis to Lewis) control aortas also
demonstrate a low, but detectable, increase in AIF-1 mRNA expression.
AIF-1 is constitutively expressed in spleen tissue and was used as a
positive control. The identity of this amplicon as human AIF-1 was
confirmed by Southern analysis of the same gel hybridized with
a probe specific for a sequence internal to the AIF-1 amplimers (Figure 1). These results indicate that AIF-1 mRNA is acutely and
transiently induced in aortas from allografted rat hearts.
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It was important to determine whether AIF-1 protein was expressed in a
fashion similar to that for AIF-1 mRNA. Protein was extracted from the
same aorta as used for RNA and subjected to Western analysis
with AIF-1–specific antibody. Figure 2
indicates that AIF-1 protein expression is also inducible by cardiac
allograft transplantation and follows a pattern similar to that for
mRNA, with protein levels peaking at 3 to 7 days and declining by 10
days after surgery. No AIF-1 was observed 1 day after transplant, which
may be due to the time necessary for mRNA to be translated into
detectable amounts of protein. Again, a small increase in AIF-1 protein
was detectable in 7-day Lewis to Lewis control rats. These results
indicate that AIF-1 protein is also acutely and transiently expressed
by immunologic insult and reflects that of mRNA expression.
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We used immunohistochemistry to localize progression of AIF-1 protein
accumulation in allografted aortas. Serial sections from Lewis to
Wistar-Furth cardiac allografts were
immunohistologically examined with AIF-1 antisera, and
Figure 3 shows rat aortas before and at
various times after transplant. Similar to protein levels determined by
Western analysis, little to no AIF-1 protein was present in
the uninjured aorta or in the aorta 24 hours after transplantation
(Figure 3A and 3B). By 3 days after surgery, however, a marked
induction of AIF-1 protein was observed in the medial VSMCs, continuing
through day 7 (Figures 3C and 3D). By day 10, AIF-1 protein
levels appeared to decline, and a modest amount of AIF-1 protein was
detectable in a 7-day Lewis to Lewis control aorta (Figures 3E and 3F, respectively).
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It is important to note that positive AIF-1 staining localizes to
SMCs, which are identified by their morphological features and positive
staining for SMC -actin. (Figure 3H
). These findings are also
consistent with the cellular pathology of this model, in which
monocytes and macrophages are frequently found only in the
allograft adventitia in early lesions and are not detected in the
media and intima until 20 days to 3 months
onward.17 Overall, these results closely mirror
those observed for AIF-1 mRNA and protein expression as detected by
RT-PCR and Western analysis and indicate that AIF-1 is
expressed acutely and transiently in aortic VSMCs by allograft
transplantation.
AIF-1 Protein Is Induced in Swine Coronary Arteries by
Balloon Angioplasty
The swine response to vascular injury presents many
similarities to that of humans, particularly in the generation of
neointimal formation. We performed immunohistochemistry on
swine coronary arteries subject to oversized-balloon
angioplasty and examined them for AIF-1 expression at several time
points after surgery. Figure 4A
demonstrates that AIF-1 protein was not detectable in uninjured
arteries but was rapidly expressed in medial VSMCs by 1 day after
injury (Figure 4B). This staining is distributed
circumferentially around the vessel, and as with the aortic allografts,
expression is almost exclusively limited to the medial VSMCs, because
these cells also stain positively for SMC -actin (Figures 4G and 4L). Intense AIF-1 expression was apparent in the medial VSMCs and
neointimal cells 3 days after injury (Figures 4C, 4H, and 4M). The most striking observation at this time point is that
AIF-1 was present in cells of the developing neointima,
whereas these same cells were negative for SMC -actin.
Neointimal VSMCs that have switched from the contractile to
the synthetic phenotype characteristically do not synthesize
-actin.4 AIF-1 expression appeared to decline by 14
days after balloon injury (Figures 4D and 4I). At this time
point, a large neointima was evident; however, the most
intense AIF-1 immunostaining localized to the medial
VSMCs compared with neointimal cells. The
neointimal cells that did stain appeared to localize to the
luminal side of the injury (Figure 4I). This
neointima is a mix of SMC -actin–positive and
–negative cells; however, not all SMC -actin–positive cells
stained positively for AIF-1 (Figure 4N). This indicates that
AIF-1 expression is not limited to rejecting allografts but is also
induced by mechanical injury of the coronary arteries as well.
These results are also similar to our findings in balloon-injured rat
carotid arteries with respect to the acute and transient kinetics of
AIF-1 expression.
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AIF-1 Protein Expression Is Induced by Cytokines in
Human VSMCs
The lack of AIF-1 in uninjured vessels and its inducible
expression in medial VSMCs after transplantation and mechanical injury
suggested that expression of this protein in vivo is regulated by
soluble factors. Because AIF-1 immunostaining localized
to VSMCs, we examined induction of AIF-1 protein expression in primary
cultured human coronary artery VSMCs stimulated with a variety
of cytokines. In these experiments, cells were starved into
quiescence by serum deprivation for 48 hours and exposed to
10% FCS, bFGF, IFN-, IL-1ß, PDGF, TGF-ß, and
T-cell–conditioned media for 48 hours. Extracts were separated by
SDS-PAGE, and AIF-1 was detected by Western analysis with
specific antisera. The results of Figure 5 indicate that human AIF-1 is
differentially induced by various soluble factors in cultured human
VSMCs. Ten percent FCS, IFN-, and IL-1ß are capable of inducing
this protein, whereas AIF-1 is not expressed in unstimulated cells.
bFGF, PDGF, and TGF-ß can induce AIF-1 protein expression to a small
degree. T-cell–conditioned media, which contains several soluble
factors, elicited the strongest induction of AIF-1.
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Because activated lymphocytes are among the first immune cells
present in the arterial lesion and because
T-lymphocyte–conditioned media elicited the strongest induction of
AIF-1 protein in cultured VSMCs, we treated cultured human VSMCs with
T-lymphocyte–conditioned media and examined extracts of these cells
for AIF-1 protein by Western analysis at different time points.
Figure 6 demonstrates that AIF-1 is not
expressed in unstimulated cells but is induced by T-cell–conditioned
media 24 hours after stimulation and peaks by 72 hours. These results
indicate that in human VSMCs, AIF-1 is a cytokine-responsive
protein and is particularly responsive to immune cell factors,
suggesting that its induction in arteriopathic lesions is primarily
dependent on inflammatory processes.
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Overexpression of AIF-1 in Human VSMCs Increases Their
Proliferative Capacity
The expression of AIF-1 in activated VSMCs led us to
investigate whether expression of this protein was linked to cell
growth. As an initial approach toward understanding the function of
AIF-1 in VSMC activation, we determined the effects of overexpression
of this protein on VSMC proliferation. Primary human VSMCs were
transfected with either no plasmid (mock control), with pBK-CMV plasmid
alone, or with pBK-CMV containing AIF-1 protein coding region cDNA
(pBK-CMV-AIF-1). Two days after transfection, cells were trypsinized
and split 1:2, with one half left to grow in the presence of growth
medium+G418 (Geneticin) for 15 days. The other half was saved for RNA
isolation. After selection, the cells were trypsinized and counted. The
results of 3 independent experiments are presented in the
Table and demonstrate an average 2.6
fold-increase in pBK-CMV-AIF-1–containing cells compared with
pBK-CMV–containing cells. Because the transfection efficiency of
primary human cells is low, it was necessary to use RT-PCR of RNA
isolated from newly transfected cells to indicate that equal amounts of
plasmid are present in control and in pBKCMV-AIF-1–transfected
cells. Figure 7 indicates that these
results are not due to differences in transfection efficiency,
suggesting that human VSMCs that overexpress AIF-1 proliferate at a
more rapid rate than do cells that do not.
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Discussion |
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Two widely used and clinically relevant models of arterial injury are rat cardiac allograft rejection and swine balloon angioplasty.15 18 We initially focused on Lewis to Wistar-Furth allografts, which consistently develop an acute graft rejection within 12 days, and aortic tissue, which is almost exclusively composed of VSMCs. It has been shown that nonimmunosuppressed rat aortic allografts are not acutely rejected but develop arteriosclerotic alterations in the vascular wall that closely and histologically resemble the pathological changes observed in clinical transplants that undergo chronic rejection.17 19 These alterations include infiltration of mononuclear cells into the adventitia, endothelial cell hypertrophy, and SMC proliferation, resulting in thickening of the intima over time. Aortic allografts also resemble chronically rejecting human transplants biochemically with respect to plasma levels of thromboxane and prostacyclin.17 At the mRNA and protein level, AIF-1 is acutely and transiently expressed in aortic allografts (Figures 1
and 2). An induction of AIF-1 mRNA is detectable 24
hours after surgery, and AIF-1 protein is detectable shortly
thereafter, at 3 days. mRNA and protein decline by 10 days after
transplant. AIF-1 mRNA and protein are detectable at low levels in the
Lewis to Lewis control animals, likely as a result of small numbers of
macrophages and the moderate expression of growth factors that
are found in isografted arteries.18
Immunohistochemical analysis of donor aortas with AIF-1
antibody closely reflects the acute and transient expression pattern
obtained by Western analysis and RT-PCR. It has been
demonstrated that in aortic allograft rejection, IL-2 receptors (T
lymphocytes) and OX42-positive cells
(monocytes/macrophages), although present in the
adventitia, are generally absent in the media and intima until 20 days
after transplant.17 Notwithstanding, it was possible that
AIF-1 expression was due at least in part to infiltrating immune cells.
Consequently, it was important to verify that the primary source of
AIF-1 expression that we observed was derived from SMCs, and Figure 3 shows that AIF-1 is expressed in SMC -actin–positive
medial VSMCs. It is also interesting to note that in allografted rat
aortas, medial thickening and cellular proliferation in medial and
intimal VSMCs does not begin until 14 days after
surgery.17 The early presence of mononuclear cells in the
adventitia of the grafted aorta may be the source of cytokines,
which activate the medial VSMCs, which in turn induce AIF-1
expression in these cells. Because AIF-1 expression is readily
detectable in VSMCs by 3 days after transplantation, expression of this
protein may be an early marker of graft rejection originating from the
vessel itself.
At the cellular and molecular level, the vascular responses to
mechanical and immune injury are similar,1 15 16 18 and we
hypothesized that balloon angioplasty–initiated injury would also
evoke AIF-1 expression. In particular, the response produced by
mechanical injury of porcine coronary arteries presents
many similarities to that of humans, including coronary
circulation and spontaneous development of
atherosclerosis.5 20
Neointimal formation subsequent to balloon angioplasty is a
dynamic process involving several different cell types and occurring in
several phases. Early events are primarily inflammatory in nature and
are initiated by T lymphocytes and macrophages, which secrete
various cytokines and growth factors seminal to the local
inflammatory response. Later events may last several weeks after injury
and involve activated VSMCs, which at this stage are primarily
fibroproliferative in nature and synthesize soluble factors and
extracellular matrix proteins. Because several studies have suggested
that the response of the injured vessel is related to the degree of
injury,21 22 we examined swine coronary arteries
at several time points after balloon angioplasty to correlate AIF-1
expression with the progression of restenosis. The rapid and
robust expression of AIF-1 in 1-day balloon angioplasty–injured swine
coronary arteries (Figures 4A to 4J) is associated with
the initial arterial response to injury. Of particular note
is the rapid and circumferential expression of AIF-1 in 1-day vessels.
This expression is localized to the VSMC, as confirmed by morphology
and SMC -actin staining. Studies have shown that very low numbers of
proliferating cells are found in the media of angioplasty-damaged swine
vessels.23 This acute expression pattern is quite similar
to that observed by us in balloon-injured rat carotid
arteries,14 indicating that AIF-1 expression is
independent from, or at least precedes, proliferation. In the more
advanced 14-day lesions, AIF-1 expression is stronger in medial VSMCs
and the luminal side of the neointima rather than in the
fibroproliferative core of the lesion, suggesting that expression of
this protein may responsive to soluble factors originating from
inflammatory cells present in the adventitia and circulation. This
is in contrast to that observed for SMC -actin–positive cells,
which are more uniformly present throughout the
neointima. Taken together, this infers that AIF-1
expression may be more of a marker of the initial VSMC response to
inflammation rather than of the progression of intimal hyperplasia.
Immune cells secrete several soluble factors that stimulate VSMC
migration, growth factor secretion, and cellular proliferation, and
numerous studies have shown that VSMCs in allografted vessels and
balloon angioplasty–injured arteries express several classes of
cytokines and inflammatory factors.4 24
Inflammatory cytokines, such as IFN- and IL-1ß, along with
T-cell–conditioned media and FCS, are proliferative as well as
inflammatory and display the most potent induction of AIF-1 compared
with other factors.4 25 Although cytokines such as
bFGF-1, PDGF, and TGF-ß have been implicated as being responsible for
the promotion of intimal hyperplasia in response to
injury,1 4 these factors were capable of inducing AIF-1
protein expression only to a minor degree. Other studies have observed
that in macrophages, AIF-1 mRNA expression is induced by T-cell
factors, such as IFN-, and that this expression could be modulated
by compounds that influence the inflammatory response.26
These studies have suggested that AIF-1 is not expressed in VSMCs;
however, the VSMCs in those experiments were not
cytokine-stimulated.13 Our results indicating that
AIF-1 is not expressed in unstimulated VSMCs corroborate that earlier
work. T-lymphocyte–conditioned medium contains several factors,
including granulocyte-macrophage colony–stimulating factor,
IFN-, IL-1, IL-6, and TNF-ß, which are known to influence the VSMC
phenotype, and it has been demonstrated that infiltration of
the artery wall by T lymphocytes induces a change in VSMC
phenotype, which is mediated by inflammatory,
mitogenic, and antiproliferative factors.27 28
In agreement with what we observed with injured vessels in vivo,
induction of AIF-1 occurs rapidly and is detectable at 24 hours of
exposure to T-cell–conditioned medium (Figure 6).
Stimulation of cells with cytokines and mitogens results in a rapid transient increase in calcium levels,29 and mitogenic stimulation of arterial SMCs involves a flux of calcium ions through the plasma membrane.30 31 Calcium and its primary receptor protein, calmodulin, are required for cellular growth and survival, because both of these are essential for the entry of quiescent cells into the cell cycle in response to mitogenic signals.32 33 Not surprisingly, calcium channel blockers and calcium antagonists inhibit restenosis subsequent to mechanical (balloon angioplasty) and immunologic (transplant vasculopathy) insult.34 35 36 37 Overexpression of calmodulin in cultured cells enhances cell proliferation, primarily through a reduction in the length of the G1 phase of the cell cycle,8 9 and stable overexpression of several other calcium-binding proteins has also been shown to influence the cell cycle.12 13 With these observations as a starting point, we hypothesized a role for AIF-1 expression in VSMC growth.
Similar to other proteins that interact with calcium, AIF-1 is involved
in cellular proliferation, because overexpression of this protein
enhances the growth of human VSMCs. Stable transfection of AIF-1 cDNA
into primary human VSMCs results in an average 2.6-fold increase in
cell number (Table). Given the role of calcium regulation in
growth control, one possible explanation for the growth-enhancing
properties of AIF-1 is through its ability to interact with and
possibly regulate the concentration of calcium ions in the cell.
The expression of AIF-1 in a localized cytokine-rich injury implies that AIF-1 may take part in inflammation-initiated activation events. The expression of AIF-1 in medial and intimal VSMCs before proliferation in vivo infers that this protein may function in the mediation of VSMC growth initiated by inflammatory stimuli. Additionally, although the vascular response to injury and development of restenosis has been shown to differ from species to species,38 the present study also indicates that AIF-1 expression is conserved, suggesting a fundamental function for this protein in vascular injury. In immune and mechanical vascular injury, immune cells play an important role in the initiation and progression of restenosis. However, the vascular pathobiology resulting in intimal hyperplasia is due to VSMC activation, making AIF-1 not only a surrogate marker of arterial injury but also a potential target of antirestenotic therapies.
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Acknowledgments |
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The authors are grateful to Dr Howard Eisen for critical reading of the manuscript and Sheri Kelemen for technical advice.
Received August 17, 1999; accepted January 31, 2000.
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