Atherosclerosis and Lipoproteins |
From the Department of Pathology (T.M.C., J.W.B.) and Department of Microbiology and Immunology (J.W.B.), Albert Einstein College of Medicine, Bronx, NY; the Department of Anatomy and Cell Biology (S.D.G.), The Hebrew University, Hadassah Medical School, Jerusalem, Israel; the Cardiovascular Division (I.J.S.), The University of Virginia Health Sciences Center, Charlottesville, Va; the Departments of Pathology and Medicine (J.A.B.), University of California School of Medicine, Los Angeles; the Zena and Michael A. Wiener Cardiovascular Institute (J.T.F., M.B.T.), Department of Pathology (J.T.F.), and Department of Medicine (J.T.F., M.B.T.), Mount Sinai School of Medicine, New York, NY.
Correspondence to Tina M. Calderon, PhD, Albert Einstein College of Medicine, Department of Pathology F727, 1300 Morris Park Ave, Bronx, NY 10461. E-mail calderon{at}aecom.yu.edu
| Abstract |
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or epidermal growth factor. IG9 expression was further
increased by pretreatment of SMCs with the proatherogenic lipid,
minimally oxidized low density lipoprotein. After balloon injury (24
hours), IG9 is induced in vascular SMCs before the detectable
accumulation of monocytes within the vessel wall. Thus, the expression
of IG9 by SMCs as well as by ECs may be an important factor in the
accumulation of foam cells in atherosclerotic plaque development after
arterial injury.
Key Words: arterial injury atherosclerosis smooth muscle adhesion molecules LDL, minimally oxidized or modified
| Introduction |
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(TNF-
),5 6 and the chemokine, monocyte
chemoattractant protein-1 (MCP-1).7 8 Chemokine expression
contributes to the recruitment of additional monocytes (and T
lymphocytes) into the developing lesion, and inflammatory mediators
produced by the infiltrating leukocytes stimulate resident cells within
the vessel wall to secrete additional proatherogenic molecules,
including growth-regulatory factors, cytokines, and other
chemokines. These molecules promote medial smooth muscle cell (SMC)
migration and proliferation and the continued influx of monocytes and T
lymphocytes, thereby amplifying this inflammatory process and promoting
neointimal hyperplasia.3 9 An understanding of
the mechanisms involved in the initial recruitment and adhesion of
monocytes to the vessel wall and their subsequent retention within the
developing atherosclerotic lesion might facilitate the design of
interventional strategies to attenuate this process. Monocyte binding is mediated, in part, by the expression of adhesion proteins that are induced on the surface of ECs by a variety of stimuli, including cytokines.10 11 12 13 P-selectin,14 intercellular adhesion molecule-1 (ICAM-1),15 16 17 18 E-selectin,18 and vascular cell adhesion molecule-1 (VCAM-1)18 19 have been detected in human atherosclerotic vessels.
All of these adhesion proteins were found to be expressed by ECs, with VCAM-1 localized mainly to vessels within neovascularized areas of plaques19 and the other adhesion proteins expressed by luminal ECs overlying the plaques.14 15 16 17 18 ICAM-1 and VCAM-1 were also shown to be focally expressed by SMCs within plaques.15 16 18 19
We identified previously a novel monocyte adhesion molecule that is
recognized by monoclonal antibody (mAb) IG9 and is expressed on the
surface of activated ECs.20 The 105-kDa adhesion
molecule, referred to as the IG9 protein, is not expressed in vitro on
untreated human umbilical vein endothelial cells but is
induced after treatment with TNF-
, interleukin-1ß,
lipopolysaccharide, or phorbol myristate
acetate.20 21 IG9 expression is first detected 3 hours
after treatment of human umbilical vein endothelial
cells with these agonists, and maximal levels are induced after 4 to 9
hours, returning to baseline after 48 hours. Minimally oxidized or
modified LDL (MM-LDL) also induces IG9 expression in human umbilical
vein endothelial cells and in human aortic ECs.
E-selectin, VCAM-1, and ICAM-1 are not induced or upregulated in
MM-LDLtreated ECs.22 Monocyte adhesion to TNF-
and
MM-LDLtreated ECs is significantly inhibited by mAb IG9, whereas
lymphocyte and granulocyte binding to TNF-
treated cultures is
unaffected.20
In human and rabbit advanced atherosclerotic plaques, IG9 protein
expression in vivo has been localized to arterial luminal
endothelium, suggesting a possible functional role for
this protein in monocyte adhesion to ECs overlying the plaques during
lesion progression.20 The present study examines the
cell typespecific expression of the IG9 adhesion molecule for
monocytes and the monocyte chemoattractant, MCP-1, after
balloon-induced arterial injury in the
cholesterol-fed rabbit models of single and double
injury.23 24 25 We found that IG9 and MCP-1 are prominently
expressed by medial SMCs very early after balloon injury (BI) and
before detectable monocyte infiltration of the tunica media. In cell
culture studies, SMCs were induced to express IG9 protein after
treatment with MM-LDL, platelet-derived growth factor (PDGF)-BB,
and either TNF-
or epidermal growth factor (EGF) for 24 hours. Thus,
vascular SMC expression of IG9, in addition to MCP-1, may be a
significant contributor to the extensive accumulation of
macrophages within the vessel wall after arterial
injury.
| Methods |
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Balloon Injury
Details of the BI protocol have been published
previously.23 24 25 Briefly, BI was performed through a
right common carotid artery incision with a 2.5-mm-diameter balloon
dilation catheter (Advanced Cardiovascular Systems Inc)
after bolus heparin administration (150 U/kg, Solopak Laboratories).
Three 1-minute 10-atm inflations were performed 1 minute apart by use
of a hand inflator. After the procedure, the catheter was removed, the
carotid artery was ligated, and the wound was sutured. Rabbits were
then fed standard rabbit chow until the time of death. Animals were
administered an overdose of sodium pentobarbital, and the femoral
arteries were perfused at 100 mm Hg with 4%
paraformaldehyde in PBS through a catheter inserted in
the carotid artery and positioned above the aortoiliac bifurcation.
Segments of femoral arteries at the site of previous BI and/or EC
injury (2 to 3 cm in length) were excised, postfixed in 4%
paraformaldehyde in PBS, cross-sectioned at 2-mm
intervals, dehydrated in ethanol and xylene, and embedded in
paraffin.
Histomorphometry
Percent luminal cross-sectional area narrowing by plaque
(%CSAN-P) was determined by computerized planimetry with use of a
CUE-2 Image Analyzer (Galai Production Ltd) in
association with an Olympus BH-2 microprojection system as
described previously.26 %CSAN-P was calculated as the
area bound by the internal elastic lamina minus luminal area times 100
divided by the area bound by the internal elastic lamina.
Immunohistochemistry
Serial sections (5 µm) were cut onto
poly-L-lysinecoated slides (Sigma Chemical Co),
deparaffinized through ethanol and xylene, and rehydrated. Sections
were blocked with 2% horse serum in Tris-buffered saline for 1 hour at
37°C, washed with Tris-buffered saline, and incubated for 2 hours at
room temperature with primary antibody. mAb IG9 ascites (IgG3, 1:1000
dilution) was used to detect IG9 protein; RAM-11 mAb (mouse anti-rabbit
monocyte/macrophage antibody, IgG1, 1:50 dilution, Dako Corp)
was used for identification of monocytes; and mAb 1A4 (mouse anti-human
-smooth muscle actin, IgG2A, 1:200 dilution, Dako Corp) was used for
identification of SMCs. MCP-1 expression was detected with the mouse
anti-human mAb, 3F11 (IgG1, 1:200 dilution; a generous gift of Dr
Charles Mackay, formerly of Leukosite Inc, Cambridge, MA). After washes
with Tris-buffered saline, sections were incubated with biotinylated
horse anti-mouse IgG (1:800 dilution) for 1 hour, followed by a
45-minute incubation with avidin-biotin alkaline phosphatase (ABC
Vectastain, Vector Laboratories). The alkaline phosphatase substrate
consisted of nitro blue tetrazolium chloride and 5-bromo-4-chloro-3
indolyl phosphate p-toluidine salt (GIBCO-BRL) and 1
mmol/L levamisole (Sigma), an inhibitor of
endogenous alkaline phosphatase activity. Positive
reactivity is indicated by a blue-purple precipitate. Serial sections
from all intervention groups were also incubated with negative control,
isotype-matched, mouse myeloma proteins (IgG1, IgG2A, and IgG3,
Cappel-Organon Teknika Corp). Nonspecific antibody reactivity was not
detected in any of the sections.
Cell Culture
Human aortic SMCs were purchased from Clonetics or isolated from
human thoracic aortas harvested from explanted hearts at the time of
cardiac transplantation. Primary cultures were established by removing
the adventitia and connective tissue, dissecting the remaining
arterial intima and media into 1-cm2
segments, and incubating the tissue fragments in tissue culture dishes
with 2.5 mg/mL collagenase and 15% FBS. Adherent cells
were washed free of debris, and cells were grown in DMEM with 10% FBS,
50 U/mL penicillin, and 50 µg/mL streptomycin (Pen-Strep). Cells were
identified as SMCs by their typical microscopic appearance and by
immunostaining with antibody to
-smooth muscle
actin. The absence of endothelial cell contamination
was confirmed by immunostaining with antibody F8/86 to
human von Willebrand factor (1:100 dilution, Dako Corp).
Growth Factors and Other Reagents
Recombinant human PDGF-BB homodimer, EGF, and interferon-
(IFN-
, 200 IU/mL; endotoxin <0.2 ng/µg) were purchased from
Genzyme. Human TNF-
(100 IU/mL, endotoxin <0.1 ng/µg) was
purchased from R&D Research Systems. DMEM, 100x Pen-Strep, and FBS
were purchased from GIBCO-BRL, and collagenase type II was
purchased from Worthington Biochemical.
Cell-Based ELISA
Human aortic SMCs were grown to confluence in 96-well culture
plates, and before treatment, SMCs (passages 7 to 12) were rendered
"quiescent" by incubation in low-serum medium consisting of
DMEM+1% FBS for 24 to 36 hours. Cells were treated (4 or 8 wells per
treatment) with TNF-
, IFN-
, EGF, or PDGF-BB for 4, 16, or 24
hours at 37°C and 5% CO2 in low-serum medium.
In addition, cells were treated with PDGF-BB in combination with
TNF-
, IFN-
, or EGF. SMCs were also pretreated with MM-LDL (250
µg/mL) for 24 hours in low-serum medium before cytokine
and/or growth factor treatment. LDL was isolated by density-gradient
centrifugation of serum and stored in PBS containing
0.01% EDTA. MM-LDL was obtained by storage of LDLs at 4°C for 3 to 6
months, as previously described,22 or by enzymatic
oxidation.27 Three to 5 separate experiments were
performed for each treatment group except for MM-LDL (n=2). Treated
SMCs were fixed in 2% formaldehyde in PBS, blocked with 1% BSA in
PBS, and incubated with mAb IG9 ascites (1:1000 dilution) for 2 hours
at 37°C. After PBS washes, alkaline phosphatasecoupled goat
anti-mouse IgG3 secondary antibody (1:1000 dilution, Southern
Biotechnologies) was added for 1 hour at 37°C, followed by the
addition of the alkaline phosphatase substrate p-nitrophenyl
phosphate disodium (Sigma). Absorbance was read at 405 nm. Results are
expressed as mean±SEM. Statistical significance was determined by
2-tailed Student t test. A value of P<0.05 was
considered significant.
| Results |
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Femoral arteries subjected to air desiccation EC injury without subsequent high-cholesterol feeding exhibited mild to moderate neointimal formation (%CSAN-P=26±6) consisting almost entirely of SMCs, with monocyte infiltration localized to focal areas in the adventitia at 28 days after EC injury (not shown). All sections from this intervention group exhibited minimal to undetectable levels of IG9 and MCP-1 expression in the vessel wall (not shown).
IG9 and MCP-1 Expression After BI in
High-CholesterolFed Rabbits (Single Injury)
Analysis of sections of arteries of
high-cholesterolfed animals excised 24 hours after BI
(single injury) showed undetectable neointimal thickening
(%CSAN-P=0±0) with monocytes localized only to small focal
areas of the adventitia (Figure 1A
).
Intense IG9 antibody reactivity was detected in medial SMCs in all
analyzed vessel sections (Figure 1B
). Adventitial
microvessel SMCs also expressed high levels of IG9
immunostaining. MCP-1 antibody reactivity closely
paralleled that of mAb IG9 in intensity and localization (Figure 1C
). IG9 and MCP-1 expression preceded detectable monocyte
infiltration of the tunica media and neointimal formation
in injured vessels. There were numerous IG9 (not shown) and
MCP-1positive cells (Figure 1D
) adherent to or just below the
internal elastic lamina, which were unreactive with the RAM-11
(monocyte) and 1A4 (SMC) antibodies. The identity of these cells is not
certain, but they could possibly be T lymphocytes, neutrophils, and/or
SMCs that have lost
-smooth muscle actin expression.
|
Twenty-eight days after BI (single injury), significant
neointimal formation (%CSAN-P=50±4) consisting
of SMCs (Figure 2A
) and monocytes (Figure 2B
) was present. Substantial numbers of monocytes and
monocyte-derived foam cells (RAM-11 positive) were seen within the
media just below the internal elastic lamina and in the adventitia as
reported previously.28 Prominent IG9 and MCP-1
immunoreactivity was present in medial SMCs and to a lesser extent
in foam cells (Figure 2C
and 2D
). Adventitial microvessel SMCs
also showed marked IG9 and MCP-1 antibody reactivity.
Neointimal SMCs and monocytes were focally IG9 and MCP-1
positive (Figure 2C
and 2D
). A continuous layer of luminal
cells, probably ECs, were seen overlying plaques at this time point.
These cells expressed moderate to high levels of IG9 and MCP-1
immunoreactivity (Figure 2E
and 2F
) and were unreactive with the
RAM-11 (monocyte) and 1A4 (SMC) antibodies (not shown).
|
IG9 and MCP-1 Expression After EC Injury and BI in
High-CholesterolFed Rabbits (Double Injury)
Substantial neointimal formation, consisting of SMCs
and monocytes, was detected in 6 of 7 animals analyzed 24 hours
after double injury (%CSAN-P=52±9), with numerous
monocytes and foam cells localized to the adventitia,
neointima, and the media below the internal elastic lamina
(Figure 3A
) as reported
previously.24 25 26 28 29 In the 6 vessels with plaque
development, IG9 (Figure 3B
) and MCP-1 (Figure 3C
)
expression was localized to neointimal, medial, and
adventitial microvessel SMCs and to monocytes and foam cells within the
vessel wall and adventitia. IG9 immunoreactivity appeared less
prominent than that of MCP-1 in all of the analyzed vessel
sections.
|
The cell typespecific expression of IG9 did not change at 7 days
(%CSAN-P=29±3, not shown) and 28 days
(%CSAN-P=61±6, Figure 3E
) after double injury,
although IG9 immunoreactivity was greater than at 24 hours. In
addition, luminal ECs overlying the plaques were also moderately to
highly reactive with mAb IG9 (28 days after BI). MCP-1 expression at 7
days (not shown) and 28 days (Figure 3F
) after BI (double
injury) was also very similar to that seen at 24 hours, with the
addition of luminal EC expression of MCP-1 28 days after BI.
Growth Factor, Cytokine, and MM-LDL Regulation of IG9
Expression in Human SMCs
The novel finding of SMC expression of IG9 after
cholesterol feeding and BI initiated a study to
characterize further the expression of the IG9 protein in this cell
type. Primary human aortic SMC cultures were treated with TNF-
,
IFN-
, EGF, or PDGF-BB. IG9 expression was analyzed by ELISA
after treatment for 4, 16, or 24 hours. Untreated SMCs expressed
minimal amounts of cell surface IG9 protein. Treatment with TNF-
(1
to 500 U/mL), IFN-
(1 to 1000 U/mL), EGF (1 to 30 ng/mL), or PDGF-BB
(1 to 50 ng/mL) for 4, 16, or 24 hours did not induce significant IG9
expression (data not shown). However, treatment with PDGF-BB (10 ng/mL)
in combination with IFN-
(100 U/mL) or EGF (3 ng/mL) for 24 hours
induced significant IG9 expression compared with no treatment
(P<0.05, Figure 4
).
|
We have previously shown that the proatherogenic oxidized lipid,
MM-LDL, at 250 µg/mL induces IG9 protein expression in human and
rabbit ECs after 24 hours and that this expression mediates a
significant portion of MM-LDLinduced monocyte
adhesion.20 To determine whether MM-LDL also induces IG9
expression in SMCs, cultures were treated with this concentration of
MM-LDL for 24 hours. Unlike ECs, IG9 expression was not significantly
induced in human aortic SMCs (Figure 5
).
However, MM-LDL pretreatment of SMCs for 24 hours increased the level
of IG9 protein induced by TNF-
, IFN-
, EGF, or PDGF-BB, with
significant synergistic increases seen after treatment with EGF or
PDGF-BB in combination with TNF-
or EGF (P<0.02 versus
no treatment, Figure 5
; IFN-
was not included in these
studies).
|
| Discussion |
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In a study by Scott et al,30 increased cell proliferation and growth factor synthesis in the adventitia were detected at early times after balloon overstretch injury of pig coronary arteries. Migration of adventitial myofibroblasts across the external elastic lamina to the developing neointima was demonstrated, and these results support the hypothesis that the adventitia may play a role in vascular lesion formation after vessel injury. In a recent report, enhanced leukocyte-endothelial cell interactions and P-selectin, ICAM-1, and VCAM-1 expression were found to occur in vivo in the rabbit mesenteric microcirculation during the first 2 weeks of hypercholesterolemia.31 The authors suggest that the microvasculature is an early site of vascular dysfunction, enabling circulating leukocytes to traffic from microvessels to large arterial vessels during plaque development. Thus, monocytes within the developing neointima and media of atherosclerotic vessels may not originate entirely from the lumen of the arterial vessel.
Neointimal formation 28 days after BI is characterized by significant monocyte accumulation throughout the vessel wall and is accompanied by prominent IG9 positivity on neointimal SMCs, monocytes, foam cells, and luminal ECs overlying the plaque. This is in contrast to the negligible monocyte accumulation that occurs 28 days after EC injury of normolipemic rabbit arteries and the minimal levels of IG9 expression in the fibrocellular lesions elicited by this type of vessel injury. The expression of IG9 on multiple cell types within the vessel wall at later time points after single BI is associated with marked monocyte infiltration into the neointima and media during injury-induced plaque progression and may result from the establishment of a lipid, cytokine, and growth factorenriched microenvironment within the developing plaque.3 9 We demonstrate that cytokines and growth factors expressed in vivo during atherogenesis32 33 34 35 are inducers of IG9 expression in cultured SMCs and ECs. Thus, the expression of IG9 by multiple cell types within the vessel wall during injury-induced plaque progression may contribute to monocyte accumulation within the developing neointima, in addition to the media.
Our finding that IG9 is expressed in vivo by SMCs in the early response
to vessel injury initiated the study of the regulation of IG9
expression in this cell type. We found that TNF-
did not induce IG9
expression in cultured human aortic SMCs, even though this
cytokine is a positive regulator of IG9 expression in
ECs.20 PDGF-BB or EGF, potent modulators of SMC
function,36 37 38 39 also did not induce significant IG9
expression in cultured SMCs. However, PDGF-BB in combination with EGF
or the activated T-lymphocyte product, IFN-
, induced
significant IG9 protein expression. The regulation of IG9 protein
expression in SMCs may be dependent on activation of signaling pathways
by multiple growth factor and cytokine receptors to provide a
threshold level of protein expression that can be detected by mAb IG9.
Cytokine and growth factorinduced IG9 expression in SMCs was upregulated by MM-LDL pretreatment. Oxidized lipoproteins are postulated to play a role in the development of atherosclerosis,40 41 42 and active oxidized phospholipids derived from MM-LDL were found to be increased in rabbit atherosclerotic lesions.43 Enhanced levels of MM-LDL within vessel walls may function as activators of SMCs and ECs because MM-LDL is mitogenic to cultured SMCs44 and induces MCP-1 in SMCs and ECs45 and macrophage colonystimulating factor46 and tissue factor47 in ECs. In addition, MM-LDL also selectively stimulates ECs to bind monocytes,22 and this adhesion is dependent on MM-LDLmediated increases in intracellular cAMP.27 MM-LDLmediated signaling mechanisms in SMCs have not been extensively characterized to date, but the present study suggests that additive signal transduction pathways activated by cytokines, growth factors, and MM-LDL may significantly contribute to the induction of IG9 expression in SMCs. Another possibility is that MM-LDL may be modifying the plasma membrane, resulting in enhanced cytokine and growth factor receptormediated induction of IG9 or in increased recognition of IG9 surface protein by mAb IG9 in treated SMCs.
IG9 expression in vivo at early time points after balloon
arterial injury in cholesterol-fed rabbits is
localized to vascular SMCs, and cytokines, growth factors, and
oxidized lipids associated with atherogenesis, including PDGF-BB,
IFN-
, and MM-LDL, are potential inducers of IG9 expression in this
cell type. Therefore, our results suggest that IG9 is a cellular
adhesion molecule that contributes to the accumulation of monocytes
within the media of arterial vessels during the initiating
phase of injury-induced atherogenesis in cholesterol-fed
rabbits.
| Acknowledgments |
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Received June 18, 1999; accepted January 18, 2000.
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