(Circulation. 2000;101:1519.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
IL-8 Is an Angiogenic Factor in Human Coronary Atherectomy Tissue
From the Department of Internal Medicine, Division of Cardiology, and the Department of Surgery, Section of Cardiothoracic Surgery, The University of Michigan Medical School, Ann Arbor, Mich (M.M., D.W.M.M., E.R.B., F.D.P.); the Atherosclerosis Research Center, Division of Cardiology, Burns and Allen Research Institute, Cedars-Sinai Medical Center, and UCLA School of Medicine, Los Angeles, Calif (A.S.); and the Department of Medicine, Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine Center for Health Sciences, Los Angeles, Calif (M.D.B., R.M.S.).
Correspondence to Robert M. Strieter, MD, Department of Medicine, Division of Pulmonary and Critical Care Medicine, UCLA School of Medicine, 900 Weyburn Place, 14-154 Warren Hall, Box 711922, Los Angeles, CA 90095-1922.
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Background—Interleukin-8 (IL-8), a CXC chemokine that induces the migration and proliferation of endothelial cells and smooth muscle cells, is a potent angiogenic factor that may play a role in atherosclerosis. Previously, IL-8 has been reported in atherosclerotic lesions and circulating macrophages from patients with atherosclerosis. Therefore, we sought to determine whether IL-8 plays a role in mediating angiogenic activity in atherosclerosis.
Methods and Results—Homogenates from 16 patients undergoing directional coronary atherectomy (DCA) and control samples from the internal mammary artery (IMA) of 7 patients undergoing bypass graft surgery were assessed for IL-8 content by specific ELISA, immunohistochemistry, and in situ hybridization for IL-8 mRNA. The contribution of IL-8 to net angiogenic activity was assessed using the rat cornea micropocket assay and cultured cells. IL-8 expression was significantly elevated in DCA samples compared with IMA samples (1.71±0.6 versus 0.05±0.03 ng/mg of total protein; P<0.01). Positive immunolocalization of IL-8 was found exclusively in DCA tissue sections, and it correlated with the presence of factor VIII–related antigen. In situ reverse transcriptase polymerase chain reaction revealed the expression of IL-8 mRNA in DCA tissue. Corneal neovascular response, defined by ingrowth of capillary sprouts toward the implant, was markedly positive with DCA pellets, but no constitutive vessel ingrowth was seen with IMA specimens. Neutralizing IL-8 attenuated both the in vivo corneal neovascular response and the in vitro proliferation of cultured cells.
Conclusions—The results suggest that, in human coronary atherosclerosis, IL-8 is an important mediator of angiogenesis and may contribute to plaque formation via its angiogenic properties.
Key Words: angiogenesis • atherosclerosis • cytokines • coronary disease
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Introduction |
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Coronary atherosclerosis continues to be the leading cause of morbidity and mortality in the United States.1 The pathogenesis of coronary atherosclerotic plaque formation is a complex process that demonstrates features of exaggerated injury and repair, including the recruitment of mononuclear cells, fibroproliferation, deposition of extracellular matrix, and angiogenesis, which lead to progressive fibrosis, calcification, and eventual luminal occlusion.2 3 4 Angiogenesis is an essential process that supports fibroplasia, the deposition of extracellular matrix during wound healing, and chronic inflammation; it contributes to progressive fibrosis in several chronic inflammatory states, such as rheumatoid arthritis, psoriasis, and idiopathic pulmonary fibrosis. Angiogenesis also occurs in atherosclerosis.5 6 7 8
Chemotactic cytokines (chemokines) are polypeptide molecules
that have proinflammatory activities. Interleukin-8 (IL-8) is an ELR
(glutamic acid-leucine-arginine) CXC chemokine that was
initially discovered using chemotaxis and the activation of
neutrophils. It has endothelial cell chemotactic
activity in vitro and induces neovascularization in the cornea of rats
and rabbits in vivo.9 10 In addition, the angiogenic
(IL-8) and angiostatic interferon-
–inducible protein 10
(IP-10) CXC chemokines were recently reported to regulate
angiogenesis associated with non–small cell lung cancer and idiopathic
pulmonary fibrosis.11 12 13
Because IL-8 has previously been reported in atherosclerotic lesions, we hypothesized that IL-8 may contribute to the pathogenesis of atherosclerosis via its angiogenic properties.14 Therefore, we analyzed human coronary atherosclerotic lesions for their IL-8 content, performed immunohistochemical examinations to localize IL-8, and determined the angiogenic activity of IL-8 in patients undergoing directional coronary atherectomy (DCA) for anginal syndromes.
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Methods |
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Tissue Collection
Atherectomy specimens were collected from 16 patients who underwent DCA at the Cardiac Catheterization Laboratories of the University of Michigan Medical Center after they presented with symptomatic coronary disease (unstable or exertional angina or angina following a recent myocardial infarction or coronary intervention). Samples of the left IMA from 7 patients undergoing elective coronary artery bypass graft surgery were used as controls. After performing each coronary atherectomy, tissue was promptly retrieved from the atherectomy catheter (Devices for Vascular Intervention Inc), rinsed in sterile saline, and either snap-frozen in liquid nitrogen for subsequent homogenization or fixed in 4% formaldehyde and imbedded in paraffin. IMA specimens were obtained in the operating room during the bypass procedure, rinsed in sterile saline, and processed in a manner similar to that used with the DCA specimens. All DCA patients had clinical and angiographic findings consistent with the clinical diagnosis of unstable angina and had coronary luminal stenoses >70%, as measured by quantitative angiographic analysis.
Tissue Homogenization
Frozen DCA or IMA tissue was homogenized and
sonicated in an "anti-protease" buffer on recovery from the cardiac
catheterization laboratory using a method that was
previously described.12 13 Specimens were
centrifuged at 1500g for 15 minutes, filtered
through 1.2-µm sterile Acrodiscs (Gelman Sciences), and kept frozen
at -70°C until they were thawed for assay by specific IL-8 ELISA. In
addition, a portion of the specimen was lyophilized (SpeedVac, Savant),
normalized to an equivalent amount of total protein, and used in the
corneal micropocket model of neovascularization for the
analysis of angiogenic activity.
Reagents
Polyclonal anti-human IL-8 specific anti-sera were produced by
the immunization of rabbits with human recombinant IL-8 (R&D Systems)
in multiple intradermal sites with complete Freund’s
adjuvant.10 15 The specificity of this antisera was
assessed by Western blot analysis against a panel of other
human recombinant cytokines.15 16 Antibodies were
specific in our sandwich ELISA, without cross-reactivity to a panel of
12 human recombinant interleukins, including interleukin-1 receptor
antagonist IL-1ra (IRAP), IL-1, IL-2, IL-4, IL-6, tumor necrosis
factor 
, interferon-, and other members of the CXC and CC
chemokine families (eg, CXC: epithelial neutrophil activating
protein-78 [ENA-78], growth-related gene [GRO]-, GRO-ß,
GRO-, neutrophil-activating peptide-2 [NAP-2], platelet factor
4 [PF-4], IP-10, and measles immune globulin; CC: monocyte
chemoattractant protein-1 [MCP-1], macrophage inflammatory
protein- and ß, and regulated on activation normal T cell
expressed and secreted [RANTES]).16 17 The
anti-protease buffer for tissue homogenization
consisted of 1xPBS with 2 mmol/L phenylmethylsulfonyl
fluoride and 1 µg/mL each of antipan, aprotinin, leupeptin,
and pepstatin A. Goat anti–factor VIII–related antigen antibodies
were purchased from Biomeda. Anti-macrophage antibodies (HAM
56) were purchased from Enzo Diagnostics.
IL-8 ELISA
IL-8 was quantitated by specific ELISA using a modification of a
double-ligand method.16 17 Briefly, flat-bottomed, 96-well
microtiter plates (Nunc) were coated with 50 µL/well of
specific anti-IL-8 polyclonal antibodies (1 ng/µL in 0.6 mol/L NaCl,
0.26 mol/L H3BO4, and 0.08
mol/L NaOH; pH 9.6) for 24 hours at 4°C and then washed with PBS and
0.05% Tween-20 (wash buffer). Nonspecific binding sites were blocked
with 2% BSA. Plates were rinsed, and samples were added (50
µL/well). This was then followed by incubation for 1 hour at 37°C.
Plates were then washed, and 50 µL/well of specific anti-IL-8
biotinylated polyclonal antibodies (3.5 ng/µL in wash buffer and 2%
FCS) was added for 45 minutes at 37°C. Plates were washed 3 times,
streptavidin-peroxidase conjugate (Bio-Rad Laboratories) was added, and
the plates were incubated for 30 minutes at 37°C. Chromogen substrate
(Dako) was then added, and the plates were incubated at room
temperature to the desired extinction. Plates were read at 490 nm in an
automated microplate reader (Bio-Tek Instruments, Inc). Standards were
half of the log dilution of recombinant IL-8 from 100 ng to 1 pg/mL (50
µL/well).
Immunohistochemistry of IL-8, Factor VIII-Related Antigen, and
HAM 56
Paraffin-embedded tissue from DCA and IMA specimens was
processed for immunohistochemical localization of IL-8, factor
VIII-related antigen, and HAM 56, as previously
described.16 17 Briefly, consecutive tissue sections were
dewaxed with xylene and rehydrated with graded concentrations of
ethanol. Tissue nonspecific binding sites were blocked using normal
goat serum (BioGenex). Tissue sections were then washed and overlaid
with a 1:500 dilution of either control (rabbit) or polyclonal rabbit
anti-IL-8 antibodies, either control (goat) or goat anti-factor
VIII-related antigen antibodies, or either control (mouse) or mouse
anti-macrophage (HAM 56) antibodies. Sections were then
washed and incubated for 60 minutes with either secondary goat
anti-rabbit, rabbit anti-goat, or goat anti-mouse biotinylated
antibodies (BioGenex). After washing twice with Tris-buffered saline,
slides were overlaid with a 1:35 dilution of peroxidase conjugated with
streptavidin (Vector) and incubated for 60 minutes. The tissue sections
were then washed twice in Tris-buffered saline and incubated with
alkaline phosphatase conjugated with streptavidin (BioGenex) for 60
minutes. Fast Red (BioGenex) reagent was used for
chromogenic localization of antigen. After optimal color
development, tissue sections were immersed in sterile water,
counterstained with Mayer’s hematoxylin, and cover-slipped using an
aqueous mounting solution.
Localization of Tissue IL-8 mRNA by In Situ Reverse Transcriptase
Polymerase Chain Reaction
IL-8 mRNA was localized to coronary arteries using a
modification of an in situ reverse transcriptase polymerase chain
reaction analysis, as previously described.17
Briefly, 3-µm-thick paraffin-embedded sections on PLUS slides
(Fisher) were dewaxed in xylene and rehydrated in graded concentrations
of ethanol. Slides were then rinsed in diethylpyrocarbonate
H2O, followed by a proteinase K digest. Slides
were then treated with DNase overnight. Reverse transcriptase
polymerase chain reaction was performed using the Promega Access System
in the presence of digoxigenin (11-dUTP; Boehringer
Mannheim) with sense (sequence: 5' AAG-CTG-GCC-GTG-GCT-CTC-TTG 3') or
antisense (sequence: 5' AGC-CCT-CTT-CAA-AAA-CTT-CTC 3') primers. Slides
were then washed in Tris-buffered solution, blocked for 30 minutes, and
then incubated for 30 minutes with anti-digoxigenin antibody conjugate.
Slides were then washed and placed for 10 minutes in a buffered
solution of 100 mmol/L Tris-HCl, 100 mmol/L NaCl, and 50
mmol/L MgCl2 with a pH of 9.5 at 20°C. Slides
were then exposed to a color solution containing nitroblue tetrazolium,
X-phosphate (Boehringer Mannheim), and levamisole and incubated
in a darkened, humidified chamber until adequate color visualization
was determined. The reaction was then terminated by immersing slides in
H2O. Slides were counterstained with Contrast Red
(KPL), rinsed, and cover-slipped.
Corneal Micropocket Assay of Angiogenesis
The angiogenic activity of DCA and IMA homogenates
was assayed in vivo in the avascular cornea of hooded Long-Evans rat
eyes, as previously described.17 18 19 Briefly, equal
volumes of lyophilized DCA or IMA tissue specimens were normalized to
total protein and combined with a sterile Hydron (Interferon Sciences
Inc) casting solution. Aliquots of 5 µL were pipetted onto the flat
surface of an inverted sterile polypropylene specimen container and
polymerized overnight in a laminar flow hood under UV light. Before
implantation, pellets were rehydrated with normal saline. Animals were
anesthetized with intraperitoneal
ketamine (150 mg/kg) and atropine (250 µg/kg). Rat corneas
were anesthetized with 0.5% proparacaine hydrochloride
ophthalmic solution, which was followed by implantation of the Hydron
pellet into an intracorneal pocket (1 to 2 mm from the limbus).
Six days after implantation, animals received 1000 U of heparin and
ketamine (150 mg/kg) intraperitoneally,
which was followed by a 10-mL perfusion of colloidal carbon via the
left ventricle. Corneas were harvested and photographed. No
inflammatory response was observed in any of the corneas treated with
the above specimens. Positive neovascularization responses were
recorded only if sustained directional ingrowth of capillary
sprouts and hairpin loops toward the implant were observed. Negative
responses were recorded when either no growth was observed or when
only an occasional sprout or hairpin loop displaying no evidence of
sustained growth was detected. All animals were handled in accordance
with the guidelines of the Unit for Laboratory Animal Medicine.
Cell Culture and DNA Synthesis
To further illustrate the role of IL-8 as an angiogenic factor,
we measured the effects of anti-IL-8 antibody on coronary
artery smooth muscle cell DNA synthesis. Human coronary artery
smooth muscle cells were cultured in Dulbecco’s modified Eagle’s
medium, supplemented with 10% fetal bovine serum, recombinant
epidermal growth factor (0.5 ng/mL), recombinant fibroblast growth
factor-B (2 ng/mL), insulin (5 µg/mL), and gentamycin (100 µg/mL).
All experiments were conducted on cells at passages 3 to 6, and cell
cultures were incubated at 37°C in a humidified atmosphere of 5%
CO2 and 95% air. DNA synthesis was determined
using the method of [3H]-thymidine (Amersham
International) incorporation using 96-well plates. Cells
(106 cells per well) were made quiescent by
starving them in serum-free media for 48 hours; then, cells were
stimulated by being incubated with Dulbecco’s modified Eagle’s medium
containing 10% fetal bovine serum and either varying titers of
anti-IL-8 antibody (1:300 or 1:1000) or control vector (normal rabbit
serum) for 24 hours. Cells were further incubated for 4 hours with 0.5
mCi [3H]-thymidine, and the radioactivity
incorporated was determined using a cell harvester and a liquid
scintillation counter.
Statistical Analysis
Comparison of IL-8 levels was made using Student’s t
test. Data were considered statistically significant at
P
0.05.
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Results |
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DCA samples came from patients with a mean age of 59.5±5 years, whereas the specimens from the internal mammary artery (IMA) that were used for comparison were harvested from patients with a mean age of 63.8±4 years.
IL-8 Protein is Present in Coronary Atherectomy
Specimens
The presence of IL-8 in DCA and IMA samples was measured by
specific ELISA. DCA tissue constitutively expressed more IL-8 than IMA
controls, as measured by specific ELISA standardized to total protein
(1.71±0.6 versus 0.05±0.03 ng/mL; P<0.01). Because IL-8
protein, as measured by ELISA, was significantly elevated in DCA tissue
homogenates when compared with IMA samples, we assessed its
presence in paraffin-embedded tissue using immunohistochemistry.
Figures 1B
and 1C represent the
result of immunolocalization using antibodies to IL-8. IL-8 was
predominantly expressed in DCA tissue (Figures 1B and 1C).
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IL-8 Relative to Factor VIII–Related Antigen
Because endothelial cell recruitment and growth is
required to support angiogenesis, we determined whether the
immunolocalization of IL-8 was associated with vascular
endothelial cells in DCA samples. Figures 1E
and 1F represent the result of immunolocalization using antibodies
to factor VIII–related antigen, an endothelial cell
marker. The presence of endothelial cells is evidenced
by the immunolocalization of factor VIII–related antigen (Figures 1E and 1F) in regions that were colocalized by antibodies to
IL-8. In addition, areas of new vessel formation, lined by
endothelial cells positively staining for factor
VIII–related antigen, were also evident in DCA specimens.
Presence of IL-8 mRNA
Because the IL-8 protein was present in atherosclerotic (DCA)
tissue, as determined by ELISA and immunolocalization of IL-8, we used
in situ hybridization for IL-8 mRNA to localize the source of IL-8.
Figure 2 represents the result of
in situ hybridization for IL-8 mRNA in DCA tissue. IL-8 mRNA was
present in DCA specimens but was undetected in IMA controls,
suggesting that the expression of IL-8 protein was associated with
increased mRNA levels in cells comprising atherosclerotic plaque. Given
the heterogeneity of our patient population and our
sample size, the predominant cellular source of IL-8 mRNA could not be
distinguished because endothelial cells,
macrophages, and smooth muscle cells were each associated with
a localization to IL-8.
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Presence of Macrophages Relative to IL-8
Mononuclear cell recruitment and activation has been postulated to
be an important event in the developed of atherosclerotic plaque; thus,
we examined whether localization of IL-8 was also associated with that
of macrophages in atherosclerotic lesions. Figure 3 represents the result of
immunolocalization using HAM 56, an anti-macrophage antibody.
The expression of IL-8 associated with neovascularization in
coronary plaques was also associated with the presence of
macrophages.
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Corneal In Vivo Angiogenesis Assay
To substantiate that IL-8 may be modulating tissue-derived
angiogenic activity in coronary atherosclerotic lesions, we
next assessed the in vivo angiogenic activity of random pooled samples
of either DCA or IMA tissue in the presence or absence of preimmune
(control) or neutralizing IL-8 antibodies using the rat cornea
micropocket model of neovascularization. These antibodies did not
contain significant quantities of lipopolysaccharide
contamination, as assessed by limulus assay, and all samples were
normalized to total protein. Corneal neovascular response, as defined
by ingrowth of capillary sprouts and hairpin loops toward the implant,
was markedly positive with DCA pellets (Figure 4A) in 6 of 6 corneas, without evidence
of leukocyte infiltration (assessed by light microscopy). In contrast,
in corneas embedded with hydron pellets containing DCA
homogenates with neutralizing IL-8 antibodies, the
angiogenic response was completely abrogated (Figure 4B). This
complete inhibition of angiogenic activity by a neutralizing antibody
to IL-8 underscores the importance of IL-8 in stimulating angiogenesis
in DCA tissue. No growth was seen with the IMA pellets in either the
presence or absence of the neutralizing antibody (Figures 4C and 4D; n=6 for each manipulation;
Table).
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Cell Culture and DNA Synthesis
We showed that the protein and mRNA of IL-8 are present in
plaque specimens, and we demonstrated the angiogenic activity of IL-8;
thus, the role of IL-8 on DNA synthesis in cultured coronary
artery smooth muscle cells was examined in the presence and absence of
a neutralizing antibody to IL-8. Compared with stimulated cells treated
with control sera, DNA synthesis, as measured by
[3H]-thymidine incorporation, was significantly
reduced in cells treated with both 1:300 and 1:1000 titers of
neutralizing antibody to IL-8 (33±10% and 55±12%, respectively;
Figure 5). These results suggest that the
antagonism of IL-8, a CXC chemokine that induces the migration and
proliferation of endothelial cells and smooth muscle
cells, may at least partly inhibit the proliferation of
coronary artery cells.
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Discussion |
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These results suggest that CXC chemokines are produced in the coronary atherosclerotic plaques of patients presenting with unstable angina. In addition, IL-8, a CXC chemokine previously shown to be an angiogenic factor in vivo and a chemotactic and mitogenic factor for vascular smooth muscle and endothelial cells in vitro, is present in elevated levels in atherectomy specimens and may be a primary signal for angiogenesis in atherosclerotic tissue. Furthermore, the presence of IL-8 in coronary plaques was associated with that of vascular endothelial cells by virtue of the colocalization with factor VIII–related antigen. These findings strongly support a role for chemokines in atherogenesis and elucidate potential mechanisms by which IL-8, a molecule only recently described in human atherosclerosis, may contribute to the development and progression of coronary atherosclerotic disease in humans.
Clinically significant coronary atherosclerosis is the result of a multifactorial process that includes the accumulation and incorporation of lipids, the recruitment of inflammatory mononuclear cells and T lymphocytes, intimal proliferation of smooth muscle cells, the production and deposition of connective tissue or basement membrane matrix, and angiogenesis. The notion of chronic inflammation and fibroproliferation as mechanisms in the pathogenesis of atherosclerotic lesions was proposed by Virchow20 in the mid-nineteenth century and evolved over the next century into the response-to-injury hypothesis described by Ross and Glomset.21 According to this model, injury to the endothelium by multiple sources, including oxidized low-density lipoproteins, mechanical stress, toxins, and viruses, results in endothelial cell dysfunction and the increased adherence and activation of mononuclear cells, platelets, and T-lymphocytes. In turn, these activated monocytes/macrophages and the other cells involved in the developing lesion elaborate growth factors and cytokines that stimulate the fibroproliferative response; this leads to the formation and progression of atherosclerotic plaques. Indeed, observations of plaque progression in coronary disease supported, at least in part, the claim that these processes featured a continuum of chronic inflammation, an influx of granulation tissue, and fibroplasia, which ultimately led to luminal occlusion.22 23
CXC chemokines are a family of recently identified cytokines
that are characteristically basic heparin-binding proteins. These
cytokines in their monomer forms are <10 kDa and seem to have
proinflammatory and reparative activities. This family displays 4
highly conserved cysteine amino acid residues, with the first 2
cysteines separated by 1 nonconserved amino acid residue (CXC cysteine
motif).24 Over the last decade, several human CXC
chemokines have been identified; they possess either angiogenic (IL-8;
ENA-78; GRO-, -ß, and -; granulocyte chemotactic protein
[GCP-2]; connective tissue activating protein III [CTAP-III];
NAP-2; and platelet basic protein [PBP]) or angiostatic (IP-10, PF-4,
and monokine induced by gamma interferon [MIG]) activity at
physiological concentrations. These
properties are based on the presence or absence of the
Glu-Leu-Arg (ELR) motif, which immediately precedes the first conserved
cysteine at the NH2 terminal portion of the primary
sequence.25 26 27 28
Angiogenesis characterized by neovascularization is a critical event in wound healing and inflammation, and it also occurs in a variety of pathological states, such as granuloma formation, chronic inflammatory disease, and tumorigenesis.5 6 7 8 29 During neovascularization, normally quiescent endothelial cells are stimulated, degrade their basement membrane and proximal extracellular matrix, migrate directionally, divide, and organize into new functioning capillaries invested by a basal lamina. This process is at least partly dependent on a number of specific interactions between endothelial cells, the extracellular matrix, and various families of adhesion molecules in the presence of growth and regulatory factors. These factors include IL-8, a CXC chemokine possessing the ELR motif, which induces in vitro endothelial cell migration and proliferation, 2 essential components of angiogenesis.9 10 The angiogenic activity of IL-8 has been shown in vitro to be equipotent to that of basic fibroblast growth factor, vascular endothelial growth factor, and other previously recognized heparin-binding proteins that promote angiogenesis.9 30
The results of the present study are consistent with the evolving paradigm of exaggerated wound repair, aberrant angiogenesis, and inflammation as mechanisms in the pathogenesis of atherosclerosis. The immunohistochemical and in situ hybridization data, together with the complete attenuation of angiogenic bioactivity with neutralizing IL-8 antibodies presented in this study, suggest that IL-8 is produced in human coronary atheromas and may function as a mediator of tissue-derived angiogenesis in atherosclerotic lesions. The expression and action of several angiogenic factors have been well described during embryogenesis, wound healing, and tumorigenesis.
Neovascularization seems to be an important process in the pathogenesis of atherosclerotic plaque and the angiogenic molecule; vascular endothelial growth factor has been a recent focus of investigation in vascular pathology for its ability to modulate endothelial repair and smooth muscle growth.31 32 33 34 Moreover, a recent report in which the attenuation of plaque growth was achieved by the use of angiogenesis inhibitors further illustrates the potential importance of neovascularization in the progression of atherosclerosis.35 In this study, IL-8 was detected almost exclusively in DCA tissue samples compared with those from the left IMA, and it was associated with a profound angiogenic response that was completely inhibited with neutralizing anti–IL-8 antibodies. In addition, IL-8 antisera also attenuated the in vitro proliferation of stimulated human coronary artery cells. These findings suggest that IL-8 is a mediator of angiogenesis that may contribute to the evolution of clinically significant coronary lesions.
Other monocyte-derived proteins have been investigated for their role in the development of atherosclerosis. MCP-1 was strongly expressed by infiltrating macrophages after acute arterial injury.36 The expression of IL-8 protein and mRNA has been reported in the plaque macrophages and circulating monocytes of patients presenting with clinically significant atheromas, as well as in their macrophages and foam cells in response to cholesterol loading.37 38 In addition, other investigators have separately demonstrated the presence of the IL-8 protein and the evidence of neovascularization in atherosclerotic plaques.14 31 32 34 Furthermore, depletion of the IL-8 receptor CXCR2 in transgenic hypercholesterolemic mice has been associated with a reduction of macrophage accumulation and plaque size in atherosclerotic lesions.39
The findings of the current study agree with the above observations. The colocalization of IL-8 with factor VIII–related antigen supports a role for IL-8 as a contributor to endothelial cell recruitment (and, hence, neovessel formation) in human coronary plaques. This evidence would, therefore, support a mechanism by which IL-8 is, at least partly, a mediator of the angiogenesis associated with atherosclerosis and by which IL-8 may support the fibroproliferation and matrix deposition that lead to plaque formation.
The lack of a longitudinal analysis of coronary plaque formation for the presence and activity of IL-8 is a limitation to our interpretation of the data presented in this study. Such a temporal analysis, however, would be impractical without an appropriate animal model because the tissue specimens analyzed were obtained from symptomatic patients with clinical indications for coronary atherectomy and, therefore, advanced disease. Although we cannot comment on the specific role of IL-8 in each of the clinical subsets represented by the specimens, the data reported do suggest that IL-8 is present in the atherosclerotic tissue of patients presenting with symptomatic coronary disease. In addition, our ability to comment on the possible regulatory interactions of other chemokines, particularly between those of the CXC family possessing and lacking the ELR motif, was limited by sample size. However, as demonstrated by our results, the content and angiogenic activity of IL-8 in the coronary plaques analyzed were overwhelming when compared with controls. A larger survey of coronary tissue would allow us to examine inherent chemokine interactions in plaque formation and to characterize their expression and activity in lesions from patients who present with the syndromes of unstable angina, chronic exertional angina, and restenosis.
In conclusion, we reported the relative concentrations of a CXC chemokine in human coronary atherosclerotic tissue and demonstrated the significant expression and angiogenic activity of IL-8. This report extends previous observations in atheromas to include a role by which IL-8 may significantly contribute to angiogenesis, which seems to be germane to the pathogenesis of coronary atherosclerosis. Thus, our findings suggest that in human atherosclerosis, IL-8 may be chemotactic and mitogenic toward endothelial cells and smooth muscle cells as well as a signal for angiogenesis. Future investigations are required to further elucidate the role of CXC chemokines in atherosclerosis as well as to identify a potential role for therapies directed against IL-8 in mitigating the development and complications of disease.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grants P50HL60289 and CA87879 (to R.M.S.).
Received June 25, 1999; revision received October 15, 1999; accepted November 2, 1999.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Nicholls E, Peruga A, Restrepo H.
Cardiovascular disease mortality in the Americas.
World Health Status Q. 1993;46:134–150.[Medline]
[Order article via Infotrieve]
- Fowler S, Berberian P, Shio H, Goldfischer S, Wolinsky
H. Characterization of cell populations isolated from aortas of rhesus
monkeys with experimental atherosclerosis. Circ
Res. 1980;46:520–523.
[Free Full Text]
- Joris I, Zand T, Nunnari J, Krolikowski F, Majno G.
Studies on the pathogenesis of atherosclerosis, I:
adhesion and emigration of mononuclear cells in the aorta of
hypercholesterolemic rats. Am J Pathol. 1983;113:341–358.[Abstract]
- Munro J, Cotran R. The pathogenesis of
atherosclerosis: atherogenesis and inflammation.
Lab Invest. 1988;58:249–261.[Medline]
[Order article via Infotrieve]
- Folkman J. Angiogenesis in cancer, vascular,
rheumatoid, and other disease. Nat Med. 1995;1:27–31.[Medline]
[Order article via Infotrieve]
- Folkman J, Brem H. Angiogenesis and inflammation. In:
Gallin JI, Goldstein IM, Snyderman R, eds. Inflammation: Basic
Principles and Clinical Correlates. 2nd ed. New York, NY: Raven
Press Ltd; 1992:821–839.
- Koch AE, Leibovich SJ, Polverini PJ. Stimulation of
neovascularization by human rheumatoid synovial tissue
macrophages. Arthritis Rheum. 1989;29:471–479.
- Clark RA. Basics of cutaneous wound repair. J
Dermatol Surg Oncol. 1993;19:693–706.[Medline]
[Order article via Infotrieve]
- Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro
LA, Elner VM, Elner SG, Strieter RM. Interleukin-8 (IL-8) as a
macrophage-derived mediator of angiogenesis.
Science. 1992;258:1798–1801.
[Abstract/Free Full Text]
- Strieter RM, Kunkel SL, Elner VM, Martonyl CL, Koch AE,
Polverini PJ, Elner SG. Interleukin-8: a corneal factor that induces
neovascularization. Am J Pathol. 1992;141:1279–1284.[Abstract]
- Arenberg DA, Kunkel SL, Polverini PJ, Glass M, Burdick
MD, Strieter RM. Inhibition of interleukin-8 reduces tumorigenesis of
human non-small cell lung cancer in SCID mice. J Clin
Invest. 1996;97:2792–2802.[Medline]
[Order article via Infotrieve]
- Arenberg DA, Kunkel SL, Polverini PJ, Morris SB,
Burdick MD, Glass M, Taub DT, Iannetoni MD, Whyte RI, Strieter RM.
Interferon--inducible protein 10 (IP-10) is an angiostatic
factor that inhibits human non-small cell lung cancer (NSCLC)
tumorigenesis and spontaneous metastases. J Exp Med.. 1996;184:981–992.
[Abstract/Free Full Text]
- Keane MP, Arenberg DA, Lynch JP III, Whyte RI,
Iannettoni MD, Burdick MD, Wilke CA, Morris SB, Glass MC, DiGiovine B,
Kunkel SL, Strieter RM. The CXC chemokines, IL-8, and IP-10 regulate
angiogenic activity in idiopathic pulmonary fibrosis.
J Immunol. 1997;159:1437–1443.[Abstract]
- Koch AE, Kunkel SL, Pearce WH, Shaw M, Parikh D,
Evanoff HL, Haines GK, Burdick MD, Strieter RM. Enhanced
production of the chemotactic cytokines interleukin-8
and monocyte chemoattractant protein-1 in human abdominal aortic
aneurysms. Am J Pathol. 1993;142:1423–1430.[Abstract]
- Strieter RM, Phan SH, Showell HJ, Remick DG, Lynch JP
III, Genord M, Raiford C, Eskandari M, Marks RM, Kunkel SL.
Monokine-induced neutrophil chemotactic factor gene expression in human
fibroblasts. J Biol Chem. 1989;264:10621–10626.
[Abstract/Free Full Text]
- Bacon KB, Westwick J, Camp RDR. Potent and specific
inhibition of IL-8, IL-1 alpha, and IL-1 beta-induced in vitro human
lymphocyte migration by calcium channel antagonists.
Biochem Biophys Res Commun.. 1989;165:349–354.[Medline]
[Order article via Infotrieve]
- Standiford TJ, Kunkel SL, Basha MA, Chensue SW, Lynch
JP III, Toews GB, Westwick J, Strieter RM. Interleukin-8 gene
expression by a pulmonary epithelial cell line: a model for
cytokine networks in the lung. J Clin Invest. 1990;86:1945–1953.[Medline]
[Order article via Infotrieve]
- Polverini PJ, Cotran PS, Gimbrone MA, Unanue ER.
Activated macrophages induce vascular proliferation.
Nature. 1977;269:804–806.[Medline]
[Order article via Infotrieve]
- Koch AE, Burrows J, Cho M, Polverini PJ, Leibovich J.
Thiol-containing compounds inhibit the production of
monocyte/macrophage-derived angiogenic activity.
Agents Actions. 1991;34:350–357.[Medline]
[Order article via Infotrieve]
- Virchow R. Phlogose und thrombose in gefassystem. In:
Gesammelte Abhandlungen zur Wissenschaftlichen Medicin.
Berlin, Germany; 1856:458–68.
- Ross R, Glomset JA. Atherosclerosis and
the arterial smooth muscle cell. Science. 1973;180:1332–1336.
[Free Full Text]
- Flugelman MY, Virmani R, Correa R, Yu ZX, Farb A, Leon
MB, Elami A, Fu YM, Casscells W, Epstein SE. Smooth muscle cell
abundance and fibroblast growth factors in coronary lesions of
patients with nonfatal unstable angina: a clue to the mechanism of
transformation from the stable to unstable clinical state.
Circulation. 1993;88:2493–2500.
[Abstract/Free Full Text]
- Schwartz S, DeBlois D, O’Brien E. The intima: soil for
atherosclerosis and restenosis. Circ
Res. 1995;77:445–465.
[Free Full Text]
- Miller MD, Krangel MS. Biology and biochemistry of the
chemokines: a family of chemotactic and inflammatory cytokines.
Crit Rev Immunol. 1992;12:17–46.[Medline]
[Order article via Infotrieve]
- Baggiolini M, Walz A, Kunkel SL. Neutrophil-activating
peptide-1/interleukin 8, a novel cytokine that
activates neutrophils. J Clin Invest. 1989;84:1045–1049.[Medline]
[Order article via Infotrieve]
- Proost P, Wolf-Peeters CD, Conings R, Opdenakker G,
Billiau A, Damme JV. Identification of a novel granulocyte chemotactic
protein (GCP-1) from human tumor cells: in vitro and in vivo comparison
with natural forms of GROa, IP-10, and IL-8. J Immunol.
1993;l150:1000–1010.
- Strieter RM, Polverini PJ, Kunkel SL, Arenberg DA,
Burdick MD, Kasper J, Dzuiba J, Van Damme J, Walz A, Marriott D, Chan
SY, Roczniak S. The functional role of the ELR motif in CXC
chemokine-mediated angiogenesis. J Biol Chem. 1995;270:27348–27357.
[Abstract/Free Full Text]
- Strieter RM, Kunkel SL, Shanafelt AB, Arenberg DA, Koch
AE, Polverini PJ. CXC chemokines in regulation of angiogenesis. In:
Koch AE, Strieter RM, eds. Chemokines in Disease. Austin,
Tex: RG Landes; 1996:195–210.
- Folkman J. Clinical applications of research on
angiogenesis. N Engl J Med. 1995;333:1757–1763.
[Free Full Text]
- Folkman J, Klagsbrun M. Angiogenic factors.
Science. 1987;235:442–447.
[Abstract/Free Full Text]
- Barger AC, Beeuwkes R, Lainey LL, Silverman KJ.
Hypothesis: vasa vasorum and neovascularization of human
coronary arteries. N Engl J Med. 1984;310:175–177.[Medline]
[Order article via Infotrieve]
- O’Brien ER, Garvin MR, Dev R, Stewart DK, Hinohara T,
Simpson JB, Schwartz SM. Angiogenesis in human coronary
atherosclerotic plaques. Am J Pathol. 1994;145:883–894.[Abstract]
- Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ,
Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis: a
single intraarterial bolus of vascular
endothelial growth factor augments
revascularization in a rabbit ischemic hind
limb model. J Clin Invest. 1994;93:662–670.[Medline]
[Order article via Infotrieve]
- Kwon HM, Sangiorgi G, Ritman EL, McKenna C, Holmes DL
Jr, Schwartz RS, Lerman A. Enhanced coronary vasa vasorum
neovascularization in experimental
hypercholesterolemia. J Clin
Invest. 1998;101:1551–1556.[Medline]
[Order article via Infotrieve]
- Wysocki SJ, Zheng MH, Smith A, Lamawansa MD, Iacopetta
BJ, Robertson TA, Papadimitriou JM, House AK, Norman PE. Monocyte
chemoattractant protein-1 gene expression in injured pig artery
coincides with early appearance of infiltrating
monocyte/macrophages. J Cell Biochem. 1996;62:303–313.[Medline]
[Order article via Infotrieve]
- Moulton KS, Heller E, Konerding MA, Flynn E, Palinski
W, Folkman J. Angiogenesis inhibitors endostatin or TNP-470
reduce intimal neovascularization and plaque growth in apolipoprotein
E-deficient mice. Circulation. 1999;99:1726–1732.
[Abstract/Free Full Text]
- Wang N, Tabas I, Winchester R, Ravalli S, Rabbani L,
Tall A. Interleukin-8 is induced by cholesterol loading of
macrophages and expressed by macrophage foam cells in
human atheroma. J Biol Chem. 1996;271:8837–8842.
[Abstract/Free Full Text]
- Apostolopoulos J, Davenport P, Tipping P. Interleukin-8
production by macrophages from atherosclerotic plaques.
Arterioscler Thromb Vasc Biol. 1996;16:1007–1012.
[Abstract/Free Full Text]
- Boisvert WA, Santiago R, Curtiss LK, Terkeltaub RA. A
leukocyte homologue of the IL-8 receptor CXCR2 mediates the
accumulation of macrophages in atherosclerotic lesions of LDL
receptor-deficient mice. J Clin Invest. 1998;101:353–363.[Medline]
[Order article via Infotrieve]
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