Original Contributions |
From Emory University School of Medicine, Department of Medicine, Division of Cardiology, Atlanta, Ga.
Correspondence to Zorina S. Galis, PhD, Emory University School of Medicine, Department of Medicine, Division of Cardiology, 1639 Pierce Dr, Woodruff Memorial Bldg, Room 311, Atlanta, GA 30322. E-mail zgalis{at}emory.edu
| Abstract |
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Key Words: foam cell atherosclerosis matrix metalloproteinase
| Introduction |
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Intracellular lipid accumulation is a characteristic feature of resident macrophages in atherosclerotic lesions.10 In individuals with familial hypercholesterolemia, as in the animal models of this disease, accumulations of macrophage-derived foam cells are not restricted to the vessel wall but also develop in other tissues, especially in mucous membranes, where they are known as xanthomas.11 In all of these locations, monocytes have a stable relationship with the tissue. In the same individuals, circulating monocytes do not contain intracellular lipid, not even in the presence of tremendously high levels of plasma cholesterol. Thus, lipid does not seem to accumulate in monocytes that do not form stable interactions with tissues, even if monocytes are differentiated into macrophages (eg, alveolar or peritoneal macrophages). There are several possible explanations for this relationship. One is that although circulating monocytes are presented with high levels of plasma lipoproteins, these are not modified to the extent they are in atherosclerotic lesions. A second, as yet underexplored possibility is that inside tissues, resident macrophages are "primed" for lipid accumulation through interaction with extracellular matrix and neighboring cells.
Cellular differentiation and activation of macrophages increase their matrix-degrading potential through increased expression of MMPs.12 Macrophages that reside in human atherosclerotic plaques elaborate MMPs, as indicated by in situ hybridization and immunocytochemistry studies of postsurgical and endarterectomy specimens.13 14 15 16 Although MMPs are secreted as latent forms, atheroma contains enzymatically active MMPs, as shown by in situ zymography of atherosclerotic tissue specimens.14 In addition, it has been reported that MMPs elaborated by monocytes can degrade the fibrous caps of atherosclerotic lesions from human abdominal aortas when cocultured with these specimens.17 Such degradation of extracellular matrix at macrophage-rich sites may lead to tissue weakening, plaque destabilization, and rupture, with acute clinical consequences.18
In this study, we investigated the hypothesis that interaction with interstitial collagen, constituting more than half of the total protein of plaques,3 modulates human monocyte differentiation, lipid loading, and matrix-degrading potential, characteristics of macrophages resident to atheroma.
| Methods |
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Isolation of Monocytes
Peripheral venous blood samples were collected from
normal donors in 3.2% sodium citrate and enriched for monocytic cells
with Cell-Flex 1077 (Atlanta Biologicals). The resulting cell
populations were further enriched for monocytes through centrifugal
counterflow elutriation using a modification of a previously published
protocol with a J6-MI centrifuge and a J.E. 5.0
rotor.19 Flow rate was regulated with a
Masterflex peristaltic pump (Cole-Parmer). After the system was loaded
with a chilled calcium-free phosphate buffer with 0.2% dextrose and
0.2% human serum albumin, pH 7.4, cells were introduced at a
flow rate of 120 mL/min into the centrifuge rotating at 2500
rpm. Fractions of 250 mL each were collected at progressively faster
counterflow rates and slower centrifugal speeds. These fractions were
pooled to obtain a final monocyte-enriched cell suspension. Cells
demonstrated >95% viability by trypan blue staining.
Flow Cytometry
To confirm cell purity and identity, cells
(2x105) were pretreated with mouse IgG1 as a
negative control for 15 minutes on ice. Cells were then incubated with
mouse antiCD-14 IgG1 coupled to phycoerythrin for 45 minutes on ice.
Cells were resuspended in Dulbecco's PBS (pH 7.4; 0.14 mol/L NaCl,
0.005 mol/L Na2HPO4
· 7H2O, 0.002 mol/L
KH2PO4) from Whittaker
Bioproducts and analyzed for fluorescence with a
FACScan (Becton Dickinson). The negative control was used for
compensation for background staining through the FL-2
fluorescence channel.
Cell Culture
Monocytes were cultured in Opti-MEM medium (Gibco BRL)
supplemented with 50 U/mL penicillin and 100 µg/mL streptomycin at
37°C and 5% CO2. Monocytes were cultured under
adherent conditions on collagen type Icoated or uncoated polystyrene
(or "plastic") tissue culture dishes. As a positive control for
cellular differentiation, some monocyte cultures were treated with PMA,
final concentration 100 ng/mL. Triplicate cell culture dishes were
processed for each condition. Monocyte differentiation was assessed
after 24 and 48 hours by immunofluorescent confocal microscopy
and by phase-contrast microscopy in several independent experiments, as
described below. To study the effect of inhibiting cytoskeletal
function and protein phosphorylation, in separate
experiments (n=3) monocytes were cultured in medium alone or medium
supplemented with colchicine (2 µmol/L) or genistein (25
µmol/L). In both sets of experiments, monocyte-conditioned culture
media were collected for assay of MMPs after 24 hours. Fresh medium
containing treatments plus DiI-acLDL was added (final concentration, 25
µg/mL). After incubation for an additional 24 hours, cells were
analyzed by confocal fluorescence microscopy as
described below. To verify viability of cells treated with genistein or
colchicine, a concurrent viability assay using uptake of calcein by
live cells and ethidium bromide homodimer-1 by dead cells (LIVE/DEAD
kit) was performed with a confocal microscope (Bio-Rad Laboratories).
Cell viability per condition was characterized by the ratio of cells
incorporating ethidium bromide homodimer-1 to the total number of
cells.
Analysis of Cell Differentiation
To study the effect of substrate on cellular differentiation, we
analyzed expression of surface markers of monocytes cultured on
collagen type Icoated dishes or on uncoated dishes. After 24 hours in
culture, culture medium was removed, and monocytes were fixed with 4%
paraformaldehyde and then simultaneously
incubated with phycoerythrin-conjugated anti-CD14 and with
fluorescein isothiocyanate-conjugated anti-CD71 antibodies
for 30 minutes. Anti-CD14 labeling was used as a general monocyte
marker,20 and detection of CD71 (the transferrin
receptor) was used as a marker for differentiated
monocytes.21 After they had been washed with cold
PBS to eliminate nonspecific binding, labeled cells were imaged by
confocal microscopy. By use of separate fluorescence filters,
anti-CD14 and anti-CD71 labeling data were collected independently for
three randomly selected microscope fields per condition. Monocyte
differentiation was characterized as percentage of CD71 expression with
respect to CD14 expression.
Cell spreading, previously recognized as an indicator of monocyte differentiation,22 was also analyzed after 24 and 48 hours in culture. Culture dishes were visualized under an inverted phase-contrast Diaphot 300 microscope (Nikon, Inc) and photographed (x100 magnification). Prints were digitally scanned and analyzed with Image-Pro Plus 2.0 software (Media Cybernetics). Total cell number and number of differentiated cells that developed in culture filopodia were counted. The effects of substrate on differentiation, expressed as ratio of morphologically differentiated cells to total cell number, were calculated in six independent experiments. We also measured the lengths of all differentiated cells in each condition and calculated mean lengths. Mean lengths of cells differentiated in the presence of genistein or colchicine were compared with those of untreated monocytes cultured on corresponding substrates.
Intracellular Lipid Accumulation Assay
Monocytes previously maintained in culture on plastic or
collagen type Icoated dishes for 24 hours were incubated in medium
containing 25 µg/mL DiI-acLDL (
excitation=555 nm,
emission=571 nm). After 24 hours, cells were visualized with the
confocal fluorescence microscope under a water immersion (x20)
objective. Image acquisition was provided through Bio-Rad Comos
software using the DiI-membranelabeling mode with a Kallman filter.
Concurrent analysis by an inverted fluorescence
microscope was used to verify the intracellular location of the
fluorescent label. Three random fields were selected per
condition, and numbers of fluorescent cells were counted per
field with Image-Pro Plus 2.0 software. These fields were averaged to
derive a mean for each condition, and the numbers of
fluorescent cells per field for each condition were compared
through a series of six independent experiments. To assess individual
cell lipid loading in each condition, all areas of fluorescent
cells were averaged and compared through all acLDL-uptake experiments
(n=6).
SDS-PAGE Zymography
SDS polyacrylamide gels containing gelatin (1 mg/mL)
were used to identify proteins with gelatinolytic
activity in monocyte-conditioned media. After electrophoresis, gels
were incubated in 2.5% Triton X-100 (2x10 minutes), then overnight at
37°C in 50 mmol/L Tris-HCl, pH 7.3, supplemented with 10
mmol/L CaCl2 and 0.05% Brij 35 (Sigma Chemical
Co). Gels were fixed with 30% methanol and 10% acetic acid for 1
hour, then stained with colloidal brilliant blue G (Sigma).
Subsequently, gels were digitally scanned and analyzed in
triplicate with NIH Image software version 1.55 (National Institutes of
Health).
Immunoblotting
After culture of monocytes under control (no-treatment),
genistein-treatment, or colchicine-treatment conditions, culture media
were collected at 24 hours for detection of the 92-kD gelatinase
(MMP-9), the main MMP produced by monocytes. Proteins were transferred
from minigels to a nitrocellulose membrane with a semidry blotting
system (Bio-Rad Laboratories) and incubated with an antiMMP-9
antibody (Oncogene Research Products). Blocking of nonspecific
binding and dilution of primary and secondary antibodies were done with
a 5% solution of dry defatted milk in PBS containing 0.1% Tween 20. A
chemiluminescent detection system (Amersham Life Sciences) was used for
antigen detection according to the manufacturer's protocol.
Statistical Analysis
Image analysis results for monocyte spreading for each
condition were expressed as percentages of differentiated cells per
microscopic field. Percentages obtained per individual condition from
all experiments were averaged to determine means and SEMs with
Microsoft Excel 5.0. In a similar fashion, percentages of cells
containing fluorescent acLDL from the subsequent experiments
(control versus genistein or colchicine; n=2 and n=3 experiments,
respectively) were combined to determine means and SEMs. Paired
two-tailed Student's t tests were used to determine
statistical significance. Three levels of significance for results are
indicated as ***P<.001, **P<.01, and
*P<.05. Data derived from each acLDL uptake experiment were
analyzed independently. In these instances, means from each
experiment were calculated from three randomly selected fields per
condition, and each SEM was derived. Conclusions were verified by
Fisher's exact test. Correlation coefficients between monocyte
spreading and acLDL uptake were calculated independently from three
separate experiments by comparing these independent variables
within substrate cohorts.
| Results |
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Collagen Type I Increases Intracellular Accumulation of Modified
Lipoproteins in Cultured Human Monocytes
Intracellular lipid accumulation is a hallmark of
macrophages in atherosclerotic plaques. This is known to occur
in vitro,23 and probably in
vivo,7 as a result of uptake of modified
lipoproteins. We questioned whether collagen type I modulates in vitro
intracellular accumulation of a modified lipoprotein by monocytes. We
allowed monocytes to adhere and differentiate for 24 hours on plastic
or collagen type I substrates and subsequently added fresh medium
containing DiI-acLDL. After a 24-hour incubation, we analyzed
monocyte cultures by confocal microscopy (Fig 3
). The collagen type I control group
showed increased numbers of lipid-laden cells compared with
unstimulated or PMA-stimulated cells cultured in plastic dishes (Fig 4A
). In addition, we observed a
significant increase in DiI-acLDL accumulation in individual cells
cultured on collagen type I compared with cells cultured on plastic
with or without stimulation by PMA (Fig 4B
). Addition of PMA variably
increased the numbers of lipid-laden cells cultured on plastic (Fig 4A
)
and had no detectable effect on monocytes cultured on collagen-coated
dishes, suggesting a maximal enhancing effect by the collagen type I
substrate alone.
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Inhibition of Monocyte Spreading Is Associated With a Decrease in
Intracellular Accumulation of Modified Lipoprotein
We therefore found that the nature of the substrate modulates
monocyte spreading, differentiation, and accumulation of modified
lipoprotein. Monocyte spreading on a matrix substrate probably requires
formation of cellular adhesion contacts through binding of integrins
and rearrangement of the cytoskeleton of the monocyte. The connection
between monocyte spreading and formation of lipid-laden
macrophages was further explored by analysis of the
effect of treating monocytes with either genistein or colchicine.
Genistein, a nonspecific tyrosine kinase inhibitor, was
used to block intracellular signaling pathways initiated by binding of
integrins, including tyrosine kinases. To impair monocyte cytoskeletal
function, we used colchicine, an inhibitor of microtubule
assembly. Image analysis of phase-contrast photomicrographs
showed that cellular differentiation at 24 hours on collagen type I
substrates was still greater than differentiation on plastic, with or
without addition of 25 µmol/L genistein or 2 µmol/L
colchicine (Fig 5A
). However, we noticed
that both treatments impaired cell spreading, and we confirmed this
observation by analysis of average maximum cell length under
each condition (Fig 5B
). In the presence of either genistein or
colchicine, smaller percentages of monocytes were able to attain the
normal spread size.
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We then studied the functional consequences of inhibiting cell
spreading by analyzing effects of the genistein treatment on
intracellular accumulation of DiI-acLDL. Image analysis of
cultures differentiated on collagen type I after 24 hours of incubation
with acLDL revealed that genistein significantly decreased the number
of cells containing fluorescent acLDL (Fig 6A
). This treatment also reduced to
50% the extent of intracellular lipid loading on a per-cell basis
(Fig 6B
). Monocytes cultured on plastic were also affected by the
genistein treatment: the total number of cells containing
fluorescent acLDL was decreased (Fig 6A
), as was the individual
intracellular lipid loading (Fig 6B
). Treatment of monocytes with
colchicine had similar effects (data not shown). We also examined the
effects of these two treatments on cell viability to account for a
possible contribution of cytotoxic effects. Decreases in viability due
to genistein treatment were comparable in cells cultured on plastic or
collagen type I substrates (14.2±2.5% and 14.5±1.2%, respectively)
and were both smaller than effects on lipid loading. The colchicine
treatment had a greater impact on the viability of monocytes cultured
on collagen type I than on those cultured on plastic (12.7±2.4%
versus 4.3±1.3%). A high degree of correlation was found between
monocyte spreading and intracellular lipid accumulation on the collagen
type I substrate (r>.98), whereas these two processes
exhibited a less consistent relationship for cells cultured
directly on plastic in uncoated dishes (r>.65). We
interpret these results as suggesting that monocyte interaction with
extracellular matrix plays a significant role in their acquisition of
the resident macrophage phenotype and that it enhances
their progression toward formation of the
macrophage-derived foam cell.
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Culture on Collagen Type I Substrate Increases the Amount of MMP-9
Secreted by Human Monocytes In Vitro
Gelatinolytic activity colocalizes with
macrophages resident in human or experimental atherosclerotic
lesions.14 24 Enhanced MMP-9 synthesis was found
to be associated with unstable angina.15 To
determine whether a collagen type I substrate modulates
production of this MMP, characteristic of cells of the
monocytic lineage,25 we investigated in vitro
secretion of MMP-9 by human monocytes. Culture media harvested from
monocytes cultured on plastic or collagen type I were analyzed
by SDS-PAGE zymography and immunoblotting (Fig 7
). The SDS-PAGE zymography technique
allows detection of the latent as well as the activated forms
of MMPs, because of molecular conformational changes that occur in the
presence of SDS, allowing digestion of the substrate incorporated into
the gel. Using this approach (Fig 7A
), we found a consistent
increase in the amount of gelatinolytic activity
associated with pro-MMP-9 released after 24 hours by monocytes cultured
on collagen type I substrates compared with those cultured on uncoated
dishes (141±10%, n=6 experiments, P=.005), indicating that
interaction with collagen type I enhances secretion of
macrophage MMP-9. Faint gelatinolytic bands
running at lower molecular weights were also detected in the gels,
suggesting processing of zymogen to activated forms. These
forms appeared to be preferentially increased in the media of monocytes
cultured on collagen type I substrates as well.
Immunoblotting using an antibody that recognizes both
latent and active MMP-9 species confirmed the identity of
gelatinolytic bands detected by zymography (Fig 7B
). Both the gelatinolytic activity and the MMP-9
immunopositive signals were decreased by treatment with 25
µmol/L genistein or 2 µmol/L colchicine (Fig 7
), conditions
that inhibited cell spreading. These results support the hypothesis
that interaction with a collagen type I substrate contributes to an
increased production of pro-MMP-9 and also could be related to
its activation by human macrophages.
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| Discussion |
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50% of the total protein content of
plaques3 ) and thus its likelihood of interacting
with monocytes infiltrating the vessel wall in vivo. Gudewicz et
al26 suggested previously that a collagen type I
substrate increases in vitro monocyte spreading compared with collagen
type IV or denatured gelatin. In contrast to their study, in which
noted effects on cell shape did not reach statistical significance,
probably because of the smaller numbers of monocytes analyzed,
collagen type I effects were found to be statistically significant in
all our experiments (n=6). The same authors showed that culturing
monocytes on collagen type I enhanced their superoxide
production. Our laboratory has shown recently that reactive
oxygens can activate latent gelatinases produced by
vascular cells.27 Thus, formation of superoxide
could be responsible for increased generation of active MMP-9, which we
detected in the culture media of monocytes cultured on collagen type I.
A similar reaction could occur in the vessel wall if monocytes bind to
collagen and simultaneously release latent MMP and reactive
oxygen species, leading to activation of these MMP precursors. This
effect of matrix would result in its degradation and could assist
infiltrating monocytes in their penetration through vascular matrix.
Supporting evidence for extracellular matrix components modulating
matrix metabolism in other types of mononuclear phagocytes
was reported previously. A recent study of transformed
monocyte/macrophages showed that laminin, a matrix component
that enhances cell migration,28 increases the
production of proteases considered to be essential in
migration, urokinase-type plasminogen activator
and pro-MMP-9. At variance with our approach, in that study the
investigators examined transformed monocytic cell lines. Although
essentially different from their nontransformed progenitors, cell lines
are frequently used to avoid the laborious procedure and the limited
yield associated with the study of monocytes from
peripheral blood. Compared with several matrix substrates,
collagen types I and III have been found to increase in vitro release
of human alveolar macrophage MMPs.29
However, at variance with our study of peripheral blood
monocytes, collagen type I increased expression of
interstitial collagenase (or MMP-1) but not
that of MMP-9. This difference substantiates previously reported
differences between the profile of MMPs produced in vitro by alveolar
macrophages and peripheral blood monocytes or
monocytic cell lines12 as well as that of in
situdifferentiated macrophages.30 Once they are established within the atheroma, a complex relationship between monocytes and matrix metabolism is suggested by previous observations and results of the present study. We present evidence to support the idea that collagen type I contributes to differentiation of monocytes into macrophages. Matrix also increases survival of a variety of cells31 and promotes progression through the cell cycle9 and thus could potentially contribute to the lasting presence of macrophages and to their previously reported capacity to divide inside atherosclerotic lesions.6 In turn, resident macrophages may modulate the metabolism of collagen and of other matrix components. Because these cells are an important source of factors that promote collagen gene expression, such as platelet-derived growth factor and transforming growth factor-ß, they might subsequently stimulate further collagen synthesis and early plaque growth.32 Conversely, pathological studies of advanced, rupture-prone human plaques have shown an increased density of macrophages, with reduced collagen and smooth muscle cell content in their fibrous caps.33 This is consistent with the hypothesis that in their lipid-laden phase, macrophages residing in the fibrous cap and shoulder regions produce important amounts of collagen-degrading enzymes, including the gelatinases MMP-9 and MMP-2 and interstitial collagenase (MMP-1).14 30 An earlier idea that macrophage production of MMPs could be tuned in response to the surrounding matrix34 recently received support from a study of atheroma showing that macrophage-derived foam cells that expressed matrilysin, a member of the MMP family, colocalized with its enzymatic substrate.35 Our experiments showed that interaction with collagen type I also increases the matrix-degrading capacity of monocytes, a feature recently recognized as essential in atherosclerotic plaque outcome. Such findings suggest a close and dynamic relationship between macrophages and matrix, especially collagen type I, in human atheroma. Induction of MMP expression may be directly modulated by the extracellular matrix composition or may be mediated via effects of substrate on cell shape and spreading,36 as suggested by the decrease in MMP-9 released in conditions in which we inhibited monocyte spreading. Welgus et al12 also showed previously that the amount of MMP-9 secreted in culture increases with differentiation of peripheral blood monocytes. In their experiments, differences in MMP-9 production were observed in monocytes cultured on regular (uncoated) cell culture dishes that were allowed to differentiate for 7 days under the stimulation of lipopolysaccharide or PMA. We have detected an increase in the production of MMP-9 in monocytes cultured on collagen type Icoated plates as early as 24 hours without exogenous stimulation, which further supports the notion of an accelerated cellular differentiation of monocytes29 when they are cultured on this particular substrate.
Our experiments also support the hypothesis that interaction with
matrix enhances intracellular lipid accumulation in
macrophages. This effect could be due to increased uptake or
decreased catabolism of acLDL, but elucidation of this process awaits
further investigation. In previous studies, el Khoury et
al37 38 found that adhesion of
macrophages to certain substrates interfered with
metabolism of modified lipoproteins and suggested that
scavenger receptors might function in macrophage adhesion.
Monocyte adhesion to collagen type I is most likely mediated through
integrins,39 a family of matrix receptors, which
provide a link between the extracellular matrix and the cell
cytoskeleton. We found that inhibition of cytoskeletal function by
colchicine decreased monocyte spreading and
macrophage-derived gelatinolytic
activity. Colchicine is also an inhibitor of endocytosis,
and treatment of monocytes decreased their intracellular accumulation
of acLDL (not shown). These effects were twofold to threefold greater
than effects on cell viability, suggesting an important correlation
between monocyte substrate-dependent spreading and monocyte
differentiation and function. Binding of ligands to integrins is known
to induce protein tyrosine phosphorylation in carcinoma
cells, lymphocytes, fibroblasts, and neutrophils.
Phosphorylation of a 76-kD protein (pp76) occurs
shortly after cross-linking of monocyte
ß1-integrins or after monocytes are allowed to
adhere to tissue culture dishes coated with fibronectin, laminin,
collagen type I, or collagen type IV.40 In our
experiments, in which monocytes were maintained for longer times in
culture, blockade of tyrosine phosphorylation signaling
inhibited monocyte spreading, essential for progression of cells
through the cell cycle,41 42 and reduced the
amount of intracellularly accumulated lipid. Our results thus suggest
that inhibition of tyrosine kinase function has lasting effects on
monocytes adherent to collagen type I, including interference with
morphological differentiation into the foam cell phenotype.
Monocytes also spread on the surface of plastic culture dishes,
probably by nonspecific recruitment of surface adhesive molecules. In
fact, in the previously cited study, at short times in culture, Lin et
al40 noted that monocytes adhering to plastic
culture dishes presented the highest level of the pp76
phosphorylation. Earlier studies have noted rapid
induction of multiple inflammatory mediator genes with monocyte
adherence to plastic tissue culture dishes.43 In
situ, however, monocytes interact with extracellular matrix. Adherence
of monocytes to matrix components seems to result in a more selective
pattern of gene induction, perhaps due to the induction of a more
specific signal transduction pathway.44 45
Previous studies have shown that engagement of monocyte integrins
triggers expression of interleukins,40 tumor
necrosis factor-
,43 45 or tissue
factor,46 all of which are thought to play
important roles in atherogenesis.5 Taken together
with previous observations, our findings support a still underestimated
role for matrix in differentiation of circulating monocytes into the
resident macrophage phenotype characteristic of
atherosclerotic lesions.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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Received June 23, 1997; accepted November 14, 1997.
| References |
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