Extracellular Matrix Modulates Macrophage Functions Characteristic to Atheroma

Collagen Type I Enhances Acquisition of Resident Macrophage Traits by Human Peripheral Blood Monocytes In Vitro

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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Abstract—Activated resident macrophages sustain atheroma, and a high macrophage content is associated with plaque vulnerability. Factors leading to differentiation and activation of these blood-derived cells remain largely uncharacterized. We investigated the contribution of interaction with collagen type I, the predominant component of atherosclerotic matrix, to differentiation and modulation of characteristic macrophage functions, including intracellular lipid accumulation and production of the typical matrix-degrading enzyme matrix metalloproteinase (MMP)-9. When used as an adhesion substrate for human peripheral blood monocytes in vitro, collagen type I increased monocyte differentiation, assessed by analysis of CD71 expression and cell spreading. Culturing on collagen type I doubled the number of differentiated monocytes at 24 hours (44.9±1.4% versus 18.4±1.7% on uncoated dishes, P<.001, n=3 independent experiments) and was a stronger stimulus for differentiation than phorbol myristate acetate, a known inducer of monocyte differentiation. The effect of substrate on intracellular accumulation of modified lipoproteins was assessed by quantitative confocal microscopy of monocytes incubated with fluorescent acetylated LDL. The collagen type I substrate also doubled the number of macrophages containing intracellular lipid and significantly increased the individual intracellular loading. Monocytes cultured on collagen type I also released more MMP-9 than did cells plated directly on plastic. The role of monocyte spreading was further assessed by treatment with colchicine, an inhibitor of cytoskeletal function, or with genistein, a nonspecific inhibitor of tyrosine kinases, shown to participate in cell adhesion. Cell spreading was inhibited in 72.3±6.7% of colchicine-treated and in 62.4±6.4% of genistein-treated monocytes (n=3, P<.01 in both cases). The same conditions also decreased secretion of MMP-9, and genistein reduced the number of acetylated LDL-containing cells (from 286±7 to 184±8 cells/mm² with genistein, n=3, P<.001). Data showed a strong correlation (r>.98) between monocyte spreading on collagen type I and intracellular lipid accumulation. Our results indicate that interaction with vascular matrix may play an important role in differentiation of peripheral blood monocytes into resident lipid-laden macrophages, which act as central stimulators throughout the natural history of atheroma.

Key Words: foam cell • atherosclerosis • matrix metalloproteinase

	Introduction

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Circulating monocytes participate in transient inflammatory responses, phagocytose foreign bodies, and produce regulatory cytokines. Early in the development of atherosclerotic lesions, activated endothelial cells recruit circulating peripheral blood monocytes, which then migrate into the subendothelial space.¹ During this invasion into the arterial wall, monocytes probably interact with components of extracellular matrix, especially collagen type I, a major constituent of the normal arterial wall matrix² and the most prominent matrix component of atherosclerotic plaques.³ These cells receive signals from factors that inhibit migration and prolong exposure to extracellular matrix, possibly contributing to their differentiation.⁴ Once established in the atherosclerotic plaque, resident macrophages accumulate lipid and maintain a continuous state of activation during which they secrete mediators of inflammation,⁵ functions that distinguish them from their circulating progenitors. Thus, from a short-lived circulating cell, the monocyte differentiates into a resident cell with the potential to divide inside the lesions.⁶ The presence of modified lipoproteins has long been recognized as a potential stimulus for recruitment and differentiation of circulating monocytes into the foam cell phenotype characteristic for atheroma.⁷ However, interaction with matrix, the main constituent of most atherosclerotic lesions, could contribute to regulation of monocyte behavior, as suggested by recent studies examining the role of signals mediated through matrix receptors called integrins.⁸ Integrin-mediated adhesion to matrix is required for many cell types to progress normally through the G₁ phase of the cell cycle.⁹ Adhesive events may allow the stabilization of integrins at cell surfaces and could enable signaling events responsible for acquiring the resident status of the macrophage and for its state of activation.

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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Collagen type I–coated and uncoated (plastic) six-well dishes were obtained from Collaborative Biomedical Products (Becton Dickinson Labware). PMA and genistein were acquired from Sigma Chemical Co. acLDL labeled with DiI, the LIVE/DEAD Viability/Cytotoxicity kit, and colchicine (demecolcine) were purchased from Molecular Probes. Fluorescently conjugated antibodies used for flow cytometry and immunofluorescent confocal microscopy were from Becton Dickinson. Anti–MMP-9 was from Oncogene Research Products. Centrifugal counterflow elutriation was done with equipment from Beckman Instruments, Inc.

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 anti–CD-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 I–coated 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 I–coated 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 I–coated 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-membrane–labeling 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 anti–MMP-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 Enhances In Vitro Differentiation of Human Monocytes
Human monocyte purity after isolation was >85% and viability >95% in all experiments (not shown). Monocytes were cultured on uncoated or on collagen type I–coated cell culture dishes. Monocytes adhered to substrate within 15 minutes after seeding, demonstrating round to ovoid cell bodies with prominent perinuclear regions within 24 hours. Subpopulations of monocytes under all conditions started to spread, as indicated by development of cellular processes (filopodia) and ramified shapes. Simultaneous fluorescent detection of monocyte surface markers after 24 hours in culture indicated that adhesion to a collagen type I substrate increases the percentage of monocytes that express the differentiation marker CD71 (Fig 1A). Differentiation was also analyzed on the basis of cell spreading (Fig 1B). In these experiments, we collected images with the confocal or phase-contrast microscopes and calculated ratios of differentiated monocytes per total number of cells in each of three randomly selected microscopic fields by computer-assisted image analysis. Both approaches showed that culturing the monocytes on collagen type I–coated dishes leads to a significant increase in the percentage of differentiated cells compared with monocytes adherent to uncoated dishes. Furthermore, values obtained by the two types of analysis were very similar, showing that cell spreading is a good indicator for monocyte differentiation. In some experiments, monocytes were stimulated with PMA for comparison. Interestingly, the effect of substrate on monocyte differentiation was significantly higher than that of PMA (44.9±1.4% versus 18.7±2.1%, respectively, P<.001). After 48 hours, we noted progressive lengthening of cellular processes (Fig 2). Image analysis confirmed that the significant advantage in monocyte differentiation on collagen type I substrate was maintained. The effect of PMA stimulation was slightly increased after 48 hours on either substrate, but it was still not statistically significant (unstimulated versus PMA-stimulated monocytes differentiated on uncoated dishes, 21.9±1.2% versus 25.0±3.1%, respectively; unstimulated versus PMA-stimulated monocytes differentiated on collagen type I–coated dishes, 42.3±0.8% versus 46.2±1.3%).

View larger version (16K): [in this window] [in a new window]   Figure 1. Collagen type I increases differentiation of human peripheral blood monocytes in vitro. Same numbers of freshly isolated cells (1x106/well) were seeded in six-well culture dishes (Plastic) or in six-well dishes coated with collagen type I (Collagen I). A, Monocyte differentiation after 24 hours in culture expressed as percentage of CD71-positive cells. Surface distribution of CD14, a general monocyte marker, and of CD71 (transferrin receptor), a monocyte differentiation marker, was investigated by double immunofluorescent labeling followed by quantitative confocal microscopy. B, Monocyte differentiation expressed by quantification of cell spreading. Data were obtained by image analysis of monocyte morphology from phase-contrast microscopy after 24 hours in culture. As a positive control for differentiation, some monocytes cultured were treated with 100 ng/mL PMA. Untreated cells are labeled 0. Total number of adherent cells and number of differentiated cells per field were calculated by computer-assisted image processing. For both approaches, images of multiple fields from each cell culture dish were acquired and analyzed. These numbers were used to generate average percentage of differentiated cells in each condition±SEM. Graphs present data quantified from three independent experiments.

View larger version (149K): [in this window] [in a new window]   Figure 2. Morphology of human monocytes after 48 hours in culture. Phase-contrast microscopy image shows representative morphology of human blood monocytes maintained in culture for 48 hours in serum-free medium on two different substrates. Same number of cells (1x106/well) were seeded in regular six-well culture dishes or in six-well dishes coated with collagen type I, with or without addition of 100 ng/mL PMA. Bar=100 µm.

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.

View larger version (65K): [in this window] [in a new window]   Figure 3. Intracellular accumulation of acLDL in human monocytes cultured on plastic or collagen type I–coated cell culture dishes (confocal microscopy image). Equal numbers of monocytes (1x106/well) seeded in uncoated six-well culture dishes (plastic) or in six-well dishes coated with collagen type I, with or without addition of 100 ng/mL PMA, were allowed to attach and differentiate for 24 hours. Fluorescently labeled acLDL was added (final concentration, 25 µg/mL), and after a subsequent incubation of 24 hours, cells were washed and imaged with a confocal microscope.

View larger version (32K): [in this window] [in a new window]   Figure 4. Adhesion to a collagen type I substrate increases accumulation of fluorescent acLDL in human monocytes in vitro, as shown by quantitative confocal microscopy. Culturing on collagen type I increased both total number of fluorescent cells per microscopic field (A) and intracellular lipid loading of individual cells, as shown by estimated average area of fluorescence per cell (B). Some monocyte cultures were treated with 100 ng/mL PMA. Untreated cells are labeled 0. After loading with fluorescently labeled acLDL, cells were examined with a confocal microscope, and data were derived by image analysis. Columns show representative values of mean±SEM from one experiment (***P<.001, **P<.01). Enhancing effect of collagen on intracellular lipid loading was consistently significant in all three independent experiments.

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.

View larger version (24K): [in this window] [in a new window]   Figure 5. Monocyte differentiation in the presence of genistein or colchicine. Freshly isolated monocytes were allowed to adhere for 2 hours on plastic or on collagen type I then were treated with genistein (25 µmol/L), a nonspecific inhibitor of protein phosphorylation, or colchicine (2 µmol/L), an inhibitor of microtubule assembly, for 24 hours. Differentiation was quantified by image-assisted microscopy. A, Differentiation was still increased by culturing monocytes on collagen type I compared with plastic. Inhibitors did not modify percentage of monocytes differentiating on plastic or on collagen type I. B, Both genistein and colchicine affected monocyte spreading: many differentiated monocytes were prevented from spreading efficiently, resulting in a decreased percentage of cells maintaining average size of differentiated cells. Data were obtained by calculating percentages of cells that had maximum lengths greater than a threshold value selected between mean maximum length of untreated and treated cells.

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.

View larger version (25K): [in this window] [in a new window]   Figure 6. Inhibition of monocyte spreading abolishes the enhancing effect of collagen type I on intracellular lipid loading in cultured monocytes. Inhibition of protein phosphorylation reduced intracellular accumulation of fluorescently labeled (DiI)-acLDL in monocytes. Genistein treatment abolished increase in lipid accumulation caused by culturing monocytes on a collagen type I substrate. Treatment reduced total number of cells that accumulated fluorescent acLDL (A) and individual cell loading, as assessed by measurement of fluorescence area per labeled cell (B). Columns show mean values obtained from image analysis of confocal microscopy data±SEM from a representative experiment. ***P<.001, **P<.01, *P<.05. Three independent experiments showed similar results.

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.

View larger version (48K): [in this window] [in a new window]   Figure 7. Secretion of MMP-9 by human monocytes is increased when cells are cultured on a collagen type I substrate. Culture media harvested from monocytes cultured for 24 hours on uncoated (plastic) or collagen type I–coated dishes were analyzed simultaneously on two 10% polyacrylamide minigels. One gel contained gelatin and was processed for SDS-PAGE zymography (A); the other was processed for immunoblotting (B) by transferring to nitrocellulose and probing with an anti–MMP-9 antibody, followed by chemiluminescent detection. Cultures of monocytes on collagen type I demonstrate increased release of gelatinolytic activity associated with pro-MMP-9 compared with cells cultured on uncoated dishes. Culture media of monocytes cultured on collagen type I substrate produced a faint, lower-molecular-weight lytic band that was visible on the gel but did not reproduce well, suggesting processing of pro-MMP-9 into active form. Treatment with either genistein or colchicine, both of which reduce monocyte spreading, also reduces gelatinolytic activity in media compared with that of untreated monocytes. Immunoblotting also indicated increased pro-MMP-9 protein release from monocytes cultured on collagen type I and inhibitory effect of genistein and colchicine. Immunopositive band running at lower apparent molecular weight probably represents a processed form of MMP-9. Conditioned culture media were collected from wells in which equal numbers of cells (106) were seeded under all conditions. Equal amounts of protein from culture media were loaded on gel. Position of same set of molecular weight markers in each gel (kD) showed that electrophoretic migration was slightly retarded in polyacrylamide gel containing gelatin (A) compared with regular gel with same polyacrylamide percentage (B).

	Discussion

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After their recruitment inside atherosclerotic vessels, peripheral blood monocytes differentiate into resident cells and participate in evolution and complication of plaques. We have examined whether adhesion to collagen type I contributes to monocyte differentiation and functions relevant to atherosclerosis. Our data show that collagen type I promotes differentiation of freshly isolated human monocytes to a greater degree than phorbol esters, soluble stimulators often used to induce monocyte differentiation.¹² Other matrix components might also modulate monocyte differentiation in vitro or in situ. However, we rationalized our approach and chose to study the effects of collagen type I on the basis of its overwhelming presence in the atherosclerotic plaques (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 situ–differentiated 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 I–coated 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 al^{37 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

 

acLDL = acetylated LDL DiI = 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate MMP = matrix metalloproteinase PMA = phorbol myristate acetate pro-MMP-9 = zymogen form of MMP-9

	Acknowledgments

 
Funds for this study were provided through grants from the American Heart Association, the Emory University Research Committee, and the Whitaker Foundation. Dr Wesley was supported through NIH training grant T32-HL-07745. The authors gratefully acknowledge Dr John Boring III, Chairman, Department of Epidemiology, Rollins School of Public Health of Emory University, for his generous advice regarding the statistical analysis of data, and Ulla Marzec, Division of Hematology, Emory University School of Medicine, for her expert assistance with the flow cytometry experiments.

Received June 23, 1997; accepted November 14, 1997.

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