© 1999 American Heart Association, Inc.
Vascular Biology |
An In Vitro Coculture Model of Transmigrant Monocytes and Foam Cell Formation
From the Department of Molecular Biology and Medicine (M. Takaku, Y.W., Y.K., M.U., T.H., T.K.) and the Department of Dynamics of Life Chemistry (Y.O., N.N., E.N.), Research Center for Advanced Science and Technology, University of Tokyo, Tokyo; the Second Department of Pathology (K.J., M. Takeya, K.T.), Kumamoto University School of Medicine, Kumamoto; the Second Department of Pathology (H.U., M.N.), Niigata University School of Medicine, Niigata; the Department of Cardiovascular Medicine (Y.W., H.K., Y.Y.), Graduate School of Medicine, University of Tokyo, Tokyo; and Chugai Pharmaceutical Co Ltd (M. Takaku), Tokyo, Japan.
Correspondence to Tatsuhiko Kodama, Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology (No. 35), University of Tokyo, 4-6-1, Komba, Meguro-ku, Tokyo 153-0061, Japan. E-mail kodama{at}med.rcast.u-tokyo.ac.jp
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Abstract—To analyze in vitro the migration of monocytes to the subendothelial space, their differentiation into macrophages, and the subsequent formation of foam cells in vitro, we have developed a 2-coculture system with rabbit aortic endothelial cells (AECs), aortic smooth muscle cells (SMCs), and a mixture of matrix proteins on polyethylene filters in chemotaxis chambers. AECs were seeded on a mixture of type I and IV collagen with or without various types of serum lipoproteins (method 1) or on matrix proteins secreted by SMCs (method 2). In these coculture systems, rabbit AECs can maintain a well-preserved monolayer for up to 2 weeks. When human CD14-positive monocytes were added to the upper medium of the system, with monocyte chemotactic protein-1 treatment
60% of the monocytes
transmigrated within 24 hours and were retained for up to 7 days,
whereas without MCP-1 treatment, <30% of monocytes transmigrated. On
day 1, transmigrant monocytes were negative for
immunostaining of type I and II macrophage
scavenger receptors but by day 3, became positive for scavenger
receptors as well as other macrophage markers. When oxidized
low density lipoprotein was added to the matrix layer of the method I
coculture, on day 4 transmigrant cells exhibited lipid deposit
droplets, and by day 7, they had the appearance of typical foam cells.
Some of the transmigrant cells recovered in the lower medium on day 7
also appeared to be foam cells, indicating foam cell motility and
escape from the coculture layer through the filter. In summary, this
coculture system is a useful in vitro tool to dissect the cellular and
molecular events that make up the process of foam cell formation.
Key Words: atherosclerosis • chemoattractants • macrophages • foam cells • oxidized LDLs
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Introduction |
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A key cellular component of atherosclerosis is the lipid-laden macrophage termed the foam cell. In animal models of atherosclerosis and in human subjects with hypercholesterolemia, increased levels of plasma cholesterol cause the migration of monocytes into the subendothelial space of the arterial wall, where they differentiate into macrophages and subsequently generate foam cells.1 2 3 4 However, many aspects of the cellular mechanisms by which foam cells are formed remain unknown. Nevertheless, several previous attempts have opened a window on approaches to this cellular process in vitro by using cultured aortic endothelial cells (AECs) and smooth muscle cells (SMCs).5 6 7 8 9 10 11 Monocyte migration into the subendothelial space in a coculture of adult human AECs and SMCs was previously reported,6 and its use was suggested for study of the cellular processes of atherogenesis. One of the major difficulties of this system, unfortunately, is the limited stability of the AEC monolayer, which can be easily disturbed by underlying matrix components and other factors, including growth factors and cytokines.12 13 This presents an obstacle: for example, in the rabbit model on a high-cholesterol diet, it takes at least 1 to 2 weeks for sufficient lipid to accumulate in the vascular wall and an additional 1 to 2 weeks for foam cells to appear.14 15 Thus, for an adequate analysis of foam cell formation, the AEC monolayer in the in vitro coculture system needs to be stable for at least 2 weeks.
In an effort to establish a more stable system, we selected rabbit AECs and SMCs as the components of the coculture. Rabbit AECs are derived from the artery, which is prone to develop atherosclerotic lesions, and lipid metabolism and atherogenesis in animal species have been studied intensively.14 15 16 17 18 We here report an improved method for primary culture of rabbit AECs and the effect of matrix proteins on the establishment of a longer-lasting culture, since type I collagen or MATRIGEL6 19 20 cannot support the AEC monolayer for 2 weeks. We used 2 different methods. Either a mixture of type I and type IV collagen (method I) or a mixture of matrix proteins secreted from rabbit SMCs (method II) was used to stabilize the continuity of the endothelial monolayer.
Using these 2 methods, we studied the effect of monocyte chemotactic protein-1 on monocyte transmigration and foam cell formation. The transmigration of fluorescently labeled monocytes was quantitatively analyzed by 3-dimensional analysis with confocal laser microscopy. Each step of this transmigration was also studied with electron microscopy and immunohistochemical analysis. Finally, we report the formation of foam cells in these systems with an appearance similar to those found in human atherosclerotic lesions.
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Methods |
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Reagents
The reagents and their suppliers are as follows: Dulbecco's modified Eagle's medium (DMEM), M199, trypsin-EDTA, penicillin-streptomycin, and other tissue culture materials from Gibco BRL, Life Technologies Inc; PBS from the Takara Co Ltd; endothelial growth medium-2, the culture medium for ECs, including EC growth supplements, from Clonetics Corp; collagenase type 2 from Worthington Biochemical Corp; 96-well and 24-well tissue-culture plates from Becton Dickinson Labware; 60-mm and 100-mm collagen type I–coated dishes from the Iwaki Co Ltd; the insert well, Chemotaxicell (filter surface area 0.48 cm2, pore size 3 µm in diameter) from the Kurabo Co Ltd; living cell fluorescent staining kit PKH2(FITC-like fluorescent pigment) and PKH26 (rhodamine-like fluorescent pigment) from Zynaxis Cell Science, Inc; collagen type I and IV solution from the Koken Co Ltd; Microbead-labeled anti-CD14 antibody, MidiMACS, and single-use, type BS separation columns from Militenyi Biotec GmbH; and recombinant human MCP-1 from Pepro Tech EC Ltd.
Lipoproteins
Lipoproteins were separated from the plasma of a normal healthy
volunteer,21 and LDLs were oxidized by a 16-hour
incubation in the presence of 10 µmol/L CuSO4
in RPMI.22
Cell Culture
Rabbit AECs
A male, Japanese White rabbit (1.0 to 1.5 kg) was
anesthetized by intravenous injection of
pentobarbital. The midline of the abdomen was incised, and almost all
of the blood was taken from the abdominal aorta at the bifurcation by
using a heparin-flushed syringe. The thoracic portion of the aorta was
dissected out. The connective tissue and fat were removed, and a
segment of aorta was dipped in DMEM (containing 5% FCS). After being
washed with serum-free DMEM, the distal end of the fragment was
fastened with a thread, and the proximal end was bound with the syringe
joint. Collagenase type 2 solution (400 U/mL, dissolved in
serum-free DMEM) was poured into the aorta, and the segment was
incubated for 30 minutes at 37°C. After collagenase
digestion, ECs were removed from the aorta by flushing with 25 mL of
DMEM (with 5% FCS) twice. Cells were collected by
centrifugation at 70g for 5 minutes and were
resuspended in 50 mL of DMEM supplemented with 5% FCS, 100 U/mL
penicillin, and 100 µg/mL streptomycin. Cells were dispersed in ten
96-well plates at 50 µL per well. To remove contaminated SMCs, after
a 4-hour incubation at 37°C, the wells of these plates were washed
twice with 300 µL of PBS in a 12-channel 96-well plate-washing
machine (Flexiwash I, Towa Lab Co). Adherent cells, almost all of which
were ECs, were cultured for 7 days with EGM-2 and 2% rabbit serum
at 37°C. When the cells reached 80% confluence, they were passed
into a 60-mm collagen type I–coated culture dish with the use of
0.05% trypsin in 0.53 mmol/L tetrasodium EDTA. Rabbit AECs from 5
wells were cultivated in one 60-mm dish for 1 week without changing
the medium. The cells were checked by morphology, DiI acetyl LDL uptake
activity, and immunocytostaining with an anti–von Willebrand
factor antibody.23 Rabbit AECs from the subculture of 3 or
4 passages were used in this study without freezing and thawing.
Rabbit Aortic SMCs
Rabbit SMCs were isolated from the aortic media by an explant
technique modified from that of Ross.24 25 After removal
of the ECs, the segments were cut open and the media separated by means
of a thin pincette. During these procedures, the segments were dipped
in DMEM with 5% FCS. The isolated medial fragments were minced into
small pieces, and these were scattered on the dish and dried for 2
to 5 minutes. DMEM (with 5% FCS) warmed at 37°C was poured slowly
into the dish. The fragments were cultivated for 7 days at 37°C in
95% air/5% CO2. Migrant SMCs were passed by using 0.05%
trypsin in 0.53 mmol/L tetrasodium EDTA. SMCs were defined by an
immunocytostaining procedure with anti-rabbit SM myosin heavy-chain
antibody (SM1, Yamasa Co Ltd). Rabbit SMCs from the subculture of 3 to
7 passages were used in this study without freezing and thawing.
Human Peripheral Blood Monocytes
Monocytes were isolated from the whole blood of a healthy donor.
Mononuclear cells were enriched by centrifugation with
a Ficoll-Paque Plus (Pharmacia Biotech) gradient. Monocytes were
isolated by a magnetic cell-sorting apparatus with the use
of microbead-labeled anti-human CD14 antibody (MACS system). More than
95% of the purified cells were positive for CD14. Most exhibited the
morphology of human monocytes and adhered immediately (within 5
minutes) to glass plates.
Construction of a Vascular Model
Method 1
Reagents for the matrix layer, ie, collagen type I solution
(0.5%, Koken), collagen type IV solution (0.3%, Koken), 10x
conditioned medium 199, and reconstruction buffer (0.05 mol/L NaOH,
200 mmol/L HEPES, and 0.26 mol/L NaHCO3) were mixed in
the following volumetric proportions at 4°C: 32:48:10:10,
respectively. The mixture was laid on the Chemotaxicell filter, and
incubated at 37°C for 1 hour to complete gel formation. Rabbit AECs
(2x105) labeled with PKH-26 were seeded on the gel. One
week later, rabbit AECs became confluent and were ready for the
following experiments.
Method 2
SMCs (106) were laid on the filter and cultivated
with EGM-2 (with 2% rabbit plasma) for >7 days. After a sufficient
amount of matrix proteins had been secreted from the SMCs, rabbit AECs
labeled with PKH26 were seeded on the SMCs and their secreted matrix
proteins and proteoglycans. One week later, they were used for
experiments.
Quantitative Analysis of Monocyte Transmigration With or
Without MCP-1 Treatment
After the rabbit AEC monolayer became confluent, both the upper
and lower media were changed to a medium containing recombinant human
MCP-1 so that cell surface heparan sulfate could sequester
chemokines.26 A preliminary study had indicated that
monocyte transmigration was stably enhanced at concentrations of 10 to
100 nmol/L, and for these experiments, a concentration of 30 nmol/L
MCP-1 was used. After a 4-hour incubation, the upper medium was
removed, and the well was washed twice with PBS. Monocytes
(105 CD14-positive cells) were then added to a single
chamber. To maintain the MCP-1 gradient, the lower medium was changed
to a fresh medium containing 30 nmol/L MCP-1, and the coculture systems
were incubated for various periods of time at 37°C. At days 1, 4, and
7, the endothelial surface of each filter was washed
independently with 400 µL of PBS 3 times and with 1 mL of PBS for the
outside of the chamber twice. The housing was then cut off, and a
quantitative assay with confocal microscopy (LEICA TCS SP, Leica
Microsystems) was performed. Also, the number of cells in the matrix
layer and the number of cells attached to the ECs were counted by using
confocal microscopy. For each count, the recovered upper medium and 1.2
mL of the PBS used for washing the endothelial layer
were collected together to count the total number of living, floating
monocytes.
Histopathological Analysis
Silver Nitrate Staining and Immunocytochemistry
After the experiments, the cell layers on the filters were fixed
with 4% paraformaldehyde fixative for 15 minutes at
4°C and washed with PBS. To confirm formation of the AEC monolayer,
the samples were stained with silver nitrate by using the method
described by Furie et al.5 Anti-catenin
antibody27 was kindly provided by Drs Akira Nagafuchi and
Sho-ichiro Tsukita. Anti–PECAM-1 (CD31) antibody28 was
kindly provided by Drs Michitaka Masuda and Keigi Fujiwara.
Anti–VE-cadherin antibody (C-19)29 was supplied by Santa
Cruz Biotechnology Inc. These antibodies were used as markers for tight
junctions and adherence junctions in the cells. For the study of
differentiation of monocytes into macrophages, the samples were
embedded in a tissue-embedding compound and frozen in LN2
for subsequent preparation of frozen sections. After
endogenous peroxidase activity was blocked, the sections
were incubated with MH-1, an anti-human type I and II scavenger
receptor monoclonal antibody,30 and KP-1 (CD68,
Dako)31 for 1 hour. The anti-macrophage
cell-surface marker antibody AM-3K32 (at the time of this
writing, the antigen for the AM-3K antibody has yet to be
characterized) was also used for immunohistochemical analysis.
The sections were then reacted with anti-mouse Ig
[F(ab')2] conjugated with peroxidase (Amersham) for 1
hour. To visualize peroxidase activity, 3,3'-diaminobenzidine was used
as the substrate. Counterstaining was done with hematoxylin. As
controls, sections were incubated with nonimmunized mouse serum or PBS
instead of primary antibody and then processed as described above.
Electron Microscopy
For scanning electron microscopic observation, the cell layers
on the filters were fixed with 1.2% glutaraldehyde for
4 hours, followed by 1% OsO4 for 1 hour. The specimens
were dehydrated with a graded ethanol series and isoamyl acetate. They
were desiccated by the critical-point method using CO2 and
were coated with metal in an ion coater (type IB-3, EIKO Engineering
Co). The specimens were observed in a Hitachi S-800 scanning electron
microscope (Hitachi) at an accelerating potential of 20 kV. For
transmission electron microscopic observation, the cell layers on
filters were fixed with 1.2% glutaraldehyde for 60
minutes, washed with 0.1 mol/L cacodylate buffer (pH 7.4), and further
fixed with 1% OsO4, as described above. Then the samples
were again dehydrated through a graded series of ethanol solutions.
After the filters were removed from the cell layers, the latter were
passed through propylene oxide and embedded in epoxy resin. Ultrathin
sections were cut with an ultramicrotome (MT-7000, Research and
Manufacturing) and examined with an electron microscope (H-7500,
Hitachi) after being counterstained with uranyl acetate and lead
citrate.
Statistics
Results are given as the mean±SEM. Statistical comparisons were
made by the 2-tailed Student's paired t test. Results were
considered significant at P<0.01.
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Results |
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Formation of the Endothelial Monolayer on the Collagen Layer
Figure 1
indicates the hematoxylin
staining of the coculture systems used in this article. Rabbit AECs
were cultivated for 7 days on either a mixture of type I and type IV
collagen (method I, Figure 1a) or matrix proteins secreted from rabbit
SMCs (method 2, Figure 1b). We optimized rabbit AEC growth with a
mixture of 0.5% type I solution and 0.3% type IV collagen solution in
a range of ratios between 1:9 and 9:1 (vol/vol), with the ratio of
4:6 (vol/vol) of type I to type IV, which yields the ratio of 9:10
(wt/wt), providing the best attachment and monolayer growth. At this
content (method 1), rabbit AECs can maintain a fully functional
monolayer with continuous tight junctions for up to 14 days, as judged
by silver nitrate staining (Figure 2a and 2d) or immunohistochemistry with
anti-catenin (Figure 2b and 2e) and anti–VE-cadherin antibodies
(Figure 2c and 2f). The rabbit AEC monolayer on SMCs (method II) also
is able to maintain a well-preserved monolayer for up to 14 days.
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Transmigration of Monocytes
As shown in Figures 3a and 3b,
rabbit AECs were labeled with PKH26 (red) and human monocytes with PKH2
(green). After the sample was fixed with 4%
paraformaldehyde solution, the coculture system on the
microporous membranes was carefully separated from the plastic housing
and placed on glass slides. The vertical sectioning observation of
samples by confocal microscope clearly indicates the position of the
monocytes. The monocytes were attached to the AEC layer (Figure 3a),
and the transmigrant monocytes (Figure 3b) are clearly
identifiable.
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For quantitative analysis, the microporous filter was divided
into 28 separate areas as indicated in the inset on the left in Figure 3
. The center of each area was scanned vertically by using a
computer-assisted scanning table, and the number of monocytes either
attached to rabbit AECs or transmigrated into the matrix layer was
counted in the 90x125-µm2 area.
For the initial attempts to analyze monocyte transmigration,
the confluent monolayer of rabbit AECs in method 1 was pretreated for 4
hours by the addition of 30 nmol/L MCP-1 to both the upper and lower
media. Before the addition of monocytes, the chamber was washed twice
with PBS. To obtain the MCP-1 concentration gradient, the upper medium
was changed to AEC growth medium without MCP-1 and the lower medium to
fresh AEC medium containing 30 nmol/L MCP-1. Then, 105
CD14-positive monocytes were added to the upper medium, and the numbers
of monocytes in the collagen layer and of floating monocytes were
counted. Figure 4 indicates the time
course of monocyte migration in the method 1 culture system. After 24
hours of incubation with the monocytes, >60% of the monocytes were
detected within the matrix layer, and this percentage did not change
for up to 7 days. However, in the absence of MCP-1 treatment, <30% of
the monocytes were detected in the matrix layer throughout the 7
days.
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) and transmigrant monocytes
()
were calculated as described in Methods. Values are mean±SEM, n=5. *,
**, ***P<0.01 vs MCP-1 (-) values on the same day.
Ultrastructure of the Transmigrant Monocytes
Examination by scanning electron microscopy of the method 1
coculture system treated with MCP-1 revealed many of the features of
monocyte transmigration previously reported1 2 in
experimental animal models (Figure 5).
Monocytes attached to the rabbit AECs (Figure 5a) displayed a variety
of plasma membrane configurations, including "ruffles"
(lamellipodia), filopodia, microvilli, and pseudopodia, suggesting that
they were already activated. Transmigrating monocytes exhibited
a well-developed, veillike structure (Figures 5b and 5c). Some
monocytes also exhibited structures known as lamellipoda (Figure 5b, arrowhead) and uropoda (Figure 5b, arrow). The position of the
endothelial junction and its relationship to the
transmigrating monocytes could not be identified in these pictures, and
further studies will be needed. During fixation, the AEC layer at times
dissociates from the collagen layer, and this may be ascertained by the
position of micropores on the filter membrane (indicated as P in Figure 5d). The monocyte in Figure 5d is moving through the basement membrane
of the AEC layer (bEC). Below the AEC layer, the fixed matrix layer can
be seen in the form of entangled fibers. During migration, the
monocytes appeared to be digesting these fibers (Figure 5e).30
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An increase in the number of transmigrant monocytes was also detected
in the matrix layer of method 2. To confirm monocyte differentiation
into macrophages, we characterized these transmigrant cells by
immunostaining for macrophage-specific
cell surface markers, including the types I and II macrophage
scavenger receptors,31 CD68 and AM-3K antigen. On day 1,
monocytes attached to rabbit AECs (Figure 6a through
6c, arrows), or monocytes at the site of
rabbit AECs (Figure 6a, arrowheads) were positive for CD68 (Figure 6a)
but negative for immunostaining of type I and II
macrophage scavenger receptors (Figure 6b) or AM-3K antigen
(Figure 6c). These results are consistent with the reported
pattern of cell surface markers of typical human
monocytes.31 32 33 On day 3, they became positive for
scavenger receptors as well as for AM-3K antigen (Figures 6d through
6f, arrowheads), indicating that they had already differentiated into
macrophage-like cells at this point. Monocytes transmigrated
into the matrix layer of method 1 also exhibited positive staining for
type I and II scavenger receptors and AM-3K antigen.
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Foam Cell Formation in the Coculture Systems
Figure 7 indicates the morphological
changes that occurred during transmigration. In method 1, oxidized LDL
was added to the collagen solution at 20 µg/mL at 4°C before it was
applied to the microporous filter. After polymerization of the collagen
gel, rabbit AECs and then monocytes were laid in the same manner as
before. At 2 hours, monocytes floating in the culture medium or
adherent to the endothelial surface had indented
nuclei, a few granules, small vesicles, and abundant microvillous
projections (Figure 7a). Cells of similar size and ultrastructure
were also found in the collagen layer 8 hours after culture (Figure 7b). The vacuoles were 0.5 to 1.0 µm in diameter and often
contained a few round, electron-lucent vacuoles bounded by a unit
membrane, suggesting that they were fat droplets.
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The size of the cells and the number of lipid droplets increased as the
days elapsed. On day 4, the transmigrant cells possessed abundant
cytoplasm containing a number of lipid droplets. They exhibited the
characteristic morphology of macrophages, such as elongated and
indented nuclei and a distorted cytoplasmic membrane with abundant
microvillous projections (Figure 7c). On day 7, the size of these
cells reached 50 µm in diameter, and they contained a large
number of lipid droplets (Figure 7d). The cytoplasmic pro-jections
were not prominent in these cells. The size of the lipid droplets
varied from 0.5 to 10 µm. These lipid droplets occasionally
contained a few crystalloid structures and electron-dense amorphous
material. The nuclei were ovoid or irregular and usually located in the
periphery of the cytoplasm. These cells were morphologically very
similar to the foam cells observed in atheromatous
plaques, which are regarded as monocyte-derived foam
cells.1 2
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Rabbit AECs and the Matrix Components Suitable for This Coculture System
The in vitro system that we report here allows the quantitative analysis of monocyte transmigration, differentiation into macrophages, and foam cell formation in a matrix layer underneath a rabbit AEC monolayer. The concept of an in vitro system for the analysis of atherogenesis was originally developed by Navab, Fogelman, and colleagues6 7 8 9 10 11 using human AECs. Because there are several difficulties with obtaining primary human cultured AECs, we tested a primary culture of rabbit AECs. The rate of transmigration in this system is affected by the continuity of the endothelial monolayer, while the need to maintain the integrity of the tight junctions as well as other intracellular apparatus comprises the most important barrier to well-executed cell transmigration. To avoid excessive disruption of intercellular junctions, several improvements were adopted. First, we avoided excessive trypsin digestion of rabbit AECs. On each passage, a concerted effort was made to maintain the rabbit AECs in a sheetlike condition, each sheet consisting of >30 rabbit AECs. Freezing and thawing of AECs were also avoided. On examination by confocal microscopy, we often found places where the AECs had not covered the matrix layer properly when AECs of low viability had been used. We also used a combination of cultivation at low rabbit serum concentrations, which profoundly inhibited the growth of SMCs, and selection with a 96-well culture dish, which enabled us to completely eliminate SMC contamination. By means of this improved method, we established a workable AEC monolayer for the study of transmigration.
The second important problem was to define the optimal components of the matrix layer that would support the accumulation of transmigrant monocytes. Previous articles6 16 17 reported collagen type I or MATRIGEL as a component of the matrix layer. However, for rabbit AECs, pure collagen type I induces detachment of AECs from the well at the edge of the filter after several days of cultivation. MATRIGEL induces a change in the conformation of rabbit AECs, especially elongation. We therefore tested various combinations of available matrix proteins for the matrix layer underneath the rabbit AECs. Among these, a mixture of types I and IV collagen yielded the best monolayer, type IV collagen being an important component of the normal vascular basement membrane.34 35 A 9:10 (wt/wt) mixture of type I collagen to type IV collagen optimally supported monolayer formation, as judged by electrical resistance studies (data not shown), the continuous monolayer image obtained by confocal microscopy, and immunostaining for junction proteins during the 2-week cultivation period.
We also tested matrix proteins secreted from rabbit SMC cultures. After 1 week of cultivation on the microporous filter, rabbit SMCs were able to secrete sufficient matrix proteins to support the growth of rabbit AECs for up to 2 weeks.
Quantitative Analysis of Monocyte/Macrophage
Transmigration
To study monocyte transmigration, monocytes were separated by a
combination of Ficoll-Paque gradient and a magnetic bead–associated
anti-CD14 antibody. We chose to avoid methods involving adhesion
molecules, which might have affected the results of the transmigration
assay. Previous reports had indicated that anti-CD14 bead separation
may only minimally affect monocytes.36 37 38 This method
also enabled us to obtain >6x106 CD14-positive cells from
200 mL of blood from a single healthy donor. Attempts were made to
develop a similar method for the separation of rabbit monocytes, but a
sufficient number of pure monocytes could not be obtained by this
method.
Because the number of spontaneously transmigrating monocytes was low, a treatment step with human recombinant MCP-1 was implemented.39 40 MCP-1 is the most potent chemoattractant for human monocytes, and expression of MCP-1 in human atherosclerotic lesions has been reported.41 Pretreatment with human MCP-1 was able to induce the transmigration of human monocytes through rabbit AECs, and many of these transmigrant human monocytes in the matrix layer survived for >7 days. Furthermore, recombinant human MCP-1 can effectively mediate the interaction between human monocytes and rabbit AECs. Compared with the survival rate of human monocytes cultivated on collagen-coated dishes, the cell survival rate improved profoundly. Extracellular matrix is known as a cell survival factor,42 and extracellular matrix can also modulate macrophage functions characteristic of atheroma, including the production rate of matrix metalloproteinases.43 Most of the monocytes that transmigrated into the matrix layer in both methods 1 and 2 differentiated into macrophage-like cells and expressed cell surface markers reported as being characteristic of foam cells in atherosclerotic lesions, including type I and type II macrophage scavenger receptors.44
A Tool for the Study of Atherogenesis
This coculture system can support both the differentiation of
transmigrant monocytes and their survival for >7 days. In a
preliminary experiment, we added a significant amount of oxidized LDL
to the matrix layer in method 1, and this resulted in an increase in
the number of monocytes retained in the matrix layer (data not shown).
The addition of oxidized LDL, as shown by the successive
ultrastructural studies (Figure 7), resulted in an increase in the size
of monocytes after transmigration, mainly reflecting the increase in
lipid droplets in their cytoplasm. By day 4, these transmigrant cells
had accumulated a significant number of lipid droplets (Figure 7c), and
these droplets appeared to be surrounded by a membranous structure. On
day 7, the transmigrant cells had accumulated a large number of lipid
droplets (Figure 7d) and appeared similar to the foam cells found in
atherosclerotic lesions in experimental animals and human subjects.
The coculture system reported in this article allows the observation and investigation of monocyte transmigration and foam cell formation, as well as quantitative analyses of these processes. However, additional improvements are still needed. At first, many of the present results were obtained using method 1, which lacks SMCs, a major cellular component in the artery wall. For the preliminary experiments, we used a coculture system with SMCs and matrix proteins secreted from SMCs (method 2). This method can also support the AEC monolayer and monocyte differentiation, but the major difference in monocytes/macrophages caused by the presence of SMCs is now under investigation. Another important problem concerned the handling of lipoproteins. Although lipid accumulation in atherosclerosis in human subjects and experimental animals has distinctive histopathological features,14 15 45 in this study we simply added CuSO4-modified, oxidized LDL to the matrix component.
The molecular mechanisms by which modification of lipoproteins in the arterial wall occurs still remain poorly understood. The lack of positional and signal information for vascular cells, including the role of shear stress, the oxygen gradient, the NO gradient, and stimulation by the nervous system, may prove to be important shortcomings of this system. In the presence of these stimuli, vascular cells may well behave differently. Nevertheless, whatever refinements of this system are developed in the future to bring it into closer approximation of the actual physiological conditions, the establishment of such a system marks an important advance, in that it now makes possible the study of molecular mechanisms of foam cell formation in vitro in the presence of the main types of artery wall cells and in a special, structural setting similar to that in the artery wall. It is well known that the interaction of the 3 cell types (ie, ECs, SMCs, and monocytes/macrophages) with each other has profound effects on the metabolism and function of vessel wall components, including matrix, cytokines, and growth factors.46
In conclusion, we have developed a vascular cell coculture system that can support an AEC monolayer for up to 2 weeks, as well as foam cell formation by transmigrant human monocytes. This coculture method can provide a technology platform for future studies addressing crucial aspects of atherosclerosis.
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Acknowledgments |
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This work was supported by the Japan Foundation for Aging and Health and the "Research for the Future" program of the Japan Society for the Promotion of Science. The authors are grateful to Dr Mohamad Navab for initial instructions of the coculture technique and thoughtful comments on this work. We also thank Kazumasa Sato and Yuko Imai for their expert technical assistance and Dr Kevin Boru for review of the manuscript. We also thank Dr Kiyoko Akagawa for technical instruction on monocyte purification.
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Footnotes |
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The first 2 authors contributed equally to this study.
Received November 2, 1998; accepted March 2, 1999.
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References |
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