© 1998 American Heart Association, Inc.
Original Contributions |
Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions
From the Department of Geriatrics, Nagoya University School of Medicine, Nagoya (M.A.R., M.K., T.E., S.S., T.A., S.K., T.H., A.I.), and the Department of Pathology, Faculty of Medicine, Mie University, Tsu (S.M.), Japan.
Correspondence to Dr Masafumi Kuzuya, Department of Geriatrics, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan. E-mail kuzuya{at}med.nagoya-u.ac.jp
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Abstract |
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Abstract—The interaction between macrophages and oxidatively modified low density lipoprotein (Ox-LDL) appears to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the induction of numerous cytokines and growth factors. The current study demonstrated that Ox-LDL upregulated vascular endothelial growth factor (VEGF) mRNA expression in RAW 264 cells, a monocytic cell line, in a time- and concentration-dependent manner and that Ox-LDL stimulated VEGF protein secretion from the cells. Lysophosphatidylcholine, a component of Ox-LDL, also enhanced VEGF mRNA expression in RAW 264 cells and VEGF secretion from RAW 264 cells, with a maximal effect at a concentration of 10 µmol/L lysophosphatidylcholine. Immunohistochemical studies showed that human early atherosclerotic lesions exhibited intense VEGF immunoreactivity in subendothelial macrophage-rich regions of the thickened intima. In atherosclerotic plaques, VEGF staining was also observed in foam cell–rich regions adjacent to the lipid core or the neovascularized basal regions of plaque consisting predominantly of smooth muscle cells. High-power-field observation revealed that VEGF was localized in the extracellular space as well as at the macrophage cell surface. These observations suggest the possible involvement of Ox-LDL in the development of human atherosclerosis through VEGF induction in macrophages.
Key Words: vascular endothelial growth factor • vascular permeability factor • atherosclerosis • macrophages • oxidized low density lipoprotein
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Introduction |
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During the process of atherogenesis, a number of growth-regulatory molecules and cytokines may be formed and released from cells within the lesions, consisting of ECs, SMCs, macrophages, and T lymphocytes.1 These molecules induce and regulate numerous critical cell functions, including cell recruitment, migration, proliferation, and matrix protein synthesis. Through a complex network of cellular interactions, these growth factors and cytokines seem to play an important role in the progression of atherosclerotic lesions.1
Recent findings indicate that Ox-LDL may play a key role in the atherosclerotic process.2 3 Immunohistochemistry studies suggest that Ox-LDL accumulates in atherosclerotic lesions.4 5 Ox-LDL is characterized by altered chemical and physical properties, including elevated levels of lipid hydroperoxide, oxidized sterols, LPC, degradation fragments of apoB-100, and electrophoretic mobility.6 Oxidative modification of LDL alters its biological properties, resulting in monocyte chemotaxis,7 foam cell formation in macrophages,8 and cytotoxicity of a variety of cells.9 10 Recent evidence also suggests that Ox-LDL can modulate growth factor or cytokine production from ECs, SMCs, or macrophages.11 12 13 Thus, Ox-LDL can act as a mediator of many cellular events relevant to atherogenesis.
VEGF, also known as vascular permeability factor, is a heparin-binding, dimeric, EC-specific mitogen and angiogenic factor that is also a potent mediator of vascular permeability.14 15 This growth factor has been demonstrated to be involved in normal and pathological processes, including tumor progression, collateral vessel formation in ischemic tissues, and inflammation.16 17 18 In addition, VEGF can induce monocyte chemotaxis.19
We hypothesized that VEGF might be involved in the process of atherogenesis and might be a possible candidate for mediating neovascularization and hyperpermeability in the atherosclerotic plaque. In fact, we and others have already demonstrated that vascular SMCs can express and secrete VEGF in response to serum, growth factors, and cytokines.20 21 22 23 24
Macrophages in the vascular wall seem to play a central role in the atherosclerotic process through their capacity to produce numerous cytokines and growth factors. In the current study, we investigated whether Ox-LDL upregulated VEGF expression in the macrophage, which is already known to have the capability to produce VEGF.25 26 In addition, we also studied whether VEGF was really present in human atherosclerotic lesions, which may be relevant to vascular remodeling during the progression of atherosclerotic lesions.
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Methods |
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Cell Culture
The mouse fibroblast-like cell line NIH 3T3 and the mouse macrophage-like cell line RAW 264 were obtained from Riken Cell Bank (Tsukuba, Japan). In the current study, we used the mouse myeloid leukemia cell line RAW 264 as the model for macrophages, because this cell line displays macrophage properties without application of any stimulus. RAW 264 cells were cultured in RPMI 1640 (Nissui Pharmaceutical) containing 10% FBS. For the experiments, RAW 264 cells at confluence in the dishes were pretreated in serum-free RPMI 1640 for 24 hours. The medium was then replaced with fresh, serum-free medium containing Ox-LDL, native LDL, or LPC and incubated for the indicated periods of time.
Northern Blotting
A VEGF cDNA fragment (0.5 kb) including the coding region of
mouse VEGF165 was cloned from NIH 3T3 cells
stimulated by phorbol 12-myristate 13-acetate (25 ng/mL) and
inserted into the pT7 Blue T-vector (Promega). This VEGF cDNA probe was
digested with BamHI and PstI and isolated by
agarose gel electrophoresis. Total RNA was prepared from the RAW 264
cells with the use of TRIzol reagent (GIBCO BRL). RNA (15 µg) was
fractionated by denaturing electrophoresis and capillary blotted to
nylon membranes (Boehringer Mannheim). The filters were
cross-linked with UV light, followed by prehybridization for 4 hours at
50°C in prehybridization solution. Hybridization was then performed
with 5 ng/mL of [
-32P]CTP–labeled VEGF cDNA
probe for 18 hours at 50°C. The VEGF cDNA probe had been labeled with
the use of the [-32P]CTP Megaprime DNA
labeling system (Amersham International plc) using the random-primer
method. After hybridization, the filters were washed, exposed to a Fuji
imaging plate (Fuji Photo Film Co, Ltd), and analyzed with
BAStation version 1.4 software (Fuji Photo Film Co, Ltd) or exposed to
Fuji RX-U film (Fuji Photo Film Co, Ltd) with intensifying screens at
-80°C. The relative mRNA level of VEGF was normalized against the
signal of GAPDH.
VEGF Protein Quantification
After incubation of the RAW 264 cells under the indicated
conditions, the conditioned medium was collected and
centrifuged at 100g for 5 minutes, and the
supernatant was used for the measurement of VEGF protein with a
Quantikine M mouse VEGF ELISA kit (R&D systems, Inc). The cells were
then trypsinized and cell number was counted with a Coulter counter.
VEGF concentration was normalized to cell number.
LDL Preparation
LDL (d=1.019 to 1.063 g/mL) was separated from normal
human plasma by preparative ultracentrifugation,
dialyzed against PBS containing 0.3 mmol/L EDTA, sterilized by
filtration through 0.45-µm Millipore membranes (Millipore), and
stored at 4°C as described previously.27 EDTA
was removed from the LDL by dialysis against PBS before oxidation or
immediately before addition to the cell culture medium for native LDL.
LDL was oxidized by incubating 500 µg/mL LDL in 10 µmol/L
CuSO4 for 16 hours at 37°C and then dialyzed in
PBS containing 0.1 mmol/L EDTA for 24 hours at 4°C, followed by
further dialysis against PBS for 24 hours at 4°C to exclude EDTA as
previously described.27 28 Fresh preparations of
LDL and Ox-LDL were used for each experiment.
Immunohistochemistry
Twenty-three human artery specimens from thoracic (n=8) and
abdominal (n=8) aortas or subclavian arteries (n=7), each containing 1
or more lesions, were obtained from 8 autopsied patients (mean age, 67
years). The autopsies were performed 4 to 8 hours after death. Tissues
were fixed in buffered 10% formalin, routinely processed, and embedded
in paraffin.
Paraffin-embedded sections were cut at 6 to 8 µm thick, and the paraffin was removed from the tissue sections with xylene and ethanol before they were stained. Endogenous peroxidase activity was reduced by preincubation of sections with 0.3% H2O2 in PBS. The sections were pretreated with diluted blocking serum and then incubated with primary antibodies diluted in PBS supplemented with 1% BSA at room temperature for 60 minutes. After being washed in PBS, species-appropriate biotinylated secondary antibodies were applied, followed by the avidin-biotin peroxidase complex immunoperoxidase system (Vectastain ABC kit, Vector Laboratories). A 3,3'-diaminobenzidine solution (Vectastain) was used as the peroxidase substrate.
Antibodies
The following polyclonal and monoclonal antibodies were used as
primary antibodies to detect VEGF and cell types. Two different
anti-VEGF antibodies were used in the current study, and identical
distributions of VEGF in arterial tissues were obtained.
Rabbit polyclonal antibody against a 20–amino acid synthetic peptide
corresponding to residues 1 to 20 and mapping at the amino terminus of
human VEGF (Santa Cruz Biotechnology Inc) and goat polyclonal antibody
against human VEGF165 (R&D Systems) were used at
a final concentration of 0.5 µg/mL and 1 µg/mL, respectively.
Monoclonal antibodies HHF35 (1:50 dilution) and HAM56 (1:50 dilution;
ENZO Diagnostics) were used as specific markers for SMCs
and macrophages, respectively. Monoclonal anti-human von
Willebrand factor (1:50 dilution; clone F8/86, Dako) was used
as a specific marker for ECs. Negative staining with nonspecific
immunoglobulin was documented for each experiment (result not
shown).
Reagents
BSA, phorbol 12-myristate 13-acetate, and palmitoyl
L--LPC were purchased from Sigma Chemical Co.
CuSO4 and EDTA were obtained from Katayama
Chemical.
Statistical Methods
Values are given as mean±SD. Analysis by ANOVA was
used, followed by post hoc testing (Scheffé's test). A value of
P<0.05 was considered significant.
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Results |
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Induction of VEGF mRNA in Macrophages by Ox-LDL
After incubation of RAW 264 cells with serum-free medium for 24 hours, the cells were exposed to Ox-LDL at concentrations between 10 and 100 µg/mL for 16 hours. As shown in Figure 1
A, Ox-LDL increased VEGF mRNA expression in
RAW 264 cells in a concentration-dependent manner. After normalization
to GAPDH mRNA, the increase in VEGF mRNA was estimated to be 3.2-fold
for 100 µg/mL Ox-LDL–treated cells versus control cells (Figure 1B).
When RAW 264 cells were exposed to 50 µg/mL of Ox-LDL for 4, 8, or 16
hours, the VEGF mRNA level increased in a time-dependent manner (Figure 2A and 2B). Native LDL had no effect on VEGF
mRNA levels, as assessed by measurement of the VEGF mRNA level of cells
exposed to 50 µg/mL of native LDL for 8 hours (Figure 3A and 3B). No evidence for a toxic effect of
Ox-LDL was observed during the incubation with RAW 264 cells, as
assessed by morphology and LDH release.
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Ox-LDL Increases VEGF Protein Levels in Conditioned Medium
To examine whether Ox-LDL stimulates VEGF production
by RAW 264 cells, VEGF levels were determined in conditioned medium
from cells that had been incubated with or without Ox-LDL or native LDL
for 24 hours by ELISA. Consistent with the results of mRNA
expression, VEGF production was increased by exposure to Ox-LDL
(50 µg/mL) for 24 hours, whereas no significant increase in VEGF
production was observed with native LDL (50 µg/mL) (Figure 4A). Ox-LDL stimulated VEGF
production from RAW 264 cells in a concentration-dependent
manner (Figure 4B). In the culture medium collected after treatment
with 100 µg/mL Ox-LDL, the VEGF concentration was 2.8-fold higher
than that of control (Figure 4B).
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Effect of LPC on VEGF Expression
To examine whether LPC, a component of Ox-LDL, might have an
effect on VEGF production and VEGF mRNA expression in RAW 264
cells, the cells were exposed to LPC between 5 and 100 µmol/L.
As shown in Figure 5A, LPC upregulated VEGF
mRNA expression of RAW 264 cells, and a maximal effect was observed at
a concentration of 10 µmol/L, whereas no stimulatory effect was
observed at higher concentrations. An increase in VEGF protein was also
detected in conditioned medium from RAW 264 cells exposed to LPC for 24
hours (Figure 5B). Concentration-response experiments demonstrated that
a maximal effect of LPC on VEGF production was obtained at a
concentration of 10 µmol/L, consistent with the result
of mRNA expression (Figure 5B).
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Presence of VEGF in Atherosclerotic Lesions
We next used immunohistochemical techniques to examine whether
VEGF was really present in macrophage-rich regions in human
atherosclerotic lesions. In nonatherosclerotic arteries composed of the
media and a zone of intimal thickening, only weak, positive staining
for VEGF in the intima was observed in 8 specimens from 17 normal
arteries. Weak, positive staining for VEGF was also obtained
consistently in the adventitia, including microvascular SMCs of
the vasa vasorum and adventitial fibroblasts, which were negative for
SMC staining. The inner media of normal arteries had no VEGF staining
(data not shown).
In contrast to nonatherosclerotic arteries, strong staining for VEGF
was observed in the subendothelial region of thickened
intimas of early atherosclerotic lesions (fatty streaks). Of 23 artery
specimens, 15 regions contained this stage of lesion, of which 12
(80%) stained positively for VEGF. Figure 6A and 6B illustrates representative positive specimens of
an early atherosclerotic lesion. Staining of serial sections indicated
that the predominant cell type in these areas was the
macrophage, as assessed by staining with a monoclonal
macrophage antibody (Figure 6C). No staining for SMCs was
observed in these areas (data not shown). In this stage of lesion, no
von Willebrand factor–positive cells were observed in the
thickened intima or inner media.
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In the atheromatous plaque, strong, VEGF-positive
regions were detected (Figure 7A and 7B). The
intense staining for VEGF was observed in atheromatous
cores of lesions, consisting of lipid-filled macrophages
beneath a fibrous cap (13 of 13 lesions), as indicated by
immunostaining of serial section with an
anti-macrophage antibody (Figure 7C). High-power-field
observation of macrophage-rich regions revealed that VEGF was
localized in the extracellular space as well as on the
macrophage cell surface (Figure 7D). The basal lesions of
plaques also stained for VEGF (9 of 13 lesions) (Figure 7B and 7E).
These regions contained a predominant population of SMCs, as suggested
by staining of serial section for the SMC marker (Figure 7F). A
high-magnification view of this lesion shows the extracellular
localization of VEGF (Figure 7G). The patchy staining for VEGF was also
frequently observed in acellular or cell-sparse core lesions (12 of 13
lesions) containing some macrophage foam cells (Figure 7H and 7I). In these cell-sparse regions, macrophage foam cells were
found as isolated cells and sometimes as small clusters of cells
(Figure 7I). As shown in Figure 7J, VEGF-positive staining in this
region was observed in the extracellular space as well as on the
macrophage cell surface.
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Microvessels were found at the base of the plaque near the junction of
the intima and media. Microvessels at the regions were often seen
within or adjacent to VEGF-positive areas (Figure 8A and 8B).
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Discussion |
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Also known as vascular permeability factor, VEGF is structurally related to platelet-derived growth factor29 30 and possesses a unique, target-cell specificity for vascular ECs.12 13 This EC growth factor is a heparin-binding growth factor that is angiogenic in vitro31 32 and in vivo.17 33 In the current study we demonstrated that Ox-LDL induced the expression of VEGF mRNA in RAW 264 cells and augmented the level of VEGF protein in supernatants of RAW 264 cells. Because no effect of native LDL on VEGF expression was observed, oxidative modification is necessary for this induction.
During oxidation of LDL, LPC is thought to be formed from PC by an intrinsic phospholipase A2.34 Recent reports have demonstrated that LPC has a predominant role in the function of Ox-LDL and that LPC indeed has been shown to accumulate in the atherosclerotic arterial wall.35 LPC impairs endothelial NO release,36 upregulates cell adhesion molecules,37 increases SMC proliferation,38 and acts as a monocyte chemoattractant.39 We demonstrated that LPC upregulated VEGF mRNA and induced VEGF production in macrophages, suggesting that the induction of VEGF expression in macrophages by Ox-LDL can be explained at least partly by LPC. However, our result that the maximal effect of LPC, which was obtained at 10 µmol/L, on both VEGF mRNA expression and VEGF protein production was less than that of Ox-LDL indicated that other components of Ox-LDL may also contribute to the induction. In fact, Ruef et al40 recently reported that 4-hydroxynonenal, another component of Ox-LDL, increases VEGF secretion by SMCs.
We have also demonstrated the presence and distribution of VEGF in human atherosclerotic arteries. In nonatherosclerotic arteries only a weak, positive staining for VEGF was observed in the intima of some sections and in the adventitia. In the early atherosclerotic lesion (fatty streak), VEGF staining was frequently observed in subendothelial macrophage-rich regions, indicating the possible role of macrophages as the source of VEGF.
Intense VEGF staining was observed in atherosclerotic plaques. Our observations that the plaque regions with abundant VEGF contained numerous macrophages or SMCs, together with in vitro findings that SMCs and macrophages have the capability to produce VEGF in response to biological stimuli, suggest that macrophages and SMCs appear to represent the major source of VEGF in atheromatous lesions. In addition, VEGF was also frequently present in acellular or cell-sparse lipid-core lesions.
High-power-field observations suggest that VEGF is localized mainly in the extracellular space surrounding the SMCs or macrophage-derived foam cells. In addition, VEGF staining was also observed on macrophage cell surfaces. These findings agree with a previous report that the predominant isoform, VEGF165, as well as VEGF189, binds to heparin proteoglycans distributed on cellular surfaces and within extracellular matrixes.41 Couffinhal and colleagues42 recently reported that extensive VEGF was observed in human atherosclerotic lesions and that activated T lymphocytes were positive for VEGF in the lesions. However, they failed to identify VEGF localization in macrophages.42 Although we do not know the reason for the discrepancy between our results and those of Couffinhal et al, Inoue et al43 recently observed VEGF localization in accumulated macrophages in human atherosclerotic lesions.
Because in the current study we did not confirm the presence and
distribution of Ox-LDL in atherosclerotic lesions, the involvement of
Ox-LDL in VEGF induction in atherosclerotic lesions remains unknown.
However, a number of reports have already provided the evidence that
Ox-LDL is really present and is localized in foam cell–rich
regions,4 5 indicating that Ox-LDL is a possible
candidate as a VEGF inducer in vivo. However, we cannot exclude the
possibility that cytokines or growth factors in the lesion may
also upregulate macrophage and SMC VEGF expression, because
various cytokines and growth factors increase VEGF expression
in SMCs,20 21 22 23 24 and inflammatory cytokines
such as interleukin-1ß and tumor necrosis factor- upregulate
macrophage VEGF expression (M.A. Ramos et al, unpublished
observations, 1997).
Although we do not know the exact role of VEGF in atherosclerotic lesions, the absence of microvessels in fatty streak lesions as well as VEGF localization adjacent to ECs at the luminal surface suggest that VEGF in subendothelial macrophage-rich regions participates in the maintenance and repair of the lumen endothelium, since the EC turnover rate increases in these atherosclerosis-prone areas of arteries.44 It is also well known that endothelium permeability increases in atherosclerotic arteries.45 VEGF in these regions subjacent to the lumen may also contribute to the increase in vascular permeability through the endothelial layer, since VEGF enhances vascular permeability.14 15
Recently it has been reported that VEGF can induce monocyte chemotaxis through interaction with its receptor.19 46 Our observation of the VEGF accumulation in macrophage-rich areas, together with previous reports,19 46 raises the possibility that VEGF may act as a chemoattractant for monocytes/macrophages by accumulating monocytes/macrophages within atherosclerotic lesions in a paracrine or autocrine manner. The intima and inner media usually have no microvessels in the wall of normal, large human arteries, although the adventitia and outer media have a network of vasa vasorum. With development of the atherosclerotic plaque, microvessels appear in the thickened intima and media.47 Although little is known about the pathogenesis of neovascularization in the atherosclerotic plaque, it has been suggested that microvessels in atherosclerotic plaques may contribute to plaque evolution or complications via leakage of various plasma proteins48 and intraplaque hemorrhage.49 Because angiogenic factors are essential for the induction of neovascularization, the presence of angiogenic factors has been speculated in atherosclerotic lesions. In fact, fragments of atherosclerotic plaques have been shown to induce angiogenesis in the rabbit model of corneal transplantation.50
Our observation of a prominent association between VEGF staining and plaque regions rich in microvessels raises the possibility that this growth factor participates in neovascularization of atherosclerotic lesions. The FGF family, especially acidic FGF, has been proposed to be an angiogenic factor in atherosclerotic plaque, because acidic FGF colocalizes with microvessels in atherosclerotic plaque.51 In addition, Ananyeva et al52 recently demonstrated that Ox-LDL induced the release of acidic FGF from acidic FGF–transfected SMCs. VEGF in plaque may cause neovascularization in atherosclerotic lesions in combination with FGF, since these 2 growth factors have a synergistic effect on the induction of angiogenesis in vitro.31 32 Microvessels in atheroma have been suggested to be permeable to plasma proteins48 and prone to rupture.49 VEGF adjacent to microvessels might also contribute to the increased permeability of microvessels and the possibility of rupture.
In summary, we have demonstrated that Ox-LDL as well as LPC, a component of Ox-LDL, induces macrophage VEGF expression in vitro. We have also shown the presence and the variation in the distribution of VEGF in human arteries. Our findings may suggest the possible involvement of Ox-LDL in the development of human atherosclerosis through VEGF induction in macrophage.
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Acknowledgments |
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This work was supported by grants from the Sandoz Foundation for Gerontology Research, Suzuken Memorial Foundation, and Kanae Foundation of Research for New Medicine (M.R.). We thank Norie Kametsuta, Yukako Asai, and Kaoru Kimura for their excellent technical assistance.
Received October 6, 1997; accepted February 19, 1998.
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