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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1027.)
© 2000 American Heart Association, Inc.

Vascular Biology

Expression of Macrophage (M) Scavenger Receptor, CD36, in Cultured Human Aortic Smooth Muscle Cells in Association With Expression of Peroxisome Proliferator Activated Receptor-, Which Regulates Gain of M-Like Phenotype In Vitro, and Its Implication in Atherogenesis

Kengo Matsumoto; Ken-ichi Hirano; Shuichi Nozaki; Akiko Takamoto; Makoto Nishida; Yumiko Nakagawa-Toyama; Mohamed Yakub Janabi; Takeshi Ohya; Shizuya Yamashita; Yuji Matsuzawa

From the Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Suita, Osaka , Japan.

Correspondence to Ken-ichi Hirano, MD, PhD, Department of Internal Medicine and Molecular Science, Graduate School of Medicine, B5, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail khirano{at}kb3.so-net.ne.jp

	Abstract

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Abstract—CD36 is one of the major receptors for oxidized low density lipoproteins belonging to macrophage (M) scavenger receptor (SR) class B and is thought to play an important role in the foam cell formation from monocyte-M in the atherosclerotic lesions. Although it has been hypothesized that smooth muscle cells (SMCs) may be the other origin of foam cells in vivo, supporting data are still very limited. In the present study, we have tested the expression of a variety of SRs, including CD36, in 8 lots of primary human aortic SMCs (HASMCs) explanted from 8 different donors. Functional CD36 was expressed in cultured HASMCs, and the levels of expression were widely ranged between the lots. SR class A (SR-A) was expressed abundantly in CD36-negative lots. Other M markers, such as CD32 and CD68, were expressed in all lots tested. These data suggest that the cultured HASMCs gained an M-like phenotype. To determine the mechanism for the above-described phenotypic change, we have tested the expression of a nuclear receptor, peroxisome proliferator activated receptor-, in those cells. This nuclear receptor was abundantly expressed in CD36-positive lots, whereas c-fms was expressed abundantly in CD36-negative/SR-A–positive lots. The synthetic ligand of peroxisome proliferator activated receptor-, troglitazone, upregulated the expression of CD36 only in CD36-positive lots. These observations demonstrate that cultured HASMCs can gain an M-like phenotype, possibly classified by the expression of CD36 or SR-A. The present study may support the possibilities of transformation of HASMCs into foam cells in vivo.

Key Words: CD36 • foam cells • oxidized LDL • peroxisome proliferator activated receptor- • scavenger receptors

	Introduction

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CD36 is an 88-kDa integral membrane glycoprotein that belongs to scavenger receptor (SR) class B (SR-B) and is expressed in many cell types, such as platelets, monocytes, and monocyte-derived macrophages (M).¹ Endemann et al² have reported that a murine homologue of human CD36 might be a receptor for oxidized LDLs (OxLDLs) in their experimental studies that made use of 293 cells overexpressing CD36. Genetic CD36 deficiency was found in 1990,³ and we have reported the molecular basis of this disorder by identifying 3 mutations in patients with CD36 deficiency.^{4 5 6} Using M from the patients, we have clarified that 1 of its major physiological functions is to act as a receptor for OxLDL, demonstrating that CD36 is responsible for 50% of the uptake of OxLDL.⁷ Recently, several laboratories, including ours, have reported that the in vitro expression of CD36 in human M is regulated by the addition of OxLDL and some cytokines.^{8 9 10} Moreover, a ligand for peroxisome proliferator activated receptor- (PPAR) upregulates the expression of CD36, subsequently causing the intracellular accumulation of lipids in human M.^{9 10} These observations suggest that CD36 may be important for the formation of foam cells in vivo.

Smooth muscle cells (SMCs) are 1 of the major components in atherosclerotic lesions. Previous studies have indicated that the change in SMCs from a differentiated contractile type to a dedifferentiated synthetic type is the critical phenotypic response for atherogenesis.^{11 12} After the phenotypic change, SMCs migrate into the intima and proliferate, which leads to the narrowing of the arterial lumen physically and the production of cytokines and proteinases, causing chemical modification of the lesions and the development of atherosclerosis.¹³ Another possible role of SMCs in atherogenesis has been postulated: SMCs can be transformed into foam cells under certain pathological conditions in vivo. However, supporting data are still very limited.¹⁴

In the present study, to determine whether human aortic SMCs (HASMCs) express the major receptor for OxLDL, CD36, we have screened its expression in HASMCs explanted from 8 different donors. We demonstrate that some cultured HASMCs express CD36 as a multifunctional receptor and that HASMCs can gain an M-like phenotype in vitro, and we discuss its implication in atherogenesis.

	Methods

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Materials
Human type-AB serum was purchased from ICN Biomedicals, Inc. Primary HASMCs and the growth media were purchased from Clonetics and Cascade. Information concerning the 8 donors who provided the serum (5 males and 3 females) are shown in the Table. The data were very limited because of privacy issues. The age of the donors ranged from 2.5 months to 29 years. All HASMCs were explanted from normal aortas without atherosclerosis. For immunohistochemical analysis, the following antibodies were used: (1) mouse monoclonal antibody against human CD36 (FA6-152, Cosmo Bio Co, Ltd), (2) mouse monoclonal antibody against human CD36 (OKM5, Ortho Diagnostic System Inc), (3) mouse monoclonal antibodies against CD11b, CD14, CD45RB, and CD68 (Dako, A/S) and against CD32 (Caltag), (4) nonimmune mouse IgG₁ (Ortho Diagnostic System Inc) and IgG_2a (Dako), and (5) rhodamine-conjugated phalloidin (Sigma Chemical Co). For the assay of long-chain fatty acids, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (Bodipy FL C16) was purchased from Molecular Probes. Troglitazone was kindly provided by Sankyo Pharmaceutical Co (Tokyo, Japan).

View this table: [in this window] [in a new window]   Table 1. Profiles of Donors of HASMCs

Cell Culture Experiments
Before the assay, HASMCs were grown to subconfluence in the growth medium according to the manufacturer’s protocol. We used HASMCs from passages 4 to 6 for all experiments. For the experiments assessing the effect of modified LDLs and troglitazone on CD36 expression, the growth medium was aspirated, and the cells were preincubated at 37°C for 16 hours in 2% FCS/DMEM; after which, the cells were incubated with 2% FCS/DMEM containing effectors as described in the figures. Monocyte-macrophages were prepared from the blood of healthy volunteers, as described previously.^{8 15}

FACS Analysis
To evaluate cell-surface expression of scavenger receptors, immunofluorescent flow cytometric analysis was performed with use of the specific antibodies. Subconfluent HASMCs were washed twice with PBS, gently scraped from the dish with a rubber scraper, and suspended in PBS (2x10⁵ cells in 100 µL). They were then incubated with FITC-conjugated FA6-152 antibody (final concentration 2.5 µg/mL) or FITC-conjugated mouse IgG₁ (final concentration 2.5 µg/mL) for 30 minutes at 4°C and immediately fixed with 1% paraformaldehyde for 30 minutes at 4°C. The cells were then washed twice with PBS and assayed by fluorescence-activated cell sorter (FACS) analysis (FACScan, Becton Dickinson and Co).

Immunofluorescence Microscopy
For CD36 immunofluorescence microscopy, cells were incubated in a 2-well glass chamber slide (Falcon), washed with PBS, fixed for 30 minutes with 4% paraformaldehyde, and washed with PBS again. The cells were then blocked with 5% normal horse serum at room temperature and incubated with FA6-152 diluted 1:200 in PBS containing 1% BSA for 1 hour, followed by sequential incubation with biotinylated horse anti-mouse IgG and avidin-FITC. After immunostaining with CD36, the cells were incubated with rhodamine-conjugated phalloidin to visualize F-actin. The cells were analyzed either by fluorescence microscopy (Provis Ax 80 TR, Olympus) or by confocal laser microscopy (Zeiss LSM 510, Carl Zeiss Co)

RT-PCR
Total RNA from HASMCs and M was prepared with Trizol reagent (GIBCO-BRL). To evaluate the expression of CD36, SR class A (SR-A), SR-B type I (SR-BI), c-fms, PPAR, and -actin, 2.0 µg of total RNA was subjected to Ready To Go reverse transcriptase (RT)–polymerase chain reaction (PCR) beading (Pharmacia Biotech). The DNA fragments of human CD36 cDNA (443 bp), human SR-A cDNA (330 bp), human SR-BI cDNA (696 bp), human c-fms cDNA (200 bp), human PPAR cDNA (353 bp), and -actin cDNA (146 bp) were amplified with the indicated primers. The PCR products were run on 2.0% agarose.

RNase Protection Assay
The partial cDNA encoding human CD36 was obtained by RT-PCR with use of human M RNA as a template (primers P13 and P14). Amplified cDNA fragments were subcloned into pGEM-T vector (Promega Co). RNase protection assay was performed as described previously.^{8 16}

Binding and Cellular Uptake of OxLDL
OxLDL was radioiodinated by using a iodine monochloride method as previously reported.⁷ Binding assays of ¹²⁵I-OxLDL (5 µg/mL) in HASMCs were performed at 4°C for 1 hour. The samples were incubated in the presence or absence of a 40-fold excess of unlabeled OxLDL or 4 µg/mL of OKM5. OKM5 (4 µg/mL) was ascertained to be adequate for saturation of the antibody. Radioactivity was measured by a gamma counter. The cholesterol ester accumulation study was performed as follows: HASMCs were cultured in 2% FCS/DMEM for 16 hours before the experiments, and OxLDL (30 µg/mL) was added to the medium; the cells were then incubated for 8 hours in the presence or absence of 4 µg/mL OKM5. The cellular lipids were extracted by incubating the cells with hexane/isopropanol (3:2 [vol/vol]) for 30 minutes at room temperature. An enzymatic fluorometric method was used to determine the cholesterol content of supernatant as described previously.¹⁷

Uptake of Fluorescent Long-Chain Fatty Acid Analogue: Bodipy-Labeled Fatty Acid
We used Bodipy FL C16 for long-chain fatty acid uptake by HASMCs. After preincubation in serum-free DMEM for 16 hours at 37°C, cells were washed twice with 0.1% BSA (fatty acid free) in PBS and incubated for 2 minutes at 37°C with PBS containing 20 µmol/L of Bodipy FL C16 and 1% BSA with or without a competitor. Cells were washed twice at 4°C with PBS containing 0.1% BSA to remove surface-associated Bodipy FL C16 and subjected to fluorescence microscopy.¹⁸

Primers
Primers used in the present study were as follows: P1, 5'-TGTAACCCAGGACGCTGAGGAC-3', human CD36 cDNA nucleotide (nt) 438 to nt 459 (GenBank accession No. L06850); P2, 5'-CTGTACCATTAATCATGTCGCAGTGAC-3', human CD36 cDNA nt 880 to nt 854 (GenBank accession No. L06850); P3, 5'-GCAGTTCTCATCCCTCTCATTGGA-3', human SR-A cDNA nt 230 to nt 253 (GenBank accession No. E05210); P4, 5'-ATTCCC- ATGTCCCTGGACTGAG-3', human SR-A cDNA nt 559 to nt 538 (GenBank accession No. E05210); P5, 5'-TGACCGGGTGGA- TGTCCAGGAAC-3', human SR-BI cDNA nt 1201 to nt 1179 (GenBank accession No. Z22555); P6, 5'-TGATGATGGAGA- ATAAGCCCAT-3', human SR-BI cDNA nt 506 to nt 527 (GenBank accession No. Z22555); P7, 5'-CGTAACGTGCTG- TTGACCAATGGT-3', human c-fms cDNA nt 2644 to nt 2667 (GenBank accession No. X03663); P8 5'-ATCTCCCAGAGG- AGGATGCC-3', human c-fms cDNA nt 2843 to nt 2824 (GenBank accession No. X03663); P9, 5'-GATGCAAGGGTTTCTTCC- GGAGAAC-3', human PPAR cDNA nt 631 to nt 655 (GenBank accession No. D83233); P10, 5'-TGGTGATTTGTCTGTTGT- CTTTCC-3', human PPAR cDNA nt 983 to nt 960 (GenBank accession No. D83233); P11, 5'-GACATCAGGAAGGACCTC- TATGCT-3', human -actin cDNA nt 915 to nt 938 (GenBank accession No. X13839); P12, 5'-GACAGAGTATTTGCGCTC- CGGA-3', human -actin cDNA nt 1060 to nt 1049 (GenBank accession No. X13839); P13, 5' GAATTCGTAACCCAGGA-CGCTG-3', human CD36 cDNA nt 439 to 454 (GenBank Accession No. L06850); and P14, 5'-GAAGCTTAATCATGTCGCAGTG-3', human CD36 cDNA nt 870 to nt 856 (GenBank accession No. L06850).

	Results

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We screened the expression of CD36 in the 8 lots of HASMCs from 8 different donors (listed in the Table) by RT-PCR. As shown in Figure 1A, CD36 mRNA was detectable with variable levels of expression in the cultured HASMCs; detection was confirmed by RNase protection assay (Figure 1B). FACS analysis with the anti-CD36 antibody, FA6-152, demonstrated the expression of CD36 immunoreactive mass, as shown in Figure 2. Immunofluorescence microscopy also showed that CD36 was detected mainly on the cell surface in CD36-positive, CD36 (+), cells (lots 1 and 6) (Figure 2D and 2E). Compared with CD36-negative, CD36 (-), HASMCs, CD36 (+) HASMCs appeared round in shape.

View larger version (45K): [in this window] [in a new window]   Figure 1. Expression of CD36 mRNA in HASMCs, determined by RT-PCR (A) and RNase protection assay (B). HASMCs were incubated to subconfluence in the growth medium. Human M was cultured as described in Methods. A, Total RNA (2 µg) was subjected to Ready to Go RT-PCR beading (Pharmacia Biotech). CD36 and -actin were amplified with primers P1 and P2 and primers P11 and P12, respectively. Cycle number was 30. B, Total RNA (5 µg) from HASMCs (lot 1) was hybridized with 32P-labeled cRNA probe for CD36 corresponding to nt 439 to nt 870 (GenBank accession No. L06850) and ß-actin overnight at 42°C, and the mixture was digested with a mixture of RNase A and RNase T1 (Ambion). After hybridization, the protected fragments were separated on an 8 mol/L urea/6% polyacrylamide gel. M mRNA served as a positive control for the expression of CD36.

View larger version (46K): [in this window] [in a new window]   Figure 2. Detection of CD36 protein in HASMCs by FACS analysis (A, B, and C) and confocal laser microscopy (D and E). A, B, and C, M (A), CD36 (+) HASMCs (lot 1, B), and CD36 (-) HASMCs (lot 4, C) were incubated with monoclonal antibody against human CD36, OKM5, or FITC-conjugated mouse IgG1 (final concentration 2.5 µg/mL) for 30 minutes at 4°C, immediately fixed with 1% paraformaldehyde for 30 minutes at 4°C, and then analyzed by FACS. M served as a positive control for expression of CD36. D and E, Cells were fixed and incubated for 1 hour with FA6-152 diluted 1:200 in PBS containing 1% BSA, followed by sequential incubation with biotinylated horse anti-mouse IgG and avidin-FITC (image II [D and E] ). After immunostaining of CD36, cells were incubated with rhodamine-conjugated phalloidin to visualize F-actin (image I [D and E]). Images D and E were acquired for each fluorescent probe by confocal laser microscopy as a single 1-µm-thick optical section by use of confocal laser microscopy (Zeiss LSM 510, magnification x200). Image III (D and E) shows color overlays of CD36 (green) and F-actin (red) patterns. Scale bar in the figure indicates 50 µm.

We have performed the following assays to determine the function of CD36 expressed in HASMCs. CD36 (+) cells (lots 1 and 6) would bind OxLDL and take up significant amounts of OxLDL-derived lipids, which were inhibited significantly by the specific neutralizing antibody, OKM5. They would also take up Bodipy FL C16, which was also blocked significantly by the antibody (data not shown). We examined the regulation of CD36 expression in HASMCs. OxLDL induced an 1.5-fold increase in CD36 protein on the cells, as we⁸ and others^{9 10} have reported in recent studies involving M (data not shown).

The above data clearly show that some HASMCs do express a functional CD36 molecule in vitro. However, previous immunohistochemical analyses, including ours, have not been able to demonstrate the immunoreactive mass of CD36 in SMCs in specimens in vivo, although we have been trying to detect the immunoreactive mass by using a variety of antibodies available. We speculated that the culture condition for HASMCs may be 1 of the keys for explaining the discrepancy between the in vitro and in vivo data. To maintain the cultured HASMCs, a variety of growth factors must be included, even though we know that some growth factors may induce phenotypic changes in SMCs, such as conversion from the contractile type into a synthetic type. Therefore, we have performed the following analyses to investigate whether the HASMCs tested gained an M-like phenotype in vitro.

We have examined the expression of other SR members as well as M-specific antigens commonly used as M markers.^{19 20} The patterns of SR expression were clearly different between the lots. As shown in Figure 3A, the most striking difference was the expression pattern of SR-A and CD36. SR-A was expressed exclusively in CD36 (-) lots (lots 2 and 4), whereas SR-A was not expressed in CD36 (+) lots (lot 1), suggesting that there may be a divergence of the expression of SR-A and CD36 in the cultured HASMCs as well as in foam cells in vivo, as reported by our group.²¹ On the other hand, all lots expressed another member of SR-B, SR-BI¹⁵ (Figure 3B), as well as CD68 and CD32, which are commonly used as M markers (Figure 3C). CD11b, CD14, and CD45RB were also detectable in all the lots (data not shown). These data show that cultured HASMCs apparently gained the M-like phenotype. To determine the mechanism for the above phenotypic change observed in cultured HASMCs, we tested the expression of a nuclear receptor, PPAR, in those cells. Our RT-PCR analyses showed that PPAR mRNA was expressed strongly in CD36 (+) lots (lot 1) and that mRNA of c-fms, a receptor for macrophage colony–stimulating factor, was abundantly detected in CD36 (-)/SR-A (+) lots (lots 2 and 4, Figure 3A); detection was confirmed by RNase protection assay (data not shown).

View larger version (32K): [in this window] [in a new window]   Figure 3. Expression of SR families, PPAR, macrophage-associated antigens, CD68 and CD32, and c-fms (A, B, and C) and effect of a ligand for PPAR on the expression of CD36 in HASMCs (D). Results for lots 1, 2, and 4 are shown in this figure. A and B, RT-PCR was used to assess the expression of human CD36, SR-A, c-fms, and PPAR (A) and SR-BI (B). Total RNA (2 µg) was reverse-transcribed and amplified with the following combinations of primers: P1 and P2 for CD36, P3 and P4 for SR-A, P5 and P6 for SR-BI, P7 and P8 for c-fms, P9 and P10 for human PPAR, and P11 and P12 for -actin. Cycle number was 30 for CD36, SR-A, SR-BI, c-fms, and PPAR; cycle number was 20 for -actin. C, FACS analyses are shown for CD68 and CD32. M (I), CD36 (+) HASMCs (lot 1, II), and CD36 (-) HASMCs (lot 4, III) were incubated with FITC-conjugated IgG1/FITC-conjugated CD68 or R-Phycoerythrin-IgG2a/R-Phycoerythrin-CD32 for 30 minutes at 4°C and analyzed by FACS. M served as a control for expression of CD68 and CD32 protein. D, HASMCs (lots 1 and 4) were incubated in the growth medium to subconfluent cultures. After preincubation with 2% FCS/DMEM for 16 hours, cells were incubated in medium containing indicated concentrations of troglitazone before FACS analyses to determine the expression of CD36.

We have tested the effect of troglitazone, a synthetic ligand of PPAR, on the expression of CD36 in CD36 (+) and CD36 (-) lots. As shown in Figure 3D, this compound induced up to a 2.2-fold expression of CD36 protein in a dose-dependent manner in CD36 (+) lots (lot 1), whereas it did not induce CD36 expression in CD36 (-) cells (lot 4). These results, at least, suggest that the expression of CD36 was regulated by PPAR in CD36 (+) HASMCs.

	Discussion

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The present study demonstrated for the first time that HASMCs express CD36 as a multifunctional receptor as well as other M-specific molecules in a culture condition, suggesting that cultured HASMCs can gain an M-like phenotype in vitro to possess the ability to transform into foam cells. We also found that there was such differential expression of SRs in those cultured HASMCs that some HASMCs (lots 1 and 6) expressed CD36, but not SR-A, and others (lots 2 and 4) expressed SR-A, but not CD36 (see Figure 3A). Furthermore, the expression of CD36 may be related to that of PPAR and that of SR-A may be related to c-fms in HASMCs.

What is the mechanism for the differential expression of SRs in cultured SMCs? As for M, it was reported that some THP-1 cells, human monocytic leukemia cell lines, were divided into 2 subtypes with and without expression of SR-A.²² Evans and colleagues^{9 10} reported an association with PPAR and CD36 in monocyte-M. Inaba et al²³ reported that some combinations of growth factors induced the expression of SR-A in human SMCs, in association with that of c-fms, a gene specific for monocyte-M, which encodes the receptor for macrophage colony–stimulating factor. In the present study, we have tried to induce the expression of CD36 in CD36 (-) lots (lots 2 and 4) and the expression of SR-A in SR-A (-) lots (lots 1 and 6) by various cytokines and growth factors; however, no interconversion between the HASMCs could be observed (K.M. et al, unpublished data, 1998). In addition, a ligand for PPAR did upregulate the CD36 in CD36 (+) lots but not in CD36 (-) lots. Judging from these data, we think that the most likely explanation for the differential expression is the clonal difference of HASMCs.

Many articles have been published showing that SMCs are originated from a couple of clones during embryogenesis and that those clones are distributed differently in blood vessels; these studies^{24 25} involved rodent SMCs. However, there are limited data concerning the lineage of SMCs and anatomic localization and distribution of SMCs from each clone in humans. We noticed that some lots (lot 8) expressed both CD36 and SR-A, suggesting that HASMCs in these lots may be derived from a couple of clones. However, we do not have the information concerning from what portions of the aorta the HASMCs were explanted because of privacy issues. To determine the molecular basis of the differential expression of SRs, it will be necessary to isolate a particular clone from the lots of HASMCs and to further analyze the biological nature in each potential subset.

What is the pathophysiological relevance of the phenotypic changes and the differential expression of SRs in HASMCs? In vitro findings in the present study were similar to our previous findings in foam cells in vivo; our recent immunohistochemical data demonstrated the differential localization of CD36 (+) and SR-A (+) foam cells in the atherosclerotic lesions of human aorta²¹ and coronary arteries (Y.N.-T. et al, unpublished data, 1999). In view of the fact that in vivo and in vitro data have shown that there might be 2 subsets of foam cells divided by the expression of CD36 or SR-A, our data support the possibility that SMCs are the other origin of foam cells in atherosclerotic lesions. Recently, it has been reported that PPAR is abundantly expressed in atherosclerotic lesions, which contained abundant oxidation-specific epitopes, such as malondialdehyde-lysine.²⁶ SMCs migrating from the media into the intima may be transformed into foam cells, which might be important in the development of atherosclerosis.

In conclusion, we have defined some biological and transforming features of cultured HASMCs. It is well known that SMCs are very heterogeneous and have abilities to change their own phenotype. Many studies have focused on the phenotypic changes, such as the conversion from the contractile type into synthetic type in vitro.¹¹ Because the SMCs used were obtained from rodents in most of those experiments, the present in vitro system that uses human HASMCs may be beneficial for studying the mechanism of transformation of human SMCs into foam cells and the changes in the expression of cell-specific antigens throughout the process. These are the issues for the future.

	Acknowledgments

 
This study was supported by research grants from the Study Group of Molecular Cardiology (Japan), the Japan Heart Foundation (Japan), the Osaka Heart Club (Japan), and Tanabe Medical Frontier Conference (Japan) to Dr Hirano. Dr Yamashita was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (No. 09671055). We are grateful to Eiko Okura-Okuda for skillful technical assistance.

Received October 5, 1999; accepted December 16, 1999.

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