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Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia
Atherosclerosis 155 (2001) 439– 444 www.elsevier.com/locate/atherosclerosis
Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia Kazuhiko Nakazato a, Toshiyuki Ishibashi a, Kenji Nagata a, Yositane Seino a, Yoko Wada a, Takayuki Sakamoto a, Reiko Matsuoka a, Tamio Teramoto b, Masayuki Sekimata c, Yoshimi Homma c, Yukio Maruyama a,* a
First Department of Internal Medicine, Fukushima Medical Uni6ersity, 1 Hikarigaoaka, Fukushima960 -1295, Japan b Department of Internal Medicine, Teikyo Uni6ersity School of Medicine, Tokyo, Japan c Department of Biomolecular Sciences, Institute of Biomedical Sciences, Fukushima Medical Uni6ersity, Fukushima, Japan Received 7 October 1999; received in revised form 18 May 2000; accepted 17 June 2000
Abstract Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1b or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Very low density lipoprotein receptor; Monocytes; Hypoxia macrophages; Scavenger receptor
1. Introduction Atherosclerotic lesions are characterized by the accumulation of lipids and lipid-loaded foam cells derived from macrophages and smooth muscle cells [1]. The very low density lipoprotein (VLDL) receptor is one of the several receptors that may contribute to foam cell formation in atherosclerotic lesions for the following reasons. The VLDL receptor is not down-regulated by excess apoE-containing lipoproteins such as intermediate density lipoprotein (IDL) or b-migrating VLDL (b-VLDL) [2,3]. In 1996, we reported that plaque macrophages express the VLDL receptor [4]. From a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) study, it was also found that the levels of VLDL receptor mRNA, as well as the class A * Corresponding author. Tel: + 81-24-5482111 ext. 2300, 2307; fax: +81-24-5481821. E-mail address: [email protected] (Y. Maruyama).
macrophage scavenger receptors (SR-As) are very high in atherosclerotic lesions [5]. However, it is not clear whether the VLDL receptor is expressed in circulating human monocytes. Modified low density lipoproteins, proinflammatory cytokines, and chemotactic factors play a role in the atherogenesis of monocytes/macrophages in the vascular wall [1]. It has also been shown that the oxygen tension of the artery wall falls immediately after emerging on the vascular wall from the lumen and gradually rising through the artery wall towards the adventitia [6], suggesting that low oxygen tension may also contribute to the atherogenic functions of monocytes/ macrophages. In this study on RT-PCR and nucleotide sequencing after subcloning the PCR product, we demonstrate the expression of VLDL receptor mRNA in human monocytes and monocyte-derived macrophages obtained from fresh peripheral blood. We also examined the
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effects of oxidized LDL (oxLDL), interleukin-1b (IL1b), monocyte chemoattractant protein-1 (MCP-1) and hypoxia on the levels of VLDL receptor mRNA compared with the class A scavenger receptors (SR-As) and the LDL receptor in these cells.
2. Materials and methods
2.1. Preparation of human monocytes and macrophages Human monocytes from fresh peripheral blood were obtained from normal healthy volunteers. Mononuclear cells were isolated by centrifugation through FicollPaque (Amersham, Uppsala, Sweden) and were resuspended in RPMI-1640 medium (GIBCO BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS, GIBCO BRL). Cells were plated in 10-cm cell culture dishes and incubated in a humidified incubator at 37°C for 2 h to allow cell adherence. Nonadherent cells were removed by washing five times with pre-warmed serumfree RPMI 1640 medium, and the remaining adherent cells were suspended in cold medium using a scraper. They were characterized by flow cytometry using monoclonal antibodies against CD3, CD19, CD14, and CD36. Isolated cells were also stained with a-naphtyl-butyrate esterase. Monocytes were cultured in 100% humidity and an atmosphere of 20% O2 and 5% CO2 in air at 37°C. Fresh isolated monocytes were cultured for up to 7 more days to differentiate them into macrophages [7]. In some experiments, monocytes were cultured in an incubator that was continuously flushed with 2% O2, 5% CO2 and 93% N2 (TABAI ESPEC Co., Tokyo, Japan) for 18 h. Table 1 PCR primersa
Sense Antisense
LDL-R
Sense Antisense
SR-As
Sense Antisense
b-actin
Sense Antisense
2.2. RT-PCR and semiquantification of mRNA Semiquantitative RT-PCR was performed as described previously [9]. In brief, total RNA of monocytes or macrophages was prepared using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. The total RNA (2 mg) was reverse transcribed using a Superscript II preamplification system (GIBCO BRL). Transcribed cDNA was then used for PCR amplification to estimate the expression of VLDL receptor, LDL receptor, SR-As, and b-actin mRNAs. Specific sense and antisense primers for human VLDL receptor, LDL receptor, SR-As, and b-actin were synthesized according to the published cDNA sequences [2,10 –12]. The sequences of the oligonucleotides are shown in Table 1. The primers for the human VLDL receptor consisted of nucleotides 112 –404 which do not contain an O-linked sugar domain [2,13]. Therefore, both splicing variants can be detected with the primers. PCR was performed using the Premix Ex Taq System (Takara Shuzo Co. LTD., Otsu, Japan) and in the following conditions. Denaturation at 92°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s. VLDL receptor, LDL receptor, and SR-As were amplified by 30 cycles of PCR, and b-actin was amplified by 22 cycles. After defined cycles of PCR, the reaction mixture was electrophoresed on a 2% agarose gel in 0.5X TBE buffer. Amplified bands were detected by ethidium bromide staining and photographed under UV light (Polaroid 665 film). Amplified products were quantified relative to the respective b-actin signals. All the samples tested were negative for genomic DNA contamination.
2.3. Cloning and nucleotide sequencing Nucleotide sequences
VLDL-R
Viable cells were counted by trypan blue exclusion as previously described [8].
Product sizes
TTCCAGTGCACAAA 294bp TGGTCGCTGTA ATCACATCTCCAGG (No. 112–405) ACACTGGGATA TGCCAAGACGGGAA486bp ATGCATCTCCT GCTACTGTCCCCTT (No. 100–585) GGAACACGTAA GCTCCGAATCTGTG 435bp AAATTTGATGC AGGTACTTAGCTGC (No. 50–484) AGAAGAATGTC CTCTGACTAGGTGT 173bp CTGAGACAGTG GCAAAGAACACGG (No. 685–857) CTAAGTGTGCTG
a VLDL-R indicates very low density lipoprotein receptor; LDL-R, low density lipoprotein receptor; and SR-As, class A scavenger receptors.
To confirm the PCR product, we ligated the amplified PCR fragment of the VLDL receptor into pCRII-TOPO vector (Invitrogen Co., Carlsbad, CA, USA) and sequenced the PCR fragment using an ABI 310 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA).
2.4. Isolation and oxidation of LDL LDL and oxLDL were prepared as described previously [14]. Briefly, native LDL (density 1.019 –1.063 g/ml) was isolated from serum of fasting normolipidemic volunteers by sequential ultracentrifugation and was stored at 4°C in 0.3 mmol/l EDTA. OxLDL was prepared by incubating LDL for 24 h at 4°C in phosphatebuffered saline (PBS) containing 5 mmol/l CuSO4. It was then extensively dialyzed against PBS and sterilized by filtration.
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3.3. Comparison of expression of lipoprotein receptor mRNAs in monocytes and monocyte-deri6ed macrophages
Fig. 1. Detection of VLDL receptor, LDL receptor, and class A scavenger receptors mRNAs in human peripheral blood monocytes and monocyte-derived macrophages by RT-PCR. VLDLR indicates the VLDL receptor; LDLR, the LDL receptor; SR, the class A scavenger receptors; Mono, monocytes; and Mf, monocyte-derived macrophages. The data are representative of four similar experiments.
2.5. Cytokine and chemokine Recombinant human IL-1b was generously provided from Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan) and recombinant human MCP-1 was purchased from Pharma Biotechnologie (Hannover, Germany).
3. Results
3.1. Cell isolation and 6iability
We compared the expressions of lipoprotein receptor mRNAs in monocytes with those in monocyte-derived macrophages by semiquantitative RT-PCR. The VLDL receptor mRNA was also detected in monocyte-derived macrophages (Fig. 1). There was no significant difference between monocytes and macrophages in the levels of VLDL receptor mRNA as well as LDL receptor mRNA. However, the levels of SR-As were increased in differentiated macrophages compared with monocytes (Fig. 1).
3.4. Effects of oxLDL, IL-1i, and MCP-1 on the le6els of lipoprotein receptor mRNAs OxLDL, IL-1b, and MCP-1 modulate monocyte/ macrophage functions in the vascular wall and their critical role in atherosclerosis is well-established [1]. Therefore, we examined the effects of these compounds on the levels of lipoprotein receptor mRNAs in monocytes and macrophages. Fig. 2 shows the expression of lipoprotein receptor mRNAs in human monocytes after 18 hours incubation with or without oxLDL (0, 5, 25 or 100 mg/ml). The SR-As mRNA levels increased in a dose-dependent manner in response to oxLDL. In contrast, the levels of LDL receptor mRNA were decreased
Flow cytometry showed that almost all of the isolated cells had CD14 (94.393.1%) and CD 36 (97.29 2.1%) antigens of monocytes and negligible contamination with CD3 (0.490.3%) or CD19 (0.49 0.3%) after density centrifugation and adherence to plastic (n =6). Cytohistochemistry revealed that 98.59 1.0% of the cells stained positive with a-naphtyl-butyrate esterase (n= 6). The viability of isolated monocytes was 99.190.8%, whereas monocyte-derived macrophages had a viability of 95.592.2% (n= 6). These monocytes and macrophages were used for all the experiments.
3.2. Expression of VLDL receptor gene in human monocytes Fig. 1 shows the expression of VLDL receptor, LDL receptor, SR-As, and b-actin mRNAs as assessed by RT-PCR. We detected VLDL receptor mRNA in human peripheral blood monocytes. To confirm the PCR product, we checked the nucleotide sequence of the PCR product using an automatic DNA sequencer after subcloning and found that the sequence of the PCR product (No. 112-405) completely matched human VLDL receptor cDNA which was previously reported by Sakai et al. [2] (data not shown).
Fig. 2. Effect of oxidized LDL (oxLDL) on the levels of mRNAs of lipoprotein receptors as determined by semiquantitative RT-PCR. Monocytes were incubated with or without oxLDL (5, 25 or 100 mg/ml) for 18 h. There was no significant change in VLDL receptor mRNA levels in human monocytes in response to oxLDL. The SR-As mRNA levels increased in a dose-dependent manner in the presence of oxLDL. In contrast, the levels of LDL receptor mRNA were decreased by incubation with oxLDL. The data are representative of four similar experiments.
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Fig. 3. Effect of IL-1b or MCP-1 on the levels of mRNAs of lipoprotein receptors in human monocytes. Monocytes were incubated in the presence or absence of IL-1b (5 ng/ml) or MCP-1 (25 ng/ml) for 18 h. There were no significant changes in the level of VLDL receptor, LDL receptor and the SR-As mRNAs by treatment with IL-1b (A) or MCP-1 (B). The data are representative of four similar experiments.
by oxLDL. These results are consistent with previous reports [2,3,16,17]. On the other hand, there was no significant change in VLDL receptor mRNA levels in human monocytes incubated with oxLDL (Fig. 2) and the addition of oxLDL to monocyte-derived macrophages also did not change the levels of VLDL receptor mRNA significantly (data not shown). We next studied the effect of IL-1b or MCP-1 on the levels of lipoprotein receptor mRNAs in human monocytes. Monocytes were incubated in the presence or absence of IL-1b (5 ng/ml) or MCP-1 (25 ng/ml) for 18 h. There was no significant change in the levels of VLDL receptor, LDL receptor, or the SR-As mRNAs after treatment with IL-1b (Fig. 3A) or MCP-1 (Fig. 3B). Similar results were obtained with monocytederived macrophages incubated with IL-1b for 18 h (data not shown).
4. Discussion Plaque macrophages are believed to originate from circulating monocytes [1,18]. Earlier we reported the expression of VLDL receptor mRNA by macrophages in early and advanced atherosclerotic lesions [4] and, therefore, hypothesized that the VLDL receptor is present in circulating monocytes. Argraves et al. also demonstrated the expression of the VLDL receptor in atherosclerotic macrophages [19]. However, Webb et al. could not detect VLDL receptor mRNA in circulating
3.5. Effect of hypoxia on the le6els of lipoprotein receptor mRNAs Since oxygen tensions fall immediately in the subendothelial space compared with the lumen in atherosclerotic lesions [6], we examined the effect of hypoxia on the levels of lipoprotein receptor mRNAs in monocytes. As shown in Fig. 4, treatment with hypoxia of 2% O2 for 18 h increased the level of VLDL receptor mRNA in cultured monocytes, whereas there were no significant changes in the level of SR-As or LDL receptor mRNA. Although the levels of VLDL receptor mRNA in macrophages was increased by hypoxia, the response was less than that in monocytes (data not shown).
Fig. 4. Effect of hypoxia on the levels of lipoprotein receptor mRNAs in human monocytes. Monocytes were incubated in 20% O2 or 2% O2 for 18 h. The levels of VLDL receptor, LDL receptor and the SR-As mRNAs were assessed by RT-PCR. The data are representative of four similar experiments.
K. Nakazato et al. / Atherosclerosis 155 (2001) 439–444
human leukocytes and monocyte-derived macrophages in vitro [20]. There is no report of the expression of the VLDL receptor in normal human circulating monocytes and monocyte-derived macrophages, although the human monocytic leukemia cell lines THP-1 and HL-60 express VLDL receptor mRNA [2,3,21]. In the current study, we demonstrated that human monocytes and monocyte-derived macrophages from peripheral blood express VLDL receptor mRNA. A major reason for the different results might be the way monocytes were isolated. The previous report showed the result of Northern blot analysis using 2 mg polyA-rich RNA from circulating leukocytes, which appears to be contaminated with cells other than monocytes [20]. They also failed to detect the VLDL receptor mRNA by RT-PCR in monocyte-derived macrophages, but they did not report on the purity of their isolated monocytes and the viability of their macrophages. We clearly show that our isolated cells were monocytes and monocytederived macrophages and that the cells were viable and expressed VLDL receptor mRNA. Nevertheless, it would be better to show whether or not the expression of VLDL receptor mRNA leads to detectable antigen on the cell surfaces of circulating human monocytes and monocyte-derived macrophages. Several lipoprotein receptors including SR-As, CD36, and LDL receptor-related protein (LRP) may be involved in the cellular uptake of native or modified lipoproteins in atherosclerotic lesions [22,23]. Recently, high plasma levels of apoE-enriched b-VLDL, remnant lipoproteins, and lipoprotein(a) [Lp(a)] have been shown to be risk factors for coronary heart disease and stroke [24,25]. However, the metabolism of these lipoproteins in the vascular wall is not well understood. The VLDL receptor can mediate the uptake of bVLDL, chylomicron remnant, and Lp(a) [2,3,19,26]. Taken together with our previous in situ data [4], it would seem that the VLDL receptor may be one of the candidate receptors which contribute to lipid metabolism of these atherogenic lipoproteins in the vascular wall. Our earlier report showed that the VLDL receptor mRNA was expressed by macrophages in the subendothelial space of early atherosclerotic lesions, termed fatty streaks. The cells emerging to the subendothelium appear to be initial macrophages in the process of foam cell formation. OxLDL is a potent inducer of foam cell formation. IL-1b is a major proinflammatory cytokine in atherosclerotic lesions and MCP-1 is a chemotactic factor for monocytes. Their critical roles in the pathogenesis of atherosclerosis are well-established [1,15]. Therefore, we hypothesized that oxLDL, IL-1b and MCP-1 may contribute to the induction of VLDL receptor mRNA in macrophages. However, none of these agents increased the level of VLDL receptor mRNA. Our findings may reflect the pathophysiologi-
443
cal significance of the VLDL receptor since the levels of VLDL receptor mRNA were not changed by these atherogenesis-related agents. OxLDL up-regulated the levels of SR-As mRNA in monocytes, which may support the observation by Hiltunen et al. that SR-As mRNA was highly induced in atherosclerotic lesions of hypertriglyceridemic rabbits [5]. From this finding, however, neither they nor we could explain the reason for the increased expression of VLDL receptor mRNA in atherosclerotic lesions [4,5]. The expression of SR-As mRNA was higher in differentiated macrophages than in fresh monocytes in vitro, whereas the expression of VLDL receptor mRNA was unaffected by differentiation. These two receptors showed different responses to oxLDL and cell differentiation. Thus, our results demonstrate different characteristics of the SR-As and the VLDL receptor. The most notable finding in this study is that the levels of VLDL receptor mRNA were up-regulated by hypoxia in monocytes. When peripheral blood monocytes emerge in the subendothelial space from the lumen, oxygen tensions rapidly fall in the vascular wall [6]. Therefore, we investigated the effect of hypoxia on the levels of lipoprotein receptor mRNAs and found that only VLDL receptor mRNA increased among the lipoprotein receptors examined. Thus, the expression of VLDL receptor mRNA in macrophages of atherosclerotic lesions may be mediated in part by oxygen tension. However, it remains to be clarified whether the result obtained with the conditions we used (2% O2) is related directly to the in vivo situation. Nevertheless, since little is known about the metabolism of lipoproteins through lipoprotein receptors under hypoxia in the vascular wall, which is associated with the development of atherosclerosis, our novel finding may stimulate research in this field. In addition, VLDL receptor mRNA is not down regulated by sterol. Taken together, our results suggest that the receptor may play a role in foam cell formation in atherosclerotic lesions. Koong et al. recently showed that the LDL receptorrelated protein (LRP) gene is induced by hypoxia in squamous cell carcinoma cell lines [27], which also may support the expression of LRP mRNA by macrophages in atherosclerotic lesions [5,23]. Their result suggests a pathophysiological role for LPR in lipoprotein metabolism in hypoxic conditions. Further study is required to elucidate these points with respect to the VLDL receptor and LRP in atherogenesis. In conclusion, we demonstrate the expression of VLDL receptor mRNA in circulating human monocytes and monocyte-derived macrophages and its upregulation by hypoxia in monocytes. The VLDL receptor in monocytes and macrophages may play an important role in the metabolism of atherogenic lipoproteins in the vascular wall and the development of atherosclerosis.
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Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia Kazuhiko Nakazato a, Toshiyuki Ishibashi a, Kenji Nagata a, Yositane Seino a, Yoko Wada a, Takayuki Sakamoto a, Reiko Matsuoka a, Tamio Teramoto b, Masayuki Sekimata c, Yoshimi Homma c, Yukio Maruyama a,* a
First Department of Internal Medicine, Fukushima Medical Uni6ersity, 1 Hikarigaoaka, Fukushima960 -1295, Japan b Department of Internal Medicine, Teikyo Uni6ersity School of Medicine, Tokyo, Japan c Department of Biomolecular Sciences, Institute of Biomedical Sciences, Fukushima Medical Uni6ersity, Fukushima, Japan Received 7 October 1999; received in revised form 18 May 2000; accepted 17 June 2000
Abstract Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1b or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Very low density lipoprotein receptor; Monocytes; Hypoxia macrophages; Scavenger receptor
1. Introduction Atherosclerotic lesions are characterized by the accumulation of lipids and lipid-loaded foam cells derived from macrophages and smooth muscle cells [1]. The very low density lipoprotein (VLDL) receptor is one of the several receptors that may contribute to foam cell formation in atherosclerotic lesions for the following reasons. The VLDL receptor is not down-regulated by excess apoE-containing lipoproteins such as intermediate density lipoprotein (IDL) or b-migrating VLDL (b-VLDL) [2,3]. In 1996, we reported that plaque macrophages express the VLDL receptor [4]. From a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) study, it was also found that the levels of VLDL receptor mRNA, as well as the class A * Corresponding author. Tel: + 81-24-5482111 ext. 2300, 2307; fax: +81-24-5481821. E-mail address: [email protected] (Y. Maruyama).
macrophage scavenger receptors (SR-As) are very high in atherosclerotic lesions [5]. However, it is not clear whether the VLDL receptor is expressed in circulating human monocytes. Modified low density lipoproteins, proinflammatory cytokines, and chemotactic factors play a role in the atherogenesis of monocytes/macrophages in the vascular wall [1]. It has also been shown that the oxygen tension of the artery wall falls immediately after emerging on the vascular wall from the lumen and gradually rising through the artery wall towards the adventitia [6], suggesting that low oxygen tension may also contribute to the atherogenic functions of monocytes/ macrophages. In this study on RT-PCR and nucleotide sequencing after subcloning the PCR product, we demonstrate the expression of VLDL receptor mRNA in human monocytes and monocyte-derived macrophages obtained from fresh peripheral blood. We also examined the
0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 0 ) 0 0 5 8 0 - 3
K. Nakazato et al. / Atherosclerosis 155 (2001) 439–444
440
effects of oxidized LDL (oxLDL), interleukin-1b (IL1b), monocyte chemoattractant protein-1 (MCP-1) and hypoxia on the levels of VLDL receptor mRNA compared with the class A scavenger receptors (SR-As) and the LDL receptor in these cells.
2. Materials and methods
2.1. Preparation of human monocytes and macrophages Human monocytes from fresh peripheral blood were obtained from normal healthy volunteers. Mononuclear cells were isolated by centrifugation through FicollPaque (Amersham, Uppsala, Sweden) and were resuspended in RPMI-1640 medium (GIBCO BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS, GIBCO BRL). Cells were plated in 10-cm cell culture dishes and incubated in a humidified incubator at 37°C for 2 h to allow cell adherence. Nonadherent cells were removed by washing five times with pre-warmed serumfree RPMI 1640 medium, and the remaining adherent cells were suspended in cold medium using a scraper. They were characterized by flow cytometry using monoclonal antibodies against CD3, CD19, CD14, and CD36. Isolated cells were also stained with a-naphtyl-butyrate esterase. Monocytes were cultured in 100% humidity and an atmosphere of 20% O2 and 5% CO2 in air at 37°C. Fresh isolated monocytes were cultured for up to 7 more days to differentiate them into macrophages [7]. In some experiments, monocytes were cultured in an incubator that was continuously flushed with 2% O2, 5% CO2 and 93% N2 (TABAI ESPEC Co., Tokyo, Japan) for 18 h. Table 1 PCR primersa
Sense Antisense
LDL-R
Sense Antisense
SR-As
Sense Antisense
b-actin
Sense Antisense
2.2. RT-PCR and semiquantification of mRNA Semiquantitative RT-PCR was performed as described previously [9]. In brief, total RNA of monocytes or macrophages was prepared using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. The total RNA (2 mg) was reverse transcribed using a Superscript II preamplification system (GIBCO BRL). Transcribed cDNA was then used for PCR amplification to estimate the expression of VLDL receptor, LDL receptor, SR-As, and b-actin mRNAs. Specific sense and antisense primers for human VLDL receptor, LDL receptor, SR-As, and b-actin were synthesized according to the published cDNA sequences [2,10 –12]. The sequences of the oligonucleotides are shown in Table 1. The primers for the human VLDL receptor consisted of nucleotides 112 –404 which do not contain an O-linked sugar domain [2,13]. Therefore, both splicing variants can be detected with the primers. PCR was performed using the Premix Ex Taq System (Takara Shuzo Co. LTD., Otsu, Japan) and in the following conditions. Denaturation at 92°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s. VLDL receptor, LDL receptor, and SR-As were amplified by 30 cycles of PCR, and b-actin was amplified by 22 cycles. After defined cycles of PCR, the reaction mixture was electrophoresed on a 2% agarose gel in 0.5X TBE buffer. Amplified bands were detected by ethidium bromide staining and photographed under UV light (Polaroid 665 film). Amplified products were quantified relative to the respective b-actin signals. All the samples tested were negative for genomic DNA contamination.
2.3. Cloning and nucleotide sequencing Nucleotide sequences
VLDL-R
Viable cells were counted by trypan blue exclusion as previously described [8].
Product sizes
TTCCAGTGCACAAA 294bp TGGTCGCTGTA ATCACATCTCCAGG (No. 112–405) ACACTGGGATA TGCCAAGACGGGAA486bp ATGCATCTCCT GCTACTGTCCCCTT (No. 100–585) GGAACACGTAA GCTCCGAATCTGTG 435bp AAATTTGATGC AGGTACTTAGCTGC (No. 50–484) AGAAGAATGTC CTCTGACTAGGTGT 173bp CTGAGACAGTG GCAAAGAACACGG (No. 685–857) CTAAGTGTGCTG
a VLDL-R indicates very low density lipoprotein receptor; LDL-R, low density lipoprotein receptor; and SR-As, class A scavenger receptors.
To confirm the PCR product, we ligated the amplified PCR fragment of the VLDL receptor into pCRII-TOPO vector (Invitrogen Co., Carlsbad, CA, USA) and sequenced the PCR fragment using an ABI 310 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA).
2.4. Isolation and oxidation of LDL LDL and oxLDL were prepared as described previously [14]. Briefly, native LDL (density 1.019 –1.063 g/ml) was isolated from serum of fasting normolipidemic volunteers by sequential ultracentrifugation and was stored at 4°C in 0.3 mmol/l EDTA. OxLDL was prepared by incubating LDL for 24 h at 4°C in phosphatebuffered saline (PBS) containing 5 mmol/l CuSO4. It was then extensively dialyzed against PBS and sterilized by filtration.
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3.3. Comparison of expression of lipoprotein receptor mRNAs in monocytes and monocyte-deri6ed macrophages
Fig. 1. Detection of VLDL receptor, LDL receptor, and class A scavenger receptors mRNAs in human peripheral blood monocytes and monocyte-derived macrophages by RT-PCR. VLDLR indicates the VLDL receptor; LDLR, the LDL receptor; SR, the class A scavenger receptors; Mono, monocytes; and Mf, monocyte-derived macrophages. The data are representative of four similar experiments.
2.5. Cytokine and chemokine Recombinant human IL-1b was generously provided from Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan) and recombinant human MCP-1 was purchased from Pharma Biotechnologie (Hannover, Germany).
3. Results
3.1. Cell isolation and 6iability
We compared the expressions of lipoprotein receptor mRNAs in monocytes with those in monocyte-derived macrophages by semiquantitative RT-PCR. The VLDL receptor mRNA was also detected in monocyte-derived macrophages (Fig. 1). There was no significant difference between monocytes and macrophages in the levels of VLDL receptor mRNA as well as LDL receptor mRNA. However, the levels of SR-As were increased in differentiated macrophages compared with monocytes (Fig. 1).
3.4. Effects of oxLDL, IL-1i, and MCP-1 on the le6els of lipoprotein receptor mRNAs OxLDL, IL-1b, and MCP-1 modulate monocyte/ macrophage functions in the vascular wall and their critical role in atherosclerosis is well-established [1]. Therefore, we examined the effects of these compounds on the levels of lipoprotein receptor mRNAs in monocytes and macrophages. Fig. 2 shows the expression of lipoprotein receptor mRNAs in human monocytes after 18 hours incubation with or without oxLDL (0, 5, 25 or 100 mg/ml). The SR-As mRNA levels increased in a dose-dependent manner in response to oxLDL. In contrast, the levels of LDL receptor mRNA were decreased
Flow cytometry showed that almost all of the isolated cells had CD14 (94.393.1%) and CD 36 (97.29 2.1%) antigens of monocytes and negligible contamination with CD3 (0.490.3%) or CD19 (0.49 0.3%) after density centrifugation and adherence to plastic (n =6). Cytohistochemistry revealed that 98.59 1.0% of the cells stained positive with a-naphtyl-butyrate esterase (n= 6). The viability of isolated monocytes was 99.190.8%, whereas monocyte-derived macrophages had a viability of 95.592.2% (n= 6). These monocytes and macrophages were used for all the experiments.
3.2. Expression of VLDL receptor gene in human monocytes Fig. 1 shows the expression of VLDL receptor, LDL receptor, SR-As, and b-actin mRNAs as assessed by RT-PCR. We detected VLDL receptor mRNA in human peripheral blood monocytes. To confirm the PCR product, we checked the nucleotide sequence of the PCR product using an automatic DNA sequencer after subcloning and found that the sequence of the PCR product (No. 112-405) completely matched human VLDL receptor cDNA which was previously reported by Sakai et al. [2] (data not shown).
Fig. 2. Effect of oxidized LDL (oxLDL) on the levels of mRNAs of lipoprotein receptors as determined by semiquantitative RT-PCR. Monocytes were incubated with or without oxLDL (5, 25 or 100 mg/ml) for 18 h. There was no significant change in VLDL receptor mRNA levels in human monocytes in response to oxLDL. The SR-As mRNA levels increased in a dose-dependent manner in the presence of oxLDL. In contrast, the levels of LDL receptor mRNA were decreased by incubation with oxLDL. The data are representative of four similar experiments.
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Fig. 3. Effect of IL-1b or MCP-1 on the levels of mRNAs of lipoprotein receptors in human monocytes. Monocytes were incubated in the presence or absence of IL-1b (5 ng/ml) or MCP-1 (25 ng/ml) for 18 h. There were no significant changes in the level of VLDL receptor, LDL receptor and the SR-As mRNAs by treatment with IL-1b (A) or MCP-1 (B). The data are representative of four similar experiments.
by oxLDL. These results are consistent with previous reports [2,3,16,17]. On the other hand, there was no significant change in VLDL receptor mRNA levels in human monocytes incubated with oxLDL (Fig. 2) and the addition of oxLDL to monocyte-derived macrophages also did not change the levels of VLDL receptor mRNA significantly (data not shown). We next studied the effect of IL-1b or MCP-1 on the levels of lipoprotein receptor mRNAs in human monocytes. Monocytes were incubated in the presence or absence of IL-1b (5 ng/ml) or MCP-1 (25 ng/ml) for 18 h. There was no significant change in the levels of VLDL receptor, LDL receptor, or the SR-As mRNAs after treatment with IL-1b (Fig. 3A) or MCP-1 (Fig. 3B). Similar results were obtained with monocytederived macrophages incubated with IL-1b for 18 h (data not shown).
4. Discussion Plaque macrophages are believed to originate from circulating monocytes [1,18]. Earlier we reported the expression of VLDL receptor mRNA by macrophages in early and advanced atherosclerotic lesions [4] and, therefore, hypothesized that the VLDL receptor is present in circulating monocytes. Argraves et al. also demonstrated the expression of the VLDL receptor in atherosclerotic macrophages [19]. However, Webb et al. could not detect VLDL receptor mRNA in circulating
3.5. Effect of hypoxia on the le6els of lipoprotein receptor mRNAs Since oxygen tensions fall immediately in the subendothelial space compared with the lumen in atherosclerotic lesions [6], we examined the effect of hypoxia on the levels of lipoprotein receptor mRNAs in monocytes. As shown in Fig. 4, treatment with hypoxia of 2% O2 for 18 h increased the level of VLDL receptor mRNA in cultured monocytes, whereas there were no significant changes in the level of SR-As or LDL receptor mRNA. Although the levels of VLDL receptor mRNA in macrophages was increased by hypoxia, the response was less than that in monocytes (data not shown).
Fig. 4. Effect of hypoxia on the levels of lipoprotein receptor mRNAs in human monocytes. Monocytes were incubated in 20% O2 or 2% O2 for 18 h. The levels of VLDL receptor, LDL receptor and the SR-As mRNAs were assessed by RT-PCR. The data are representative of four similar experiments.
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human leukocytes and monocyte-derived macrophages in vitro [20]. There is no report of the expression of the VLDL receptor in normal human circulating monocytes and monocyte-derived macrophages, although the human monocytic leukemia cell lines THP-1 and HL-60 express VLDL receptor mRNA [2,3,21]. In the current study, we demonstrated that human monocytes and monocyte-derived macrophages from peripheral blood express VLDL receptor mRNA. A major reason for the different results might be the way monocytes were isolated. The previous report showed the result of Northern blot analysis using 2 mg polyA-rich RNA from circulating leukocytes, which appears to be contaminated with cells other than monocytes [20]. They also failed to detect the VLDL receptor mRNA by RT-PCR in monocyte-derived macrophages, but they did not report on the purity of their isolated monocytes and the viability of their macrophages. We clearly show that our isolated cells were monocytes and monocytederived macrophages and that the cells were viable and expressed VLDL receptor mRNA. Nevertheless, it would be better to show whether or not the expression of VLDL receptor mRNA leads to detectable antigen on the cell surfaces of circulating human monocytes and monocyte-derived macrophages. Several lipoprotein receptors including SR-As, CD36, and LDL receptor-related protein (LRP) may be involved in the cellular uptake of native or modified lipoproteins in atherosclerotic lesions [22,23]. Recently, high plasma levels of apoE-enriched b-VLDL, remnant lipoproteins, and lipoprotein(a) [Lp(a)] have been shown to be risk factors for coronary heart disease and stroke [24,25]. However, the metabolism of these lipoproteins in the vascular wall is not well understood. The VLDL receptor can mediate the uptake of bVLDL, chylomicron remnant, and Lp(a) [2,3,19,26]. Taken together with our previous in situ data [4], it would seem that the VLDL receptor may be one of the candidate receptors which contribute to lipid metabolism of these atherogenic lipoproteins in the vascular wall. Our earlier report showed that the VLDL receptor mRNA was expressed by macrophages in the subendothelial space of early atherosclerotic lesions, termed fatty streaks. The cells emerging to the subendothelium appear to be initial macrophages in the process of foam cell formation. OxLDL is a potent inducer of foam cell formation. IL-1b is a major proinflammatory cytokine in atherosclerotic lesions and MCP-1 is a chemotactic factor for monocytes. Their critical roles in the pathogenesis of atherosclerosis are well-established [1,15]. Therefore, we hypothesized that oxLDL, IL-1b and MCP-1 may contribute to the induction of VLDL receptor mRNA in macrophages. However, none of these agents increased the level of VLDL receptor mRNA. Our findings may reflect the pathophysiologi-
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cal significance of the VLDL receptor since the levels of VLDL receptor mRNA were not changed by these atherogenesis-related agents. OxLDL up-regulated the levels of SR-As mRNA in monocytes, which may support the observation by Hiltunen et al. that SR-As mRNA was highly induced in atherosclerotic lesions of hypertriglyceridemic rabbits [5]. From this finding, however, neither they nor we could explain the reason for the increased expression of VLDL receptor mRNA in atherosclerotic lesions [4,5]. The expression of SR-As mRNA was higher in differentiated macrophages than in fresh monocytes in vitro, whereas the expression of VLDL receptor mRNA was unaffected by differentiation. These two receptors showed different responses to oxLDL and cell differentiation. Thus, our results demonstrate different characteristics of the SR-As and the VLDL receptor. The most notable finding in this study is that the levels of VLDL receptor mRNA were up-regulated by hypoxia in monocytes. When peripheral blood monocytes emerge in the subendothelial space from the lumen, oxygen tensions rapidly fall in the vascular wall [6]. Therefore, we investigated the effect of hypoxia on the levels of lipoprotein receptor mRNAs and found that only VLDL receptor mRNA increased among the lipoprotein receptors examined. Thus, the expression of VLDL receptor mRNA in macrophages of atherosclerotic lesions may be mediated in part by oxygen tension. However, it remains to be clarified whether the result obtained with the conditions we used (2% O2) is related directly to the in vivo situation. Nevertheless, since little is known about the metabolism of lipoproteins through lipoprotein receptors under hypoxia in the vascular wall, which is associated with the development of atherosclerosis, our novel finding may stimulate research in this field. In addition, VLDL receptor mRNA is not down regulated by sterol. Taken together, our results suggest that the receptor may play a role in foam cell formation in atherosclerotic lesions. Koong et al. recently showed that the LDL receptorrelated protein (LRP) gene is induced by hypoxia in squamous cell carcinoma cell lines [27], which also may support the expression of LRP mRNA by macrophages in atherosclerotic lesions [5,23]. Their result suggests a pathophysiological role for LPR in lipoprotein metabolism in hypoxic conditions. Further study is required to elucidate these points with respect to the VLDL receptor and LRP in atherogenesis. In conclusion, we demonstrate the expression of VLDL receptor mRNA in circulating human monocytes and monocyte-derived macrophages and its upregulation by hypoxia in monocytes. The VLDL receptor in monocytes and macrophages may play an important role in the metabolism of atherogenic lipoproteins in the vascular wall and the development of atherosclerosis.
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