CD40L expression on human umbilical vein endothelial cells induced by oxidized low-density lipoprotein

Clinica Chimica Acta 370 (2006) 94 – 99 www.elsevier.com/locate/clinchim

Pioglitazone decreased CD40/CD40L expression on human umbilical vein endothelial cells induced by oxidized low-density lipoprotein De-qian Jiang ⁎, Luo-xiang Chu, Zhao-yun Liu, Shun-bao Zhang Department of Cardiology, The Second Xiangya Hospital of Central South University, Middle Ren-Min Road No. 86, ChangSha Hunan 410011, PR China Received 5 September 2005; received in revised form 21 January 2006; accepted 24 January 2006 Available online 20 March 2006

Abstract Background: The CD40/CD40 ligand pathway mediated inflammatory processes are important in atherogenesis and the formation of the intraplaque lipid pool. We tested the hypothesis that pioglitazone could decrease lectin-like oxLDL receptor-1 (LOX-1) and CD40/CD40L expression on human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (oxLDL). Methods: HUVECs were incubated with oxLDL for 24 h with or without pretreated by pioglitazone. Expression of CD40/CD40L on the cell surface was detected by flow cytometry. CD40/CD40L and LOX-1 mRNA expression were evaluated by RT-PCR. The expression of LOX-1 on HUVECs was determined by cell immunohistochemistry. Results: OxLDL increased the expression of CD40 and CD40L in a dose- and time-dependent manner. Pretreatment of HUVECs with pioglitazone (1 and 10 μmol/l) for 60 min decreased the expression of CD40 mRNA induced by oxLDL by 16% and 52%, respectively (both P < 0.05). Pretreatment of HUVECs with pioglitazone (1 and 10 μmol/l) for 60min decreased the expression of CD40L mRNA induced by oxLDL by 16% and 43% (both P < 0.05). Also, pretreatment of HUVECs with pioglitazone (1 and 10 μmol/l) for 60 min also significantly decreased CD40 and CD40L expression on HUVECs induced by oxLDL in a concentration-dependent manner. Pretreatment of HUVECs with pioglitazone (1 and 10 μmol/l) decreased oxLDL induced upregulation mRNA of LOX-1 by 11% and 28%, respectively. Furthermore, through immunohistochemistry, we found that pioglitazone could decrease the LOX-1 expression on HUVECs induced by oxLDL. Conclusion: Pioglitazone inhibited the upregulation of LOX-1 on HUVECs elicited by oxLDL and subsequently decreased HUVECs CD40/ CD40L expression induced by oxLDL. These observations provided novel insight into a potential novel anti-inflammatory pathway of thiazolidinediones. © 2006 Elsevier B.V. All rights reserved. Keywords: Pioglitazone; Human umbilical endothelial vein cells; Oxidized LDL; CD40/CD40L; LOX-1

1. Introduction Atherosclerosis is considered an immuno-inflammatory disease. Mounting evidence suggested a central role of the CD40–CD40L signaling pathway in the pathogenesis of this disease [1]. CD40 is a member of the TNF receptor family. In addition to antigen-presenting cells, expression of CD40 has been documented in a variety of non-immune cells including endothelial cells, fibroblasts and vascular smooth muscle cells. CD40L is a member of the TNF superfamily, which is expressed on T lymphocytes, activated platelets and smooth muscle cells

⁎ Corresponding author. Tel.: +86 0731 5550350. E-mail address: [email protected] (D. Jiang). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.01.026

[2]. CD40L can also be expressed on endothelial cells under certain proinflammatory conditions [3]. Recently, two reports demonstrated that hypercholesterolemia (HC), one of the major cardiovascular risk factors, increased CD40 and CD40L expression on platelets, endothelial cells and monocytes [4] as well as plasma levels of soluble CD40L [5]. In addition, it has been shown that blocking CD40–CD40L interactions significantly prevented the development of atherosclerotic plaques and reduced already pre-established lesions [6]. Both oxLDL and CD40/CD40L have been identified to colocalize in atherosclerotic plaques [7]. OxLDL has been implicated in the pathophysiology of atherosclerosis. Recent studies showed that LOX-1, a novel lectin-like receptor for oxLDL expressed primarily in endothelial cells, facilitated the uptake of oxLDL and mediated several

D. Jiang et al. / Clinica Chimica Acta 370 (2006) 94–99

of the biological effects of oxLDL, such as apoptosis in endothelial cells [8]. Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily of ligand-activated transcription factors[9]. PPAR and retinoid X receptor contain the heterodimer to bind regulatory elements in the promoter region of a number of adipocyte-specific genes and to stimulate transcription in response to PPARγ-specific and retinoid X receptor-specific ligands. Studies showed that PPARs might protect against generation and progression of atherosclerosis not only by modifying metabolic disorders but also by inhibiting inflammatory reaction in vasculature. The antidiabetic drug pioglitazone is a synthetic ligand for PPARγ. In the present study, we tested the hypothesis that oxidatively modified LDL induced the expression of CD40/CD40L on HUVECs. Pioglitazone was capable of interfering with oxLDL induced CD40/CD40L and LOX-1 expression on HUVECs, which illustrated a potential novel anti-inflammatory pathway of thiazolidinediones. 2. Methods The fourth generation HUVECs were incubated with oxLDL for different time periods and vary concentrations as indicated below to determine the expression of CD40, CD40L. To determine the effects of pioglitazone on the expression of CD40/CD40L, we pretreated HUVECs with pioglitazone (1 and 10 μmol/l) for 60 min and then the cells were exposed to oxLDL for 24 h. To explore the possible molecular basis of the effects of the pioglitazone, we also studied the expression of LOX-1. We pretreated HUVECs with and without pioglitazone (1 and 10 μmol/l) for 60 min, then exposed the cells to oxLDL (80 μg/ ml) for 24h, and then we determined the expression of LOX-1. Concentrations of all reagents and the duration of incubation were chosen on the basis of previous studies [10,11]: 80μg/ml oxLDL were incubated with HUVECs for 24h, and 1 and 10 μmol/l pioglitazone were used in this study. 2.1. Cell culture Human umbilical vein endothelial cells were isolated by collagenase (0.25 mg/ml) digestion from fresh umbilical cords obtained at normal deliveries, which was in accordance with the ethical standards formulated in the Helsinki Declaration. The HUVECs were cultured (37 °C, 5% CO2) in medium DMEM containing 10% fetal calf serum, 3.2mmol/l glutamine and 100 U/ml penicillin–streptomycin (primary culture medium). Twenty-four hours before an experiment, the complete medium was changed to serum-free DMEM. Experiments were performed in passage 4 and 70–80% confluent cells. 2.2. Preparation of lipoproteins Native LDL and oxLDL were prepared as described earlier [12]. Briefly, human native LDL was isolated from human blood plasma by discontinuous centrifugation. LDL was

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oxidized by exposure to 10 μmol/l CuSO4 phosphate-buffered saline (PBS) at 37°C for 24 h. The extent of oxidation was determined by measuring thiobarbituric reactive substances. LDL and oxLDL were kept in 50mmol/l Tris–HCl, 0.15 mol/ l NaCl and 2 mmol/l EDTA at pH 7.4 and were used within 10 days of preparation. 2.3. Flow cytometry The fourth generation HUVECs (1 × 106/ml) in all conditions were washed with ice-cold PBS, harvested by trypsinization and fixed (PBS/4% paraformaldehyde, 15 min). Subsequently, the cells were washed once with PBS/2% BSA before being incubated (30 min, 4°C) with either buffer alone or mouse antihuman CD40, or mouse anti-human CD40L antibody (1 μg/ml, Bioscience). Then the cells were washed twice with PBS/2% BSA, centrifuged at 800 rpm × 3 min before being incubated (15 min, 4°C) with FITC-conjugated second antibody (Fluorescein isothiocyanate rat anti-mouse IgG). Finally, cells were washed with PBS/2% BSA and analyzed in FACSort flow cytometer using ModFitLT soft. At least 10,000 cells per condition were analyzed. 2.4. Reverse transcription–polymerase chain reaction (RT-PCR) We used a semi-quantitation RT-PCR. Total RNA was isolated from HUVECs using Trizol reagent according to manufacturer's recommendations. RNA (1 μg) was reversetranscripted with Oligo dT and RevertAid™M-MuLV reverse transcriptase (both purchased from Fermentas) at 42 °C for 1 h. The primer sequences used for PCR were as follows: CD40: sense primer: 5′-TGCCAGCCAGGACAGAAACT-3′, antisense primer: 5′-GGGACCACAGACAACATCAG-3′; CD40L: sense primer 5′-CACAGCATGATCGAAACATACAACC-3′, antisense primer: 5′-ATCCTTCACAAAGCCTTCAAACTGG-3′; LOX-1: sense primer: 5′-TTACTCTCCATGGTGGTGCC-3, antisense primer: 5′-AGCTTCTTCTGCTTGTTGCC-3′; β-actin: sense primer: 5′-AACCGCGAGAAGATGACCCAGATCATGTTT-3′, antisense primer: 5′-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3′. PCRs were carried out in a 25μl reaction volume containing 1.5 μl of cDNA, 20pmol of each specific primer, 200 μmol/l dNTPs, 2mmol/ l MgCl2, 1 unit of Taq DNA Polymerase and 1× PCR buffer (Fermentas). After an initial denaturation for 4 min at 94 °C, each cycle consisted of denaturation at 94 °C for 1min, primer annealing at 55°C for 1 min and primer extension at 72 °C for 1min 20s. A final extension step at 72°C for 10min was performed. Semi-quantitative PCR studies using 25, 30, 35 and 40 cycles verified that the conditions used yielded PCR products within the exponential range of amplification and were optimized for signal to background ratios (30 cycles for CD40, 35 cycles for CD40L, 38 cycles for LOX-1). The sizes of PCR products were 444 bp for CD40, 297 bp for CD40L, 193 bp for LOX-1 and 350 bp for human β-actin. Eight microliters of each PCR products were subjected to 1.5% agarose gel electrophoresis containing ethidium bromide and were scanned

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and the F-test indicated the presence of significant differences. A P-value <0.05 was considered significant.

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3.1. Oxidized LDL and CD40/CD40L expression in HUVECs

Fig. 1. OxLDL and CD40 expression. Incubation of HUVECs with oxLDL (20 to 80μg/ml) for 24h increased expression of CD40 and CD40L in a dose-dependent manner (left). Upregulation of CD40 and CD40L by oxLDL (80μg/ml) occurred in a time-dependent manner (right). CD40 was detected by flow cytometric technology; MFI represents mean fluorescence intensity. Both panels are summary of data, which are presented as mean ± S.D. from three independent experiments.

Incubation of HUVECs with oxLDL (20 to 80 μg/ml) increased the expression of CD40 and CD40L (which was demonstrated by flow cytometric technology) in a dosedependent manner. Incubation of HUVECs with oxLDL (80 μg/ml) also increased the expression of CD40, CD40L in a time-dependent manner (Fig. 1).

by the Bio-Rad Gel Doc 2000 Imaging System and analyzed optical density through Quantity One 4.03 analysis software.

3.2. The effects of pioglitazone on the expression of CD40 and CD40L in HUVECs induced by oxLDL

2.5. Immunohistochemistry

Pretreatment of HUVECs with pioglitazone (1 and 10μmol/l) for 60 min decreased the expression of CD40 mRNA induced by oxLDL by 16% and 52%, respectively (both P < 0.05). Pretreatment of HUVECs with pioglitazone (1 and 10μmol/l) for 60 min decreased the expression of CD40L mRNA induced by oxLDL by 16% and 43% (both P < 0.05). A high concentration of pioglitazone (10 μmol/l) had a greater effect than the low concentration (1 μmol/l) (Fig. 2). In parallel experiments, incubation of HUVECs with pioglitazone (10 μmol/l) alone did not affect CD40 and CD40L mRNA expression. In addition to CD40 and CD40L mRNA expression, pretreatment of HUVECs with pioglitazone (1 and 10μmol/l) for 60min also decreased CD40 and CD40L expression on HUVECs induced by oxLDL in a concentration-dependent manner, which was demonstrated by flow cytometric technology (Table 1).

For immunohistochemical staining, HUVECs were cultured on chamber slides with or without pretreatment by pioglitazone (10 μmol/l) for 1h, then cells were stimulated with oxLDL (80 μg/ml) for 24h. The cells were fixed with 4% paraformaldehyde for 30 min and washed with PBS. Staining for LOX-1 was performed with a rabbit anti-human LOX-1 polyclonal antibody. After blocking with PBS containing 2% BSA (Sigma) for 20min, sections were incubated with the primary antibody diluted in PBS (1:200 dilution) for 1 h. Thereafter, the cells were incubated with peroxidase-Conjugated Affini Pure Goat AntiRabbit IgG (second antibody) for 30 min. Antibody binding was visualized by DAB Kit. 2.6. Data analysis All data represent the mean of several independently performed experiments. Data are presented as mean ± S.D. Statistical significance was determined in multiple comparisons among independent groups of data in which analysis of variance

3.3. Pioglitazone and the expression of LOX-1 Incubation of HUVECs with oxLDL (80 μg/ml) for 24 h increased the expression of LOX-1 mRNA (the expression of

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Fig. 2. The effects of pioglitazone on the expression of CD40/CD40L mRNA induced by oxLDL. Upregulation of CD40/CD40L mRNA by oxLDL (80μg/ml, 24 h) was significantly decreased by pioglitazone. CD40/CD40L mRNA band density (determined by RT-PCR) was normalized by β-actin. Left panel is representative of four separate experiments. Right panel is the summary of data (mean ± S.D.) from these four experiments.

D. Jiang et al. / Clinica Chimica Acta 370 (2006) 94–99 Table 1 Effect of pioglitazone on HUVECs CD40/CD40L expression Stimuli

HUVECs expression

Control Ox-LDL Ox-LDL + 10μmol/l Piog Ox-LDL + 1μmol/l Piog 10μmol/l Piog

CD40 (MFI)

CD40L (MFI)

9.37 ± 0.51 13.4 ± 0.49 a 9.92 ± 0.86 b 11.23 ± 0.98 b 9.14 ± 0.59

4.07 ± 0.18 7.27 ± 0.36 a 4.63 ± 0.24 b 5.53 ± 0.45 b 4.29 ± 0.31

Shown is the MFI of CD40/CD40L expression on HUVECs cultured with oxLDL (80μg /ml), OxLDL + 10μM pioglitazone, OxLDL + 1μM pioglitazone or 10 μM pioglitazone alone. CD40/CD40L MFI was determined by FACS analysis. Data are presented as mean ± S.D. from three separate experiments. a Represents P-value <0.01 compared with control group. b Represents P-value <0.05 compared with oxLDL group.

LOX-1 mRNA on HUVECs was detected by RT-PCR, P < 0.01 compared with the control). Pretreatment of HUVECs with pioglitazone (1 and 10 μmol/l) decreased oxLDL induced upregulation mRNA of LOX-1 by 11% and 28%, respectively, a high concentration of pioglitazone (10 μmol/l) had a more pronounced effect than the low concentration (1 μmol/l) (P < 0.05). In parallel experiments, incubation of HUVECs with pioglitazone (10 μmol/l) alone did not affect the expression of LOX-1 mRNA in cultured HUVECs (Fig. 3). We also detected HUVECs LOX-1 expression by cell immunohistochemistry. Untreated control cells had little positively expression of LOX-1. The cells were cultured with oxLDL (80 μg /ml) for 24 h showed intensively positively LOX-1 expression. HUVECs pretreated with pioglitazone (10 μmol/l) showed decreased positively LOX-1 expression when incubated by oxLDL (Fig. 4). 4. Discussion Current views regard atherosclerosis as a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation. When endothelial cells undergo inflammatory activation, the increased expression of selectins, VCAM-1, ICAM-1 promotes the adherence of monocytes. Adhesion molecule expression is induced by proinflammatory cytokines such as IL-1 and TNF-α, by oxLDL uptake via LOX1 and by CD40/CD40 ligand interaction [13–15]. There is ample evidence that oxLDL plays an important role in atherosclerosis and associated inflammatory reactions, in addition to promoting the formation of form cells in atheroscle-

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rotic plaques. OxLDL stimulates expression of proinflammatory signals including free radical generation, release of the cytokines TNF-α and interleukin-6, leukocyte chemokines (MCP-1 and interleukin-8) and angiotensin II, and the expression of adhesion molecules (ICAM-1, VCAM-1 and E-selectin) and upregulating tissue factor and plasminogen activator inhibitor-1 expression [16]. All of these contribute to the inflammatory process that may initiate or accelerate atherosclerosis. CD40 signaling plays a critical role in the initiation of the immune response by inducing inflammatory responses with secretion of proinflammatory responses cytokines and chemokines. These chemokines probably attract and direct T lymphocytes and macrophages to the atheroma, thus sustaining chronic inflammation. There are multiple lines of evidence supporting the view of atherosclerosis as a chronic inflammatory disease involving certain components of the immune system. Recently, enhanced expression of CD40 and CD40L has been reported in experimental and human atherosclerotic lesions [17,18] and the concentration of sCD40L are increased in the plasma of acute coronary syndrome patients [19], Patients with moderate hypercholesterolemia reveal an upregulated CD40/CD40L system that may contribute to the known proinflammatory/proatherogenic milieu found in these patients [4]. OxLDL provides an initial signal for the expression of the CD40/CD40L in atherosclerotic plaques and may initiate and augment progression of atherosclerosis. In the present study, we demonstrate that oxLDL upregulates the expression of CD40 and CD40L in cultured HUVECs. The effects of oxLDL on CD40 and CD40L expression are concentration and time dependent. Other studies have shown that activation of the CD40 and CD40L pathway in endothelial cells results in the upregulation of ICAM-1, VCAM-1 and E-selectin, which are associated with macrophage recruitment in the plaque, expansion of the lipid core, inhibition of collagen synthesis and degradation of the fibrous cap in vivo [20]. Activation of the CD40/CD40L system may create a proinflammatory and prothrombotic milieu, aggravating the development of atherosclerosis in moderate HC. The present study provides the direct link between oxLDL and the CD40/CD40L signaling pathway. These observations provide a clue how oxLDL promotes activation of endothelial cells and initiation of inflammatory pathways, because the upregulation of CD40/CD40L system is involved in the pathogenesis of atherosclerosis.

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Fig. 3. The effects of pioglitazone on the expression of HUVECs LOX-1 mRNA induced by oxLDL. Upregulation of LOX-1 mRNA by oxLDL (80 μg/ml, 24h) was significantly decreased by pioglitazone. LOX-1 mRNA band density (determined by RT-PCR) was normalized by β-actin. Left panel is representative of four separate experiments. Right panel is the summary of data (mean ± S.D.) from these four experiments.

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Fig. 4. Representative micrographs showing immunostaining of human LOX-1-1 on HUVECs. Cells were fixed with 4% paraformaldehyde and incubated with antibodies as described in Section 2. The untreated control cells had little positively expression of LOX-1 (A); oxLDL L (80 μg/ml, 24 h) showed intensively positively LOX-1 expression (B), pretreated with pioglitazone (10μM) showed decreased positively LOX-1 expression induced by oxLDL (C). Magnification ×400.

Several investigators [21,22] have demonstrated that the uptake of oxLDL by endothelial cells is mediated by LOX-1. Studies show that LOX-1 mediates oxLDL induced expression of adhesion molecules and MCP-1 and monocyte adhesion to endothelial cells [23]. The endothelial expression of LOX-1 is induced by several stimuli relevant to atherosclerosis, such as TNF-α and phorbol 12-myristrate 13-acetate (PMA). Also, oxygen radicals and fluid shear stress also induce endothelial LOX-1 expression. In the present study, we show that oxLDL also upregulated the expression of LOX-1, which are in agreement with other reports [24]. Several lines of evidence suggest that PPAR-γ may exert anti-inflammatory effects by negatively regulating the expression of proinflammatory genes. We found that the PPAR-γ activator pioglitazone inhibited the expression of CD40 and CD4OL induced by oxLDL in HUVECs. We also found that pioglitazone decreased the expression of LOX-1 elicited by oxLDL in a concentrationdependent manner, where a high concentration of pioglitazone would almost completely blocked the expression of LOX-1. Several studies have examined the effect of PPAR ligands on LOX-1 expression. Chiba et al. have shown that 15d-PGJ2, a PPAR-γ ligand, but not the PPAR-α ligands WY14643 and fenofibric acid, inhibits TNF-α induced LOX-1 expression in bovine aortic endothelial cells [25]. Jawahar et al. has demonstrated that the PPAR-γ ligand pioglitazone inhibits intracellular superoxide radical generation and subsequently expression of the redox-sensitive transcription factor. This results in the downregulation of LOX-1 in response to a number of proinflammatory and proatherosclerotic stimuli, such as oxLDL, Ang II and TNF-α [10]. A recent study has shown that oxLDL upregulated the expression of CD40 through its own endothelial receptor, LOX1, because a specific LOX-1-blocking antibody decreased the expression of CD40 in response to oxLDL in human coronary artery endothelial cells [11]. Taken together, these findings suggest that oxLDL through its receptor LOX-1 activates the CD40 signal pathway and pioglitazone inhibited the expression of CD40/CD40L through inhibiting LOX-1 expression in vascular endothelial cells. A work has shown that oxLDL causes injury to endothelial cells through activation of PKC [26]. Another study demonstrated that activation of PKC plays a critical role in oxLDL induced gene expression of CD40/CD40L, because the PKC inhibitor markedly reduced CD40/CD40L expression elicited by oxLDL. The LOX-1 antibody blocked PKC activation in

response to oxLDL [11]. Pioglitazone may also inhibit PKC activation, which is located downstream of LOX-1, and subsequently decrease CD40/CD40L expression induced by oxLDL. Actually, recently, a study found that ciglitazone, another PPAR-γ activator, reduced oxLDL induced CD40 expressions of dendritic cells [27]. Several studies showed that other PPARγ activators rosiglitazone and troglitazone can both diminish sCD40L levels in patients with type 2 diabetes and coronary artery disease [28,29]. In conclusion, this study shows that oxLDL induces gene expression of CD40/CD40L in HUVECs. The effect of oxLDL is mediated through its newly described receptor LOX-1. These observations indicate that inhibition of LOX-1 by pioglitazone may downregulate activation of the CD40/CD40L system, which may be effective in blocking inflammation-related vascular injury associated with oxLDL and inhibiting the development of atherosclerosis in moderate HC. References [1] Lutgens E, Daemen MJ. CD40–CD40L interactions in atherosclerosis. Trends Cardiovasc Med 2002;12:27–32. [2] Mach F, Schönbeck U, Suhkova G, et al. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40–CD40 ligand signaling in atherosclerosis. Proc Natl Acad Sci U S A 1997;94:1931–6. [3] Reul RM, Fang JC, Denton MD, et al. CD40 and CD40 ligand (CD154) are coexpressed on microvessels in vivo in human cardiac allograft rejection. Transplantation 1997;64:1765–74. [4] Garlichs CD, John S, Schmeisser A, et al. Upregulation of CD40 and CD40 ligand (CD154) in patients with moderate hypercholesterolemia. Circulation 2001;104:2395–400. [5] Cipollone F, Mezzetti A, Porreca E, et al. Association between enhanced soluble CD40L and prothrombotic state in hypercholesterolemia. Effects of statin therapy. Circulation 2002;106:399–402. [6] Schonbeck U, Sukhova GK, Schonbeck U, Mach F, Libby P. Inhibition of CD40 signaling limits evolution established atherosclerosis in mice. Proc Natl Acad Sci U S A 2000;97:7458–63. [7] Mach F, Schönbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 1998; 394:200–3. [8] Mehta JL, Li DY. Identification and autoregulation of receptor for OXLDL in cultured human coronary artery endothelial cells. Biochem Biophys Res Commun 1998;248:511–4. [9] Lemberger T, Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology. Annu Rev Cell Dev Biol 1996;12:335–63. [10] Mehta JL, Hu B, Chen J, Li D. Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular

D. Jiang et al. / Clinica Chimica Acta 370 (2006) 94–99

[11]

[12] [13]

[14]

[15]

[16] [17]

[18] [19]

[20]

superoxide radical generation. Arterioscler Thromb Vasc Biol 2003; 23:2203–8. Li D, Liu L, Chen H, Sawamura T, Mehta JL. LOX-1, an oxidized LDL endothelial receptor, induces CD40/CD40L signaling in human coronary artery endothelial cells. Arterioscler Thromb Vasc Biol 2003;23:816–21. Hessler JR, Morel DW, Lewis LJ, Chisolm GM. Lipoprotein oxidation and lipoprotein-induced cytotoxicity. Atherosclerosis 1983;3:215–22. Gearing AJ, Hemingway I, Pigott R, Hughes J, Rees AJ, Cashman SJ. ESelectin, ICAM-1, VCAM-1: pathological significance. Ann N Y Acad Sci 1992;667:324–31. Chen M, Masaki T, Sawamura T. LOX-1, the receptor for oxidized lowdensity lipoprotein identified from endothelial cells: implications in endothelial dysfunction and atherosclerosis. Pharmacol Ther 2002; 95:89–100. Yellin MJ, Brett J, Baum D, et al. Functional interactions of T cells with endothelial cells: the role of CD40L–CD40-mediated signals. J Exp Med 1995;182:1857–64. Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherogenesis. Free Radic Biol Med 1996;20:707–27. Hakkinen T, Karkola K, Yla-Herttuala S. Macrophages, smooth muscle cells, endothelial cells, and T-cells express CD40 and CD40L in fatty streaks and more advanced human atherosclerotic lesions: colocalization with epitopes of oxidized low-density lipoprotein, scavenger receptor, and CD16 (Fc gammaRIII). Virchows Arch 2000;437:396–405. Schönbeck U, Libby P. CD40 signaling and plaque instability. Circ Res 2001;89:1092–103. Peng DQ, Zhao SP, Li YF, Li J, Zhou HN. Elevated soluble CD40 ligand is related to the endothelial adhesion, molecules in patients with acute coronary syndrome. Clin Chim Acta 2002;319(1):19–26. Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995;91:2844–50.

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[21] Li DY, Zhang YC, Phillips MI, Sawamura T, Mehta JL. Upregulation of endothelial receptor for oxidized low-density lipoprotein (LOX-1) in cultured human coronary artery endothelial cells by angiotensin II type 1 receptor activation. Circ Res 1999;84:1043–9. [22] Kume N, Murase T, Moriwaki H, et al. Inducible expression of lectin-like oxidized LDL receptor-1 in vascular endothelial cells. Circ Res 1998; 83:322–7. [23] Li D, Mehta JL. Antisense to LOX-1 inhibits oxidized LDL-mediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells. Circulation 2000; 101:2889–95. [24] Mehta JL, Li DY. Identification, regulation and function of LOX-1, a novel receptor for ox-LDL. J Am Coll Cardiol 2002;39:1429–35. [25] Chiba Y, Ogita T, Ando K, Fujita T. PPARγ ligands inhibit TNF-α-induced LOX-1 expression in cultured endothelial cells. Biochem Biophys Res Commun 2001;286:541–6. [26] Li DY, Yang BC, Mehta JL. Ox-LDL induces apoptosis in human coronary artery endothelial cells: role of PKC, PTK, bcl-2, and Fas. Am J Physiol 1999;275:H568–76. [27] Luo Y, Liang C, Xu C, et al. Ciglitazone inhibits oxidized-low density lipoprotein induced immune maturation of dendritic cells. J Cardiovasc Pharmacol 2004;44(3):381–5. [28] Marx N, Imhof A, Froehlich J, et al. Effect of rosiglitazone treatment on soluble CD40L in patients with type 2 diabetes and coronary artery disease. Circulation 2003;107:1954–7. [29] Varo N, Vicent D, Libby P, et al. Elevated plasma levels of the atherogenic mediator soluble CD40 ligand in diabetic patients: a novel target of thiazolidinediones. Circulation 2003;107:2664–9.