ATP-binding cassette transporter G1 protects against endothelial dysfunction induced by high glucose

diabetes research and clinical practice 101 (2013) 72–80

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ATP-binding cassette transporter G1 protects against endothelial dysfunction induced by high glucose§ Jiahong Xue *, Congxia Wang, Canzhan Zhu, Yongqin Li Department of Cardiovascular Medicine, Second Affiliated Hospital of Medical School, Xi’an Jiaotong University, Xi’an, Shaanxi 710004, China

article info

abstract

Article history:

Aims: ATP binding cassette transporter G1 (ABCG1), a regulator of cholesterol efflux to HDL,

Received 1 February 2013

has been shown to decrease in macrophages and smooth muscle cells under high glucose

Received in revised form

conditions. Endothelial cells have a high capacity to efflux sterols and express ABCG1. In the

28 March 2013

present study we explored the role of ABCG1 in high glucose-induced endothelial dysfunc-

Accepted 25 April 2013

tion.

Published on line 18 May 2013

Methods: Human aortic endothelial cells (HAECs) were cultured under high glucose condi-

Keywords:

Western blot. Cholesterol efflux and NO synthesis (NOS) activity were determined by means

tions. ABCG1 mRNA and protein expression in HAECs were measured by real time PCR and ATP binding cassette transporter G1

of scintillation counting. Total intracellular cholesterol was determined by gas-liquid

Endothelial dysfunction

chromatography. The secretion of IL-6 and ICAM-1 was measured using ELISA. The genera-

High glucose

tion of intracellular reactive oxygen species (ROS) was measured using a fluorescence

Cholesterol efflux

microscope.

Oxidative stress

Results: We observed that high glucose suppressed ABCG1 expression and intracellular cholesterol efflux to HDL. Furthermore, high glucose increased the secretion of IL-6 and ICAM, as well as decreased phospho-eNOS protein expression and NOS activity. These processes were reversed by the up-regulation of ABCG1 using the liver X receptor (LXR) agonist T0901307 and an ABCG1 expression vector. In addition, high glucose-induced oxidative stress was reduced by the upregulation of ABCG1. In contrast, knock-down of ABCG1 in HAECs significantly increased the secretion of IL-6 and ICAM, as well as decreased phospho-eNOS protein expression and NOS activity. Conclusions: The present results suggest that ABCG1 plays an important role in protecting against endothelial dysfunction induced by high glucose. # 2013 Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

Endothelial dysfunction is a key feature of early atherosclerotic lesions in both humans and animal models. It is

characterized by decreased endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) bioavailability and increased expression of inflammatory chemokines and cytokines [1–3]. It has been reported that hyperglycemia

§ This work was supported by National Natural Science Foundation of China (81100210, 81273878); supported by the Doctoral Fund of the Ministry of Education of China (1896008); supported by the Special Fund for Scientific Research Personnel Training of Second Affiliated Hospital of Medical School, Xi’an Jiaotong University. * Corresponding author at: Department of Cardiovascular Medicine, Second Affiliated Hospital of Medical School, Xi’an Jiaotong University, 157 West Five Road, Xi’an, Shaanxi 710004, China. Tel.: +86 29 8767 9346; fax: +86 29 8767 9346. E-mail address: [email protected] (J. Xue). Abbreviations: ABCG1, ATP binding cassette transporter G1; HAECs, human aortic endothelial cells; NOS, nitric oxide synthase; eNOS, endothelial nitric oxide synthase; ROS, reactive oxygen species; LXR, liver X receptor; ICAM, intracellular adhesion molecules; HDL, high density lipoprotein; BSA, bovine serum albumin. 0168-8227/$ – see front matter # 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.diabres.2013.04.009

diabetes research and clinical practice 101 (2013) 72–80

contributes to defects in endothelial function [4,5]. Although it is known that this mechanism involves the overproduction of reactive oxygen species (ROS) [6,7], increased expression of proinflammatory chemokines and increased apoptosis [4,8], the exact mechanisms of high glucose-induced endothelial dysfunction is still unclear. ATP binding cassette transporter G1 (ABCG1) has been wellstudied in macrophages and has been shown to promote cholesterol efflux to high-density lipoprotein (HDL) particles in reverse cholesterol transport. Deficiency of ABCG1 in macrophages leads to reduced cholesterol efflux and increased macrophage cholesterol ester accumulation, promoting foam cell formation [9,10]. ABCG1 is also highly expressed in endothelial cells [11], and likely aids in cholesterol homeostasis to prevent endothelial activation in vessel walls [12,13]. Thus, ABCG1 may have an important function in endothelial function. It has been reported that high glucose can significantly reduce ABCG1 expression and function in both macrophages and smooth muscle cells, potentially contributing to accelerated formation of atherosclerotic lesions [14–16]. We speculated that endothelial dysfunction induced by high glucose may partially due to the reduced expression of ABCG1. Therefore, ABCG1 expression in endothelial cells may contribute to the prevention of endothelial dysfunction in diabetes.

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gel and then transferred onto nitrocellulose membranes using a Biorad transfer blotting system. The membranes were incubated with antibodies against ABCG1 (Proteintech, USA) and phospho-eNOS (S1177, BD Transduction Laboratories). Proteins were visualized using an enhanced chemiluminescence detection system (ECL, Cell Signaling Technology Inc.). Anti-b-actin (Santa Cruz, USA) was used to control for equal protein loading.

2.4.

Cholesterol efflux

2.

Methods

HAECs were plated in 12-well plates and radiolabeled for 48 h in serum-free medium containing 1 mCi/mL [3H] cholesterol (Sigma–Aldrich). After 48 h, cell layers were rinsed and treated with different concentrations of glucose for different times. Cholesterol efflux was conducted for 12 h at 37 8C in media containing: (1) 0.2% BSA; (2) 0.2% BSA + 15 mg/mL lipid-free human apoA1; or (3) 0.2% BSA + 50 mg/mL of human HDL. At the end of this incubation, the supernatant was collected and centrifuged at 13,000 rpm for 10 min to remove debris. Cells were lysed with 0.5 mL of 0.1 N NaOH. The radioactivity in both the supernatant and cellular lipid was measured by scintillation counting. Specific efflux to apoA-I or HDL was calculated by subtracting non-specific efflux in the presence of 0.2% BSA only. The data were normalized by total [3H]-cholesterol radioactivity in the supernatant and cell pellet.

2.1.

Cell culture

2.5.

Sterol mass analysis

Human aortic endothelial cells (HAECs, ScienCell) were cultured in Endothelial Cell Medium (ScienCell, No. 1001) in a CO2/O2 incubator at 37 8C. Cells were subcultured every 72 h. HAECs were incubated in 0.2% bovine serum albumin (BSA)/DMEM medium containing D-glucose of 5.6 mM (normal glucose) and 30.0 mM (high glucose) for 24–72 h. Where indicated, cells were pretreated for 24 h with the liver X receptor (LXR) agonist T0901307. Also, where indicated, HAECs were transfected with ABCG1 siRNA or ABCG1 expression plasmid. Twenty four hours after transfection, cells were cultured in 5.6 mM or 30.0 mM glucose. All experiments were repeated at least three times.

NO synthesis activity was determined by quantifying the rate of conversion of [3H]L-arginine to [3H]L-citrulline with kits obtained from Calbiochem-Novabiochem, San Diego, USA.

2.2.

2.7.

RNA extraction and quantitative real-time PCR

Total RNA and first-strand cDNA was produced using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas). Realtime quantitative PCR analysis was used to measure the relative levels of ABCG1 mRNA expression. The amplification was performed on a Biorad IQ5.0. The nucleotide sequences of the primers were as follows: ABCG1, forward primer 50 and reverse primer 50 GGTGATGCCGAGGTGAAC-30 0 CAATGTGCGAGGTGATGC-3 ; b-actin, forward primer 50 ATCGTGCGTGACATTAAGGAGAAG-30 and reverse primer 50 AGGAAGGAAGGCTGGAAGAGTG-30 . Levels of ABCG1 mRNA were subsequently normalized to b-actin mRNA levels.

2.3.

Western blot analysis

Whole cell lysates were generated with RIPA buffer. Equal amount of protein extracts were separated by 10% SDS-PAGE

The lipid fractions of HAECs were extracted using hexane/ isopropanol (3:2, v/v). Total intracellular cholesterol was determined by gas–liquid chromatography as previously described [12,17].

2.6.

NOS activity assay

Intracellular ROS

The generation of intracellular ROS was estimated by incubating cells with 6-carboxy-20,70-dichlorodihydrofluorescein diacetate,di(acetoxymethyl ester) (CDCFHDA-AM, 20 mM) for 20 min at room temperature in the dark as described previously [14,18]. CDCFH oxidation was measured with excitation at 488 nm and emission at 530 nm using a fluorescence microscope.

2.8.

Secretion of IL-6 and ICAM

Upon collection of the supernatant from endothelial cells, samples were centrifuged at 15,700  g for 10 min. The supernatants were kept at 80 8C until analysis. The secretion of IL-6 and ICAM-1 was measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (InvitrogenTM, Camairllo, USA).

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2.9.

diabetes research and clinical practice 101 (2013) 72–80

RNA interference

The following ABCG1 siRNA sequences were designed: forward, 50 -GAGUCUUUCUUCGGGAACATT-30 and reverse, 50 UGUUCCCGAAGAAAGACUCTT-30 (Shanghai GenePharma Co., Ltd.). siRNA for ABCG1 or random siRNA were transfected into HAECs using TurboFect siRNA Transfection Reagent (Fermentas) for 24 h. Then, the siRNA-targeted cells were subjected to various treatments.

2.10.

Plasmid

The OmicsLinkTM ORF Expression plasmid of ABCG1 (pReceiver-ABCG1 Expression vector) and the control vector with eGFP were obtained from GeneCopoeiaTM.

2.11.

Statistical analysis

The results were reported as means  standard deviation (SD) of at least three measurements. One-way analysis of variance (ANOVA) was used to compare the means, and the least significant difference (LSD) test showed the statistical significance of differences. Differences were considered significant at P < 0.05. All statistical analyses were performed with SPSS19.0.

3.

Results

3.1. High glucose conditions suppressed ABCG1 expression and decreased cholesterol efflux to HDL in cultured HAECs To determine the effect of high glucose conditions on endothelial ABCG1 expression, we measured the mRNA and protein expression of ABCG1 in cultured HAECs. The results indicated that conditions of high glucose levels decreased ABCG1 mRNA and protein expression in a time- and dosedependent manner (for reference see [19]). ABCG1 is the primary protein regulating cholesterol efflux to HDL, whereas lipid-free apoA-I is the preferred cholesterol acceptor from ABCA1, another ABC transporter [9,10]. Here, we examined the cholesterol efflux from HAECs to either lipidfree apoAI or to HDL. The results showed that under high glucose conditions, no change was observed in cholesterol efflux to lipid-free apoAI (data not shown), but cholesterol efflux to HDL was reduced. The maximal observed reduction was 33% in 30 mM glucose for 72 h when compared to controls cell in normal glucose medium (Fig. 1A). However, decreased cholesterol efflux to HDL was not accompanied by increased intracellular lipid loading. As shown in Fig. 1B, the intracellular total cholesterol content was only moderately but not significantly increased in 30.0 mM glucose when compared with normal glucose controls.

3.2. High glucose induced endothelial dysfunction, which was reversed by the up-regulation of ABCG1 Endothelial dysfunction is characterized by decreased eNOS activity and increased expression of proinflammatory

Fig. 1 – High glucose decreased cholesterol efflux to HDL in HAECs. HAECs were incubated for 24–72 h with 5.6 mM or 30.0 mM glucose. At the end of the incubation, cholesterol efflux to HDL was measured by scintillation counting (A). No significant differences in intracellular cholesterol content in cells exposed to high glucose and normal glucose conditions (B). Data represent means W SD (n = 3). **P < 0.001 vs. 5.6 mM glucose controls.

chemokines [1–3]. To further understand whether the down-regulation of ABCG1 induced by high glucose was associated with endothelial dysfunction, we measured the expression and activity of eNOS. As shown in Fig. 2A, we found that high glucose decreased phosphorylated eNOS protein expression when compared with HAECs exposed to normal glucose conditions. Furthermore, NOS activity in the high glucose group was decreased by 20–40% in a time dependent manner (Fig. 2B). However, when HAECs in high glucose were treated with the LXR agonist T0901307, phosphorylated eNOS protein expression and NOS activity were normalized. This was accompanied by increased ABCG1 expression and cholesterol efflux to HDL. Furthermore, phosphorylated eNOS protein expression and NOS activity were increased in HAECs

diabetes research and clinical practice 101 (2013) 72–80

after enhanced ABCG1 expression by transfection of an ABCG1 expression vector. A decrease in phosphorylated eNOS expression and NOS activity was also observed in HAECs deficient in ABCG1 (Fig. 3A–E). The current results suggest that ABCG1 plays an important role in endothelial cell biology.

3.3. Up-regulation of ABCG1 reversed the endothelial inflammatory state induced by high glucose To further understand whether the down-regulation of ABCG1 was associated with endothelial activation by high glucose, we

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measured the secretion of ICAM and IL-6 in the culture media. Untreated endothelial cells released very low levels of ICAM1and IL-6 into the media. A marked increase in the secretion of IL-6 and ICAM-1 (2- and 3-fold, respectively) was observed after 72 h when compared with cells exposed to normal glucose levels. This response was reversed by treatment with the LXR agonist T0901307. In addition, ABCG1 transfected cells did not increase the secretion of IL-6 and ICAM after exposure to high glucose levels, while an increase in IL-6 and ICAM secretion was observed in ABCG1-deficient HAECs (Fig. 4A and B). However, changes in vascular cell adhesion molecule-1 and E-selectin were not observed between the groups (data not shown).

3.4. High glucose-induced oxidative stress was reversed by the up-regulation of ABCG1 It is widely known that high glucose can promote oxidative stress in many cell types [4–6]. We observed that high glucose conditions promoted the generation of ROS in cultured HAECs (Fig. 5A and B). However, when HAECs were pretreated with the LXR agonist T0901307, intracellular ROS production was significantly reduced under high glucose conditions (Fig. 5D). Furthermore, ABCG1 overexpression, reduced ROS production in HAECs under high glucose conditions (Fig. 5F). Suppression of ABCG1 by siRNA technology increased ROS production, both under normal or high glucose conditions (Fig. 5G and H). The current results suggest that ABCG1 has an important role in decreasing oxidative stress in endothelial cells.

4.

Fig. 2 – High glucose decreased phosphorylated eNOS protein expression and NOS activity in HAECs. HAECs were incubated for 24–72 h with 5.6 mM or 30.0 mM glucose. At the end of the incubation, phosphorylated eNOS protein expression (A) and NOS activity (B) were measured by western blot and scintillation counting, respectively. **P < 0.001 vs. 5.6 mM glucose controls.

Discussion

Endothelial dysfunction is thought to be the major cause of vascular disease in hyperglycemia and diabetes [4,5]. ABCG1, a regulator of cholesterol efflux to HDL, is found to be expressed in the endothelium and its deficiency in endothelial cells promotes monocyte–endothelial interactions and vascular inflammation [11,12,20]. In addition, previous studies have shown that high glucose can inhibit ABCG1 expression and function in both macrophages and smooth muscle cells, potentially promoting significant amounts of cholesterol accumulation, as well as accelerated atherosclerosis [14–16]. We therefore investigated the role of ABCG1 in endothelial dysfunction induced by high glucose conditions. The findings of the current study showed that high glucose levels (30 mM) suppressed the expression of endothelial ABCG1 and intracellular cholesterol efflux to HDL. Furthermore, high glucose levels increased the secretion of IL-6 and ICAM, while decreasing phosphorylated eNOS expression and activity. Both of these responses were reversed by the up-regulation of ABCG1. This regulation was accompanied by increased cholesterol efflux to HDL. In addition, we observed that high glucose-induced oxidative stress was reduced by the upregulation of ABCG1. Taken together, these results suggest that ABCG1 provided endothelial protection against high glucose conditions. Members of the ABC transporter superfamily are known regulators of cholesterol efflux. The ‘half transporter’ ABCG1

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diabetes research and clinical practice 101 (2013) 72–80

Fig. 3 – Effect of ABCG1 on high glucose-induced phosphorylated eNOS expression and NOS activity in HAECs. (A) HAECs were transfected with scrambled siRNA, ABCG1 siRNA and either pcDNA (empty vector) or a pReceiver-ABCG1 expression vector. Western blotting for ABCG1 is shown. The image is representative of three experiments. (B) Phosphorylated eNOS protein expression. Decreased phosphorylated eNOS protein expression in 30.0 mM glucose-treated HAECs was reversed by pretreating HAECs with T0901307 and an ABCG1 expression vector. However, phosphorylated eNOS protein expression

diabetes research and clinical practice 101 (2013) 72–80

Fig. 4 – Increased secretion of IL-6 and ICAM following high glucose reversed by ABCG1 upregulation. (A) IL-6 production. (B) ICAM production. HAECs were pretreated with T0901307 and transfected with either ABCG1 siRNA or a pReceiver-ABCG1 expression vector for 24 h and then exposed to 5.6 mM or 30.0 mM glucose for 72 h. At the end of the incubation, supernatants were collected for measurement of endothelial IL-6 and ICAM secretion by ELISA. *P < 0.05, **P < 0.001 vs. HAECs cultured in 5.6 mM glucose; $P < 0.05, ##P < 0.001 vs. HAECs cultured in 30 mM glucose.

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can facilitate cholesterol efflux to HDL as an acceptor [9,10,21]. It has been demonstrated that ABCG1 plays a prominent role in regulating sterol homeostasis in endothelial cells [11–13,20]. In particular, ABCG1 knock-down in endothelial cells reduced the efflux capacity of cholesterol to HDL [12]. In the current study, we demonstrated that high glucose could inhibit ABCG1 expression and cholesterol efflux to HDL in HAECs. This is similar to previous reports on macrophages and smooth muscle cells [14–16], in which high glucose reduced the expression of ABCG1, but not ABCA1, which is a primary regulator of cholesterol efflux to apoA1. This has been shown to promote foam cell formation and atherosclerosis progression. However, we did not measure the expression of ABCA1. In our study, cholesterol efflux to lipid-free apoA-I was not changed, suggesting that the ABCA1 expression did not change in endothelial cells under high glucose conditions. In contrast to other cells that form atherosclerotic plaque (smooth muscle cells and macrophages), vascular endothelial cells do not accumulate cholesterol or transform into foam cells. Despite the fact that we observed decreased cholesterol efflux to HDL in endothelial cells under high glucose conditions, we did not observe a significant increase in intracellular cholesterol mass, as compared with control cells. This observation confirms a similar report by Hedrick et al. [20], in which the authors observed the redistribution of cholesterol esters into discrete punctuate subcellular structures. It has been postulated that vascular wall endothelial cells tightly regulate their sterol homeostasis by downregulating genes involved in cholesterol synthesis and the LDL receptor [13]. Endothelial dysfunction is thought to be an early feature of vascular disease. It is characterized by decreased eNOS activity and NO bioavailability [1–3]. Many studies have demonstrated that high glucose can interfere with eNOS expression and activity, as well as decrease the endothelial vascular relaxation response [4,5]. In the present study, we also found that high glucose decreased phosphorylated eNOS protein expression, the protein that has been reported to enhance eNOS activity and exert a protective effect in the endothelium [22]. However, up-regulation of ABCG1 by the LXR agonist T0901307 or by ABCG1 transfection significantly reversed the decrease in phosphorylated eNOS expression under high glucose conditions. This was also accompanied by increased cholesterol efflux to HDL. Furthermore, deficiency in ABCG1 obviously decreased phosphorylated eNOS expression and activity, suggesting a role for ABCG1 in promoting cholesterol efflux associated with endothelial function. This result confirms the study by Terasaka et al. [12], which reported that total eNOS and phosphorylated eNOS levels were moderately decreased in ABCG1 knockout mice on a

was reduced in ABCG1 siRNA HAECs cultured in 5.6 mM or 30.0 mM glucose. (C) NOS activity in HAECs. High glucoseinduced decreased NOS activity was reversed by pretreating HAECs with T0901307 and by overexpression of ABCG1. NOS activity was reduced following ABCG1 knock-down by siRNA in both 5.6 mM and 30.0 mM glucose concentrations. (D) ABCG1 mRNA expression. Decreased ABCG1 mRNA expression induced by 30.0 mM glucose was partly reversed by pretreating cells with T0901307 and ABCG1 overexpression. (E) Cholesterol efflux to HDL. High glucose-induced decrease of cholesterol efflux to HDL was partly reversed by pretreatment with T0901307 and by ABCG1 overexpression. Cholesterol efflux to HDL was decreased in ABCG1 deficient HAECs. Data are means W SD (n = 3). *P < 0.05, **P < 0.001 vs. HAECs cultured in 5.6 mM glucose; #P < 0.01, ##P < 0.001 vs. HAECs cultured in 30 mM glucose.

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Fig. 5 – High glucose-induced oxidative stress was reduced by the up-regulation of ABCG1. Intracellular ROS production was observed by fluorescence microscopy. (A) HAECs were cultured for 72 h with 5.6 mM glucose. (B) HAECs were cultured for 72 h with 30.0 mM glucose. (C) and (D) HAECs were pretreated with T0901307 and then cultured for 72 h with 5.6 mM or 30.0 mM glucose, respectively.(E) and (F) HAECs were transfected with a pReceiver-ABCG1 expression vector and then cultured for 72 h with 5.6 mM or 30.0 mM glucose. (G) and (H) HAECs were transfected with ABCG1 siRNA and then cultured for 72 h with 5.6 mM glucose or 30 mM glucose, respectively. Representative images are shown for three independent experiments (magnification 100T).

high-cholesterol diet. This study, as well as other studies, observed decreased cholesterol efflux to HDL and increased aortic accumulation of cholesterol and 7-oxysterols [12,18,23– 25]. T0901307, a pharmacological activator of liver X receptor, has been demonstrated to prevent atherosclerosis progression [26,27]. This associated with LXR agonist promoting effect on the expression of the ABC transporters ABCA1 and ABCG1, and the reverse cholesterol transport [26–28]. Here, we further demonstrated that T0901307 treatment prevented high glucose-induced endothelial dysfunction. This clearly indicates that ABCG1 is important for the protection of endothelial function and is associated with cholesterol efflux. In addition, we also showed that high glucose increased the secretion of inflammatory molecules such as ICAM and IL-6. This process was reversed following the up-regulation of ABCG1. Similarly, the study by Whetzel et al. [20] reported that aortic endothelial cells from ABCG1 knock-out mice increased their production of chemokines, such as IL-6 and MCP-1, and adhesion molecules, such as ICAM-1 and E-selectin. Restoration of ABCG1 expression in ABCG1 knock-out endothelial cells resulted in the reduction in these inflammatory chemokines. Furthermore, inflammatory gene expression increased in ABC transporter-deficient macrophages [29–31]. The elevation of proinflammatory cytokines in ABCG1 knock-out lung macrophages resulted in severe pulmonary lipidosis [29,30]. Moreover, peritoneal macrophages from ABCG1 knock-out mice increased their secretion of a variety of inflammatory cytokines (TNFa, IL-6, IL-1b and IL-12p70) and chemokines (MIP-1a, MIP-2, MCP-1), which was thought to be secondary to cholesterol accumulation [31]. ABCG1, therefore, appears to be a critical link between lipid homeostasis and inflammatory responses. Although we did not report a significant increase in the amount of intracellular cholesterol in high glucoseexposed endothelial cells, very small changes in cholesterol

content within specific cellular compartments can greatly influence cellular function [20]. Moreover, the increased expression of ICAM and IL-6 observed in the current study suggested there was increased monocyte recruitment in vessel walls under high glucose conditions. It is known that ICAM is important for regulating monocyte rolling and firm adhesion to the endothelial lining and that IL-6 is also involved in monocyte recruitment in the vessel wall [20]. Taken together, these data suggest that ABCG1 expressed in endothelial cells is associated with endothelial activation. Prolonged exposure of cells to high glucose in vitro or in vivo generates ROS [4]. There is considerable evidence that increased ROS can inhibit eNOS formation and increase the inflammatory state in diabetic mice [7,32,33]. In the present study, the upregulation of ABCG1 significantly inhibited ROS production induced by high glucose. However, knock-down of ABCG1 resulted in a significant increase in ROS production, suggesting that ABCG1 plays a key role in decreasing oxidative stress. This result was further supported by Terasaka et al. [12], who showed that ABCG1 decreased ROS production and preserved the active dimeric form of eNOS, which is associated with ABCG1mediated efflux of 7-oxysterols from endothelial cells to HDL. Therefore, ABCG1 may protect against endothelial dysfunction under conditions of high glucose. In conclusion, the present results indicate that ABCG1 plays a key role in preventing vascular inflammation and endothelial dysfunction induced by high glucose conditions. This may be attributable in part to the ABCG1-mediated delivery of cholesterol from endothelial cells to HDL.

Conflict of interest The authors declare that they have no competing interests.

diabetes research and clinical practice 101 (2013) 72–80

Acknowledgments This study was supported by National Natural Science Foundation of China (81100210, 81273878); supported by the Doctoral Fund of the Ministry of Education of China (1896008); supported by the Special Fund for Scientific Research Personnel Training of Second Affiliated Hospital of Medical School, Xi’an Jiaotong University.

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