- Email: [email protected]
Angiotensin II increases the cholesterol content of foam cells via down-regulating the expression of ATP-binding cassette transporter A1
Biochemical and Biophysical Research Communications 353 (2007) 650–654 www.elsevier.com/locate/ybbrc
Angiotensin II increases the cholesterol content of foam cells via down-regulating the expression of ATP-binding cassette transporter A1 Wang Yanfu a, Chen Zhijian a
b
a,*
, Liao Yuhua a, Mei Chunli a, Peng Hongyu a, Wang Min a, Guo Heping a, Lu Han b
Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tong Ji Medical College, Hua Zhong University of Science and Technology, 1277 Jie-Fang Avenue, Wuhan, 430022, People’s Republic of China Department of Geratology, Union Hospital, Tong Ji Medical College, Hua Zhong University of Science and Technology, 1277 Jie-Fang Avenue, Wuhan, 430022, People’s Republic of China Received 1 December 2006 Available online 20 December 2006
Abstract ATP-binding cassette transporter A1 (ABCA1) as one kind of membrane protein was found recently to play a major role in cholesterol homeostasis. Angiotensin II (AngII) has been shown to possess several atherogenic properties. The aim of the study is to investigate the influence of AngII on the expression of ABCA1 and the content of cholesterol in THP-1 derived foam cells. Our study showed that: (1) reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting demonstrated that AngII down-regulated the expression of ABCA1 in a dose-dependent manner. (2) The content of cholesterol was negatively correlated with ABCA1. The results suggest the promoting effects of AngII on the forming of foam cells are in a dose-dependent manner via down-regulating the expression of ABCA1. 2006 Elsevier Inc. All rights reserved. Keywords: Angiotensin II; ATP-binding cassette transporter A1; Atherosclerosis; Cholesterol content
Atherosclerosis (AS) is a common clinical fundamental disease. Patients with AS are confronted with lots of risks including hypertension, coronary artery disease (CAD), renal inadequacy etc. The detailed pathogenesy of AS is still undiscovered. ABCA1 as a critical gate-keeper was found recently at many tissues of human being [1], it’s the critical channel of cholesterol efflux in macrophages and conduces to scavenge redundant lipid, which protects against the formation of foam cells [2,3]. Mutations in the ABCA1 are associated with Tangier disease (TD) and a defect in cellular cholesterol efflux [4]. AngII possess several atherogenic properties including its ability to introduce the production of adhesion, promote SMC propagation, anti-fibrolysis and to attenuate the endothelial cell dependent vasodilatation, etc [5,6]. *
Corresponding author. Fax: +86 27 85727140. E-mail address: [email protected] (Z. Chen).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.067
Nevertheless, limited data are available on the relationship between AngII and ABCA1, which plays an essential role in cellular cholesterol efflux and helps to prevent macrophages from becoming foam cells. Thus, we hypothesize that AngII can increase cellular cholesterol content via down-regulating the expression of ABCA1. In the study, we examined the expression of ABCA1 and the cholesterol content of foam cells. We also investigated the relationship between cellular cholesterol content and ABCA1. Materials and methods LDL isolation and oxidization. LDLs (d = 1.0061.063 g/ml) were purified from human plasma obtained from healthy volunteers according to published standard protocols [7]. The preparation was performed in a Beckman L8-M ultracentrifuge (70 Ti rotor) at 4 C, and densities were adjusted with solid KBr. Lipoprotein fractions were dialyzed repeatedly in phosphate-buffered saline (PBS) containing 5 mM EDTA. After the final
Y. Wang et al. / Biochemical and Biophysical Research Communications 353 (2007) 650–654 dialysis step (0.15 M NaCl), LDL lipoproteins were sterilized using a 0.45 lm sterile filter (Sartorius). The isolated LDL was oxidized with CuSO4 10 lmol/L for 18 h at 37 C. Oxidation of LDL was measured by the thiobarbituric acid-reactive substances assay. Cell culture. THP-1 cells, a human monocytic leukemia cell line, were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cultured in RPMI 1640 medium (Sigma); supplemented with 10% (v/v) fetal calf serum (Gibco); 100 U of penicillin per ml, and 100 lg of streptomycin per ml; and a humidified atmosphere of 5% CO2 and 95% air at 37 C. To induce monocyte-to-macrophage differentiation, THP-1 cells were cultured in the presence of 160 nM phorbol 12-myristate 13-acetate (PMA). THP-1 derived macrophages were preincubated with anti CD36 (20 lg/ml, 37 C, 4 h). To induce foam cell formation of differentiated cells ox-LDL (100 lg/ml) with different concentration of AngII (0, 108, 107, 106 mM) was added for 48 h, respectively. Then the cells were washed once with PBS and incubated for an additional 12 h in serum-free media containing 0.2% BSA/RPMI 1640 and apolipoprotein A-I (apoA-I, 10 lg/ml). Before assay, cultured cells were harvested, washed once again with PBS. Electron microscopy. The cells were harvested and then fixed by 3% Glutaral for 3 h in 4 C. After being washed in 0.1 M cacodylic acid buffer solution five times for 4 h, the cells were fixed with 1% osmic acid for 30 min, washed in 0.1 M cacodylic acid buffer solution five times for 2 h. Then soaked into 50%, 70%, 80%, 90% alcohol for 10 min separately to be dewatered, then into 90% acetone for 10 min, and 100% acetone three times for 10 min. The cells were saturated in mixed liquor of acetone and extemporized epoxide resin embedding medium with the ratio of 1:1 for 1 h, and then in the 1:3 mixtures for 3 h and at last in pure embedding medium for 1 h. In the end, the cells were embedded in capsules. Next they were put in a thermostat oven at the temperature of 65–70 C overnight for the imbedding medium to polymerize. Then 50-nm extrathin sections were cut out. The sections were soaked in 3% uranyl acetate saturated solution heated by microwave for 30 s, then washed in buffer solution. After that, the sections were soaked in lead citrate solution heated by microwave for 20 s, and then washed in buffer solution. Last, the sections were dried in air for observation. RNA extraction and RT-PCR. Total RNA was extracted from the cultured cells using Trizol reagent according to the protocol provided by the manufacturer (Bio Basic Inc., Shanghai). The RNA was treated with DNase (Bio Basic Inc., Shanghai) before analysis by RT-PCR. Oligonucleotide primers were designed using Primer Express software (DNAStar) and were synthesized by Bio Basic Inc. Sequences of primers are as follows, using 5 0 -ACAACCAAACCTCACACTACTG-3 0 , and 5 0 -ATAG ATCCCATTACAGACAGCG-3 0 , for ABCA1, Primers for GAPDH were purchased from Bio Basic Inc. sequences are 5 0 -TCACCATCTTCC AGGAGCGAG-3 0 , and 5 0 -TGTCGCTGTTGAAGTCAGAG-3 0 . Firststrand cDNA was synthesized from the total RNA (2 lg) in a SuperScript preamplification system (Takara). The cDNA of ABCA1 was amplified by polymerase chain reaction with primers as described for 38 cycles using the Taq polymerase (Takara). DNA was visualized by ethidium bromide gel stain (UVP gel image analysis system) after 2% agarose gel electrophoresis. Protein extraction and Western blotting. The cells were harvested and suspended in 5 mM Tris–HCl buffer (pH 8.5) containing 1% protease inhibitor phenylmethyl sulfonylfluoride (Sigma) and placed on ice for 30 min with occasional mixing by a vortex. The cell suspension was centrifuged at 650g for 5 min, and then the supernatant was centrifuged at 105,000g for 30 min. Total membrane precipitated was suspended in the same buffer. After determination of protein content by a Bradford method, the membrane protein preparations were stored at 80 C until use. Total membrane protein (60 lg) was dissolved in 0.9 M urea, 0.2% (v/v) Triton X-100, and 0.1% (w/v) dithiothreitol and supplied with 10% (w/v) lithium dodecylsulfate and then analyzed by electrophoresis in 7% (w/v) polyacrylamide gel containing 0.1% (w/v) sodium dodecylsulfate (SDS) followed by blotting to a nitrocellulose membrane (Invitrogen). The membrane was blocked in 5% skim milk and incubated with rabbit antihuman ABCA1 antisera (1:200 dilution) [8] for 2 h. After washing three times with 0.02 M Tris-buffered saline containing 0.05% Triton X-100 (pH 7.5), the membrane was incubated with horseradish peroxidase-conjugated
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Table 1 Composition of solution for assay of cellular cholesterol content Composition
Concentration (10·)
Relative proportion
Potassium phosphate buffer Cholesterol oxidase Peroxidase (horseradish) Cholesterol ester hyddrolase Triton X-100 Sodium cholate p-Hydroxyphenylacetic acid
0.1 M, pH 7.4 2 U/ml, in buffera 30 U/ml, in buffera 0.4 U/ml, in buffera 1% 20 mM 4 mg/ml
8 2 2 2 1 1 3
a Buffer is potassium phosphate, 0.1 M, pH 7.4. The assay solutions were prepared on the day of use and a total of 10 volumes of phosphate buffer were added.
anti-rabbit IgG antibody (1:5000 dilution) for 1 h. ABCA1 was visualized by a chemiluminescence’s method (ECL Western blotting detection system). Cellular cholesterol content analysis. Cholesterol content was measured by zymochemistry as described [9]. The cells were harvested and washed three times with PBS; each sample was sonified for 3 min using the microtip of the sonifier (Branson Instruments Inc.). Samples (usually 100 ll) of the sonicates were transferred to 12 · 75-mm tubes for determination of total cholesterol (Other samples were used for assay of protein), the assay solution as shown in Table 1 was then added, and after incubation for 60 min at 37 C fluorescence was measured in HITACHI 650-60 Spectrophotometer (excitation, 325 nm; emission, 415 nm). Reference cholesterol (1–10 mg/l) was used to draw standard curve. Statistical analysis. Statistical analysis was carried out by SPSS13.0. Results are given as mean ± SD. Relationship between cholesterol content and ABCA1 was examined by linear correlation analysis, p < 0.05 was considered statistically significant.
Results Electron microscopy In order to obtain direct-viewing of the effect of AngII on cellular cholesterol content, observation by electron microscopy was down (Fig. 1). As shown in Fig. 1, the difference of cellular cholesterol content in different sample is significant. RT-PCR and Western blotting To examine whether ABCA1 mRNA and protein level is regulated by AngII, ABCA1 in the cultured cells was examined in the presence of various concentration of AngII (Fig. 2). Dose-dependent change of ABCA1 is demonstrated. As shown in Fig. 2, ABCA1 mRNA level was downregulated accompanied with the increased concentration of AngII (Fig. 2A). Simultaneously, ABCA1 protein level took on the same result (Fig. 2B). Cellular cholesterol content analysis To confirm that cellular cholesterol effluxion is retarded by AngII via down-regulating the expression of ABCA1, we detected the cellular cholesterol content and examined the linear correlation of cholesterol content and ABCA1. As shown in Fig. 3, cholesterol content in
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Y. Wang et al. / Biochemical and Biophysical Research Communications 353 (2007) 650–654
Fig. 1. Significant differences can be seen on the amount of vacuoles in different groups with various concentration of AngII (80000·). The difference of cellular cholesterol content in different groups with different concentration of AngII (A, AngII 106; B, AngII 107; C, AngII 108; and D, control) is significant.
Fig. 3. Cellular cholesterol effluxion was retarded by AngII. Cellular cholesterol content was detected by zymochemistry as described under Materials and methods. It’s shown that cellular cholesterol content was increased following the augmentation of AngII. Compared with control group, *p < 0.01, differences were significant.
Fig. 2. ABCA1 mRNA and protein level were down-regulated accompanied with the increased concentration of AngII. ABCA1 mRNA (A) and its protein (B) were analyzed by RT-PCR and Western blotting, respectively, as described under Materials and methods. The expression of ABCA1 was down-regulated.
Discussion
control group was taken as base line, increased of cholesterol content was investigated following the augmentation of AngII (compared with control group, p < 0.01). Further more, expression of ABCA1 was negatively correlated with cellular cholesterol content (ABCA1 mRNA: r = 0.971, p < 0.01; ABCA1 protein: r = 0.956, p < 0.01) (Fig. 4).
Many studies have demonstrated AngII may have important effects on atherosclerotic plaque development [10–12]. Hypertensive patients with elevated plasma levels of AngII show a 5-fold increased incidence of myocardial infarction (MI), compared with hypertensives with normal or decreased levels of AngII [13]. These data reveal that AngII may have other potency besides raising blood pressure. In this study, we investigated the atherogenic effect
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cholesterol efflux via CD36, but also inhibits the uptake of ox-LDL. This study demonstrated that the expression of ABCA1 could be inhibited markedly, in the same way, cellular cholesterol content was also accumulated following the augmentation of AngII. After investigated the linear correlation of ABCA1 and cellular cholesterol content, we come to the conclusion that cellular cholesterol content could be increased by AngII via down-regulating the expression of ABCA1. From this study, we summarized an important cue that the accumulation of cellular cholesterol was not only concerned with lipid’s intake, but also correlated with lipid’s efflux. Fig. 4. Linear correlation of the expression of ABCA1 and the cholesterol content. The less expression of ABCA1, the more content of cholesterol, and they are negatively correlated. Correlation coefficient R1 (cholesterol content and ABCA1mRNA) = 0.971; correlation coefficient R2 (cholesterol content and ABCA1 protein) = 0.956.
of AngII and confirmed the relationship between AngII and ABCA1. Previously, the majority studies about AngII on pathogenesis of atherosclerosis were concentrated on how to reduce the influx of cholesterol into cells. However, the efflux was unfrequented. Thus, we detected the expression of ABCA1 interfered by AngII and observed the dose-dependent change of ABCA1 and cholesterol content. It’s known that the removal of excess free cholesterol from cells by HDL or its apolipoproteins is important for maintaining cellular cholesterol homeostasis. At present, there are four main pathways of cellular cholesterol efflux: (1) passive diffusion; (2) CD-36; (3) ABCA1; and (4) apoliprotein E [14]. Among them, the most important is ABCA1 [15]. ABCA1 is a member of a large family of ATP-binding cassette transporters that have common structural motifs and use ATP as an energy source to transport a variety of substrates, including ions, lipids, and cytotoxins [16]. Some data indicated that, the morbidity of CAD in Tangier disease (TD) with homozygote mutation is 6-fold higher than health adult. The gene phenotype of ABCA1/ mouse is analogical with TD; transgenic study has shown that the overexpression of ABCA1 could promote cellular cholesterol efflux and inhibit the formation of AS plaque [17,18]. Therefore, how to regulate the expression of ABCA1 has become the focal point. Many factors could regulate the expression of ABCA1, including cholesterol, cAMP and interferon. However, much research was focused on the point of the relationship between AngII and cholesterol influx, whereas, little attention was given to the relationship between AngII and ABCA1—the critical gate-keeper in the process of cholesterol efflux. Whether cellular cholesterol content was increased by AngII via down-regulating the expression of ABCA1 has not been discovered. In this study, in order to exclude the influence of CD36, anti-CD36 was added. It not only prevents the cellular
Acknowledgments This work was supported in part by the laboratory of cardiovascular immunology, union hospital. We thank you for reading the manuscript. References [1] J.F. Oram, R.M. Lawn, ABCA1. The gatekeeper for eliminating excess tissue cholesterol, J. Lipid Res. 42 (2001) 1173–1179. [2] G. Schmitz, T. Langmann, Structure, function, and regulation of the ABC1 gene product [J], Curr. Opin. Lipidol. 12 (2001) 129–140. [3] S. Santamarina-fojo, K. Pterson, C. Knapper, et al., Complete genomic sequence of the human ABCA1: analysis of the human and mouse ATP-binding cassatte A promoter [J], Proc. Natl. Acad. Sci. USA 97 (2000) 7987–7992. [4] S. Mott, L. Yu, M. Marcil, et al., Decreased cellular cholesterol efflux is a common cause of familial hypoalphalipoproteinemia: role of the ABCA1 gene mutations, Atherosclerosis 152 (2000) 457–468. [5] M. Grafe, W. Auch-Schwelk, A. Zakrzewicz, et al., Angiotensin IIinduced leukocyte adhesion on human coronary endothelial cells is mediated by E-selectin, Circ. Res. 81 (1997) 804–811. [6] M. Browatzki, D. Larsen, C.A. Pfeiffer, et al., Angiotensin II stimulates matrix metalloproteinase secretion in human vascular smooth muscle cells via nuclear factor-kappaB and activator protein 1 in a redox-sensitive manner, J. Vasc. Res. 42 (2005) 415–423. [7] J. Klucken, C. Buchler, E. Orso, ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport, Proc. Natl. Acad. Sci. USA 97 (2000) 817–822. [8] K. Kojima, S. Abe-Dohmae, R. Arakawa, Progesterone Inhibits Apolipoprotein-mediated cellular lipid release: a putative mechanism for the decrease of HDL, Biochim. Biophys. Acta 1532 (2001) 173–184. [9] W. Gamble, M. Vaughan, H.S. Kruth, Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells, J. Lipid Res. 19 (1978) 1068–1070. [10] U.N. Das, AngiotensinII behaves as an endogenous pro-inflammatory molecule, J. Assoc. Physicians India 53 (2005) 472–476. [11] J. Zhuo, D. Casley, C. Murone, F.A. Mendelsohn, Acute and chronic in vivo inhibition of angiotensin-converting enzyme by perindopril in the endothelium and adventitia of large arteries and organs of the rabbit, J. Cardiovasc. Pharmacol. 29 (1997) 297–310. [12] J.L. Mehta, D.Y. Li, H. Yang, et al., Angiotensin II and IV stimulate expression and release of plasminogen activator inhibitor-1 in cultured human coronary artery endothelial cells, J. Cardiovasc. Pharmacol. 39 (2002) 789–794.
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[13] I. Njolstad, E. Arnesen, Preinfarction blood pressure and smoking are determinants for a fatal outcome of myocardial infarction: a prospective analysis from the Finnmark Study, Arch. Intern. Med. 158 (1998) 1326–1332. [14] Z.H. Huang, C.Y. Lin, J.F. Oram, T. Mazzone, Sterol efflux mediated by endogenous macrophage apoE expression is independent of ABCA1, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 2019–2025. [15] P.G. Yancey, A.E. Bortnick, G.K. Weibel, et al., Importance of different pathways of cellular cholesterol efflux, Arterioscler. Thromb. Vasc. Biol. 23 (2003) 712–719.
[16] M. Dean, Y. Hamon, G. Chimini, The human ATP-binding cassette (ABC) transporter superfamily, J. Lipid Res. 42 (2001) 1007–1017. [17] R.R. Singaraja, V. Bocher, E.R. James, Human ABCA 1BAC transgenic mice show increased high density lipoprotein cholesterol and apo A1dependent efflux stimulated by an internal promoter containing liver X receptor response element in intron1, J. Biol. Chem. 276 (2001) 33969–33979. [18] B.L. Vaisman, G. Lambert, M. Amar, ABCA 1 overexpression leads to hyperalpha lipoproteinemia and increased biliary cholesterol excretion in transgenic mice, J. Clin. Res. 108 (2001) 303–309.
Angiotensin II increases the cholesterol content of foam cells via down-regulating the expression of ATP-binding cassette transporter A1 Wang Yanfu a, Chen Zhijian a
b
a,*
, Liao Yuhua a, Mei Chunli a, Peng Hongyu a, Wang Min a, Guo Heping a, Lu Han b
Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tong Ji Medical College, Hua Zhong University of Science and Technology, 1277 Jie-Fang Avenue, Wuhan, 430022, People’s Republic of China Department of Geratology, Union Hospital, Tong Ji Medical College, Hua Zhong University of Science and Technology, 1277 Jie-Fang Avenue, Wuhan, 430022, People’s Republic of China Received 1 December 2006 Available online 20 December 2006
Abstract ATP-binding cassette transporter A1 (ABCA1) as one kind of membrane protein was found recently to play a major role in cholesterol homeostasis. Angiotensin II (AngII) has been shown to possess several atherogenic properties. The aim of the study is to investigate the influence of AngII on the expression of ABCA1 and the content of cholesterol in THP-1 derived foam cells. Our study showed that: (1) reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting demonstrated that AngII down-regulated the expression of ABCA1 in a dose-dependent manner. (2) The content of cholesterol was negatively correlated with ABCA1. The results suggest the promoting effects of AngII on the forming of foam cells are in a dose-dependent manner via down-regulating the expression of ABCA1. 2006 Elsevier Inc. All rights reserved. Keywords: Angiotensin II; ATP-binding cassette transporter A1; Atherosclerosis; Cholesterol content
Atherosclerosis (AS) is a common clinical fundamental disease. Patients with AS are confronted with lots of risks including hypertension, coronary artery disease (CAD), renal inadequacy etc. The detailed pathogenesy of AS is still undiscovered. ABCA1 as a critical gate-keeper was found recently at many tissues of human being [1], it’s the critical channel of cholesterol efflux in macrophages and conduces to scavenge redundant lipid, which protects against the formation of foam cells [2,3]. Mutations in the ABCA1 are associated with Tangier disease (TD) and a defect in cellular cholesterol efflux [4]. AngII possess several atherogenic properties including its ability to introduce the production of adhesion, promote SMC propagation, anti-fibrolysis and to attenuate the endothelial cell dependent vasodilatation, etc [5,6]. *
Corresponding author. Fax: +86 27 85727140. E-mail address: [email protected] (Z. Chen).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.067
Nevertheless, limited data are available on the relationship between AngII and ABCA1, which plays an essential role in cellular cholesterol efflux and helps to prevent macrophages from becoming foam cells. Thus, we hypothesize that AngII can increase cellular cholesterol content via down-regulating the expression of ABCA1. In the study, we examined the expression of ABCA1 and the cholesterol content of foam cells. We also investigated the relationship between cellular cholesterol content and ABCA1. Materials and methods LDL isolation and oxidization. LDLs (d = 1.0061.063 g/ml) were purified from human plasma obtained from healthy volunteers according to published standard protocols [7]. The preparation was performed in a Beckman L8-M ultracentrifuge (70 Ti rotor) at 4 C, and densities were adjusted with solid KBr. Lipoprotein fractions were dialyzed repeatedly in phosphate-buffered saline (PBS) containing 5 mM EDTA. After the final
Y. Wang et al. / Biochemical and Biophysical Research Communications 353 (2007) 650–654 dialysis step (0.15 M NaCl), LDL lipoproteins were sterilized using a 0.45 lm sterile filter (Sartorius). The isolated LDL was oxidized with CuSO4 10 lmol/L for 18 h at 37 C. Oxidation of LDL was measured by the thiobarbituric acid-reactive substances assay. Cell culture. THP-1 cells, a human monocytic leukemia cell line, were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cultured in RPMI 1640 medium (Sigma); supplemented with 10% (v/v) fetal calf serum (Gibco); 100 U of penicillin per ml, and 100 lg of streptomycin per ml; and a humidified atmosphere of 5% CO2 and 95% air at 37 C. To induce monocyte-to-macrophage differentiation, THP-1 cells were cultured in the presence of 160 nM phorbol 12-myristate 13-acetate (PMA). THP-1 derived macrophages were preincubated with anti CD36 (20 lg/ml, 37 C, 4 h). To induce foam cell formation of differentiated cells ox-LDL (100 lg/ml) with different concentration of AngII (0, 108, 107, 106 mM) was added for 48 h, respectively. Then the cells were washed once with PBS and incubated for an additional 12 h in serum-free media containing 0.2% BSA/RPMI 1640 and apolipoprotein A-I (apoA-I, 10 lg/ml). Before assay, cultured cells were harvested, washed once again with PBS. Electron microscopy. The cells were harvested and then fixed by 3% Glutaral for 3 h in 4 C. After being washed in 0.1 M cacodylic acid buffer solution five times for 4 h, the cells were fixed with 1% osmic acid for 30 min, washed in 0.1 M cacodylic acid buffer solution five times for 2 h. Then soaked into 50%, 70%, 80%, 90% alcohol for 10 min separately to be dewatered, then into 90% acetone for 10 min, and 100% acetone three times for 10 min. The cells were saturated in mixed liquor of acetone and extemporized epoxide resin embedding medium with the ratio of 1:1 for 1 h, and then in the 1:3 mixtures for 3 h and at last in pure embedding medium for 1 h. In the end, the cells were embedded in capsules. Next they were put in a thermostat oven at the temperature of 65–70 C overnight for the imbedding medium to polymerize. Then 50-nm extrathin sections were cut out. The sections were soaked in 3% uranyl acetate saturated solution heated by microwave for 30 s, then washed in buffer solution. After that, the sections were soaked in lead citrate solution heated by microwave for 20 s, and then washed in buffer solution. Last, the sections were dried in air for observation. RNA extraction and RT-PCR. Total RNA was extracted from the cultured cells using Trizol reagent according to the protocol provided by the manufacturer (Bio Basic Inc., Shanghai). The RNA was treated with DNase (Bio Basic Inc., Shanghai) before analysis by RT-PCR. Oligonucleotide primers were designed using Primer Express software (DNAStar) and were synthesized by Bio Basic Inc. Sequences of primers are as follows, using 5 0 -ACAACCAAACCTCACACTACTG-3 0 , and 5 0 -ATAG ATCCCATTACAGACAGCG-3 0 , for ABCA1, Primers for GAPDH were purchased from Bio Basic Inc. sequences are 5 0 -TCACCATCTTCC AGGAGCGAG-3 0 , and 5 0 -TGTCGCTGTTGAAGTCAGAG-3 0 . Firststrand cDNA was synthesized from the total RNA (2 lg) in a SuperScript preamplification system (Takara). The cDNA of ABCA1 was amplified by polymerase chain reaction with primers as described for 38 cycles using the Taq polymerase (Takara). DNA was visualized by ethidium bromide gel stain (UVP gel image analysis system) after 2% agarose gel electrophoresis. Protein extraction and Western blotting. The cells were harvested and suspended in 5 mM Tris–HCl buffer (pH 8.5) containing 1% protease inhibitor phenylmethyl sulfonylfluoride (Sigma) and placed on ice for 30 min with occasional mixing by a vortex. The cell suspension was centrifuged at 650g for 5 min, and then the supernatant was centrifuged at 105,000g for 30 min. Total membrane precipitated was suspended in the same buffer. After determination of protein content by a Bradford method, the membrane protein preparations were stored at 80 C until use. Total membrane protein (60 lg) was dissolved in 0.9 M urea, 0.2% (v/v) Triton X-100, and 0.1% (w/v) dithiothreitol and supplied with 10% (w/v) lithium dodecylsulfate and then analyzed by electrophoresis in 7% (w/v) polyacrylamide gel containing 0.1% (w/v) sodium dodecylsulfate (SDS) followed by blotting to a nitrocellulose membrane (Invitrogen). The membrane was blocked in 5% skim milk and incubated with rabbit antihuman ABCA1 antisera (1:200 dilution) [8] for 2 h. After washing three times with 0.02 M Tris-buffered saline containing 0.05% Triton X-100 (pH 7.5), the membrane was incubated with horseradish peroxidase-conjugated
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Table 1 Composition of solution for assay of cellular cholesterol content Composition
Concentration (10·)
Relative proportion
Potassium phosphate buffer Cholesterol oxidase Peroxidase (horseradish) Cholesterol ester hyddrolase Triton X-100 Sodium cholate p-Hydroxyphenylacetic acid
0.1 M, pH 7.4 2 U/ml, in buffera 30 U/ml, in buffera 0.4 U/ml, in buffera 1% 20 mM 4 mg/ml
8 2 2 2 1 1 3
a Buffer is potassium phosphate, 0.1 M, pH 7.4. The assay solutions were prepared on the day of use and a total of 10 volumes of phosphate buffer were added.
anti-rabbit IgG antibody (1:5000 dilution) for 1 h. ABCA1 was visualized by a chemiluminescence’s method (ECL Western blotting detection system). Cellular cholesterol content analysis. Cholesterol content was measured by zymochemistry as described [9]. The cells were harvested and washed three times with PBS; each sample was sonified for 3 min using the microtip of the sonifier (Branson Instruments Inc.). Samples (usually 100 ll) of the sonicates were transferred to 12 · 75-mm tubes for determination of total cholesterol (Other samples were used for assay of protein), the assay solution as shown in Table 1 was then added, and after incubation for 60 min at 37 C fluorescence was measured in HITACHI 650-60 Spectrophotometer (excitation, 325 nm; emission, 415 nm). Reference cholesterol (1–10 mg/l) was used to draw standard curve. Statistical analysis. Statistical analysis was carried out by SPSS13.0. Results are given as mean ± SD. Relationship between cholesterol content and ABCA1 was examined by linear correlation analysis, p < 0.05 was considered statistically significant.
Results Electron microscopy In order to obtain direct-viewing of the effect of AngII on cellular cholesterol content, observation by electron microscopy was down (Fig. 1). As shown in Fig. 1, the difference of cellular cholesterol content in different sample is significant. RT-PCR and Western blotting To examine whether ABCA1 mRNA and protein level is regulated by AngII, ABCA1 in the cultured cells was examined in the presence of various concentration of AngII (Fig. 2). Dose-dependent change of ABCA1 is demonstrated. As shown in Fig. 2, ABCA1 mRNA level was downregulated accompanied with the increased concentration of AngII (Fig. 2A). Simultaneously, ABCA1 protein level took on the same result (Fig. 2B). Cellular cholesterol content analysis To confirm that cellular cholesterol effluxion is retarded by AngII via down-regulating the expression of ABCA1, we detected the cellular cholesterol content and examined the linear correlation of cholesterol content and ABCA1. As shown in Fig. 3, cholesterol content in
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Y. Wang et al. / Biochemical and Biophysical Research Communications 353 (2007) 650–654
Fig. 1. Significant differences can be seen on the amount of vacuoles in different groups with various concentration of AngII (80000·). The difference of cellular cholesterol content in different groups with different concentration of AngII (A, AngII 106; B, AngII 107; C, AngII 108; and D, control) is significant.
Fig. 3. Cellular cholesterol effluxion was retarded by AngII. Cellular cholesterol content was detected by zymochemistry as described under Materials and methods. It’s shown that cellular cholesterol content was increased following the augmentation of AngII. Compared with control group, *p < 0.01, differences were significant.
Fig. 2. ABCA1 mRNA and protein level were down-regulated accompanied with the increased concentration of AngII. ABCA1 mRNA (A) and its protein (B) were analyzed by RT-PCR and Western blotting, respectively, as described under Materials and methods. The expression of ABCA1 was down-regulated.
Discussion
control group was taken as base line, increased of cholesterol content was investigated following the augmentation of AngII (compared with control group, p < 0.01). Further more, expression of ABCA1 was negatively correlated with cellular cholesterol content (ABCA1 mRNA: r = 0.971, p < 0.01; ABCA1 protein: r = 0.956, p < 0.01) (Fig. 4).
Many studies have demonstrated AngII may have important effects on atherosclerotic plaque development [10–12]. Hypertensive patients with elevated plasma levels of AngII show a 5-fold increased incidence of myocardial infarction (MI), compared with hypertensives with normal or decreased levels of AngII [13]. These data reveal that AngII may have other potency besides raising blood pressure. In this study, we investigated the atherogenic effect
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cholesterol efflux via CD36, but also inhibits the uptake of ox-LDL. This study demonstrated that the expression of ABCA1 could be inhibited markedly, in the same way, cellular cholesterol content was also accumulated following the augmentation of AngII. After investigated the linear correlation of ABCA1 and cellular cholesterol content, we come to the conclusion that cellular cholesterol content could be increased by AngII via down-regulating the expression of ABCA1. From this study, we summarized an important cue that the accumulation of cellular cholesterol was not only concerned with lipid’s intake, but also correlated with lipid’s efflux. Fig. 4. Linear correlation of the expression of ABCA1 and the cholesterol content. The less expression of ABCA1, the more content of cholesterol, and they are negatively correlated. Correlation coefficient R1 (cholesterol content and ABCA1mRNA) = 0.971; correlation coefficient R2 (cholesterol content and ABCA1 protein) = 0.956.
of AngII and confirmed the relationship between AngII and ABCA1. Previously, the majority studies about AngII on pathogenesis of atherosclerosis were concentrated on how to reduce the influx of cholesterol into cells. However, the efflux was unfrequented. Thus, we detected the expression of ABCA1 interfered by AngII and observed the dose-dependent change of ABCA1 and cholesterol content. It’s known that the removal of excess free cholesterol from cells by HDL or its apolipoproteins is important for maintaining cellular cholesterol homeostasis. At present, there are four main pathways of cellular cholesterol efflux: (1) passive diffusion; (2) CD-36; (3) ABCA1; and (4) apoliprotein E [14]. Among them, the most important is ABCA1 [15]. ABCA1 is a member of a large family of ATP-binding cassette transporters that have common structural motifs and use ATP as an energy source to transport a variety of substrates, including ions, lipids, and cytotoxins [16]. Some data indicated that, the morbidity of CAD in Tangier disease (TD) with homozygote mutation is 6-fold higher than health adult. The gene phenotype of ABCA1/ mouse is analogical with TD; transgenic study has shown that the overexpression of ABCA1 could promote cellular cholesterol efflux and inhibit the formation of AS plaque [17,18]. Therefore, how to regulate the expression of ABCA1 has become the focal point. Many factors could regulate the expression of ABCA1, including cholesterol, cAMP and interferon. However, much research was focused on the point of the relationship between AngII and cholesterol influx, whereas, little attention was given to the relationship between AngII and ABCA1—the critical gate-keeper in the process of cholesterol efflux. Whether cellular cholesterol content was increased by AngII via down-regulating the expression of ABCA1 has not been discovered. In this study, in order to exclude the influence of CD36, anti-CD36 was added. It not only prevents the cellular
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