This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McCrohon, J. A. Articles by Celermajer, D. S. Search for Related Content PubMed PubMed Citation Articles by McCrohon, J. A. Articles by Celermajer, D. S. Related Collections Mechanism of atherosclerosis/growth factors

(Circulation. 2000;101:224.)
© 2000 American Heart Association, Inc.

Brief Rapid Communications

Androgen Receptor Expression Is Greater in Macrophages From Male Than From Female Donors

A Sex Difference With Implications for Atherogenesis

Jane A. McCrohon, MBBS, FRACP; Alison K. Death, PhD; Shirley Nakhla, BSc; Wendy Jessup, PhD; David J. Handelsman, MBBS, PhD; Keith K. Stanley, PhD; David S. Celermajer, MBBS, PhD

From the Heart Research Institute (J.A.M., S.N., W.J., K.K.S., D.S.C.) and Department of Cardiology (J.A.M., D.S.C.), Royal Prince Alfred Hospital in Missenden Road, and the Department of Medicine, University of Sydney (A.K.D., D.J.H., D.S.C.), Sydney, Australia.

Correspondence to Dr David S. Celermajer, Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, Sydney, 2050, Australia. E-mail davidc{at}card.rpa.cs.nsw.gov.au

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Male sex is an independent risk factor for the extent and severity of atherosclerosis. The influence of androgens on foam cell formation, a key event in atherogenesis, has not yet been investigated.

Methods and Results—Primary human monocytes were allowed to differentiate into macrophages. RNA was then extracted from healthy male-donor (n=8) and premenopausal female-donor (n=8) macrophages, and message for the androgen receptor (AR) was examined by RT-PCR. There was a significantly higher level of AR mRNA in macrophages isolated from men than in those from women (0.64±0.06 versus 0.15±0.02 amol/µg total RNA; P<0.001). AR mRNA levels were similar in macrophages from postmenopausal and premenopausal women (P=0.16). The functional consequence of this sex difference was then explored. Lipid-loading studies were performed on male (n=9) macrophages treated with the androgen dihydrotestosterone (DHT) and/or the AR antagonist hydroxyflutamide. These showed that DHT caused a dose-dependent and receptor-mediated increase in macrophage cholesteryl ester content (109±10%, 117±3%, and 120±4% for 4, 40, and 400 nmol/L DHT, respectively, as a percentage of control, P=0.002; 95±8% for DHT with hydroxyflutamide, P=0.58 versus controls). By contrast, there was no significant effect of androgen on lipid loading in female-donor macrophages (P>0.2 versus controls).

Conclusions—Sex differences in androgen-mediated macrophage lipid loading may contribute to the greater prevalence and severity of atherosclerosis in men.

Key Words: atherosclerosis • cells • hormones

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Men have an earlier onset and higher incidence of atherosclerosis than women.¹ The potential protective effects of estrogens on vascular structure and function have been studied extensively. Much less work, however, has addressed the possible effects of androgens on atherogenesis. In animal models, androgen treatment may increase plaque formation in the aorta and coronary vessels.^{2 3} Furthermore, in humans, we have recently demonstrated that androgens enhance monocyte adhesion to endothelial cells⁴ and are associated with impaired vascular reactivity.⁵

Foam cell formation is a key early event in atherosclerosis and is largely due to the uptake of modified lipoproteins, principally LDL, by peripheral blood monocyte-derived macrophages (MDMs) in the arterial wall.^{6 7} The possibility that androgen receptors (ARs) and androgen-related intracellular pathways may be involved in the process of lipid loading and foam cell formation in atherosclerosis has not been explored previously. We therefore investigated whether the AR is expressed in primary human MDMs and whether there is a sex difference in its expression and have examined the biological effects of androgen stimulation on cholesteryl ester (CE) formation/lipid loading in these cells.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Isolation of Human Monocytes
White cell concentrates (Red Cross Blood Bank) were obtained from the peripheral blood of individual healthy men (20 to 58 years old) and premenopausal (18 to 45 years old) and postmenopausal (45, 55, and 72 years old) women. The postmenopausal women all had had no menstruation for >3 years and had follicle-stimulating hormone levels >40 U/L. Monocytes were removed by density gradient separation and counterflow centrifugation elutriation.⁸ Monocyte purity was >90% and viability >95% (trypan blue) in all experiments.

Culture and Analysis of Human MDMs
Monocytes isolated by elutriation were resuspended in phenol red–free RPMI (Life Technology) and allowed to adhere for 1.5 hours at 37°C under 5% CO₂ in air. Thereafter, phenol red–free RPMI containing 10% postmenopausal female human serum (17ß-estradiol <75 pmol/L, progesterone <0.8 nmol/L, and testosterone <2 nmol/L), penicillin G (50 U/mL), and streptomycin (50 µg/mL) was added. Changes of medium occurred every 2 to 3 days, and hormone treatments (dihydrotestosterone, DHT, Sigma, at 4 to 400 nmol/L with or without the AR antagonist hydroxyflutamide, HF, 400 nmol/L) were added from days 3 through 10 with each change of medium. In the control conditions, no DHT or HF was added. Serum testosterone levels in healthy men range from 4 to 40 nmol/L. Lipid loading was achieved on days 8 through 10 during a 48-hour incubation with 50 µg/mL of acetylated LDL in medium containing 10% (vol/vol) lipoprotein-deficient human serum (d>1.25). Cell viability for each treatment condition was 90% to 95%. LDL was isolated from plasma from healthy, normolipidemic subjects, and macrophage cell extracts were analyzed by high-performance liquid chromatography according to previously described methods.⁹

RNA Extraction and Measurement of AR mRNA Levels
After 7 to 8 days of culture in RPMI supplemented with postmenopausal serum, male- and female-donor MDMs were assessed for AR message.

Total RNA was extracted from each male- and female-donor experiment (1.5x10⁶ macrophages per well), and RNA was quantified with the SYBR Green II assay (Molecular Probes).¹⁰ Androgen and ß-actin (control) primers were designed from the published sequence of the human genes^{11 12} : AR (sense), 5'-AGATGGGCTTGACTTTCCCAGAAAG-3'; AR (antisense), 5'-ATGGCTGTCATTCAGTACTCCTGGA-3'.

Competition-based quantitative RT-PCR was performed with an AR competitor template as described elsewhere.¹²

Statistical Analysis
AR mRNA values are expressed as mean±SEM. Statistical significance was analyzed with Fisher’s exact test. Lipid-loading results are also expressed as mean±SEM of at least triplicate wells per experiment per condition for each male and female donor. Results for each condition in each donor were calculated as a percentage of the control values (100%). Statistical significance by an independent-samples t test, Fischer’s exact test where n6, or a 1-way ANOVA for trend, as appropriate, was inferred at a 2-sided value of P<0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
AR Expression in MDMs
Male-donor MDMs (n=8) expressed levels of AR mRNA >4 times greater than those isolated from premenopausal female-donor cells (n=8) (0.64±0.06 versus 0.15±0.02 amol/µg total RNA; P<0.001). This was evident on semiquantitative RT-PCR (Figure 1A), in which representative male samples showed distinct AR bands (545 bp), whereas the female samples showed less visible bands under the same conditions, and was confirmed by quantitative competition-based RT-PCR. AR mRNA levels were also low in cells from 3 postmenopausal females (0.18, 0.24, and 0.26 amol/µg RNA, respectively, P<0.001, versus male levels, P=0.16 versus premenopausal female levels) (Figure 1B).

View larger version (40K): [in this window] [in a new window]   Figure 1. A, AR expression in male- and female-donor MDMs by RT-PCR. Top lanes 1 through 6 (left) show AR PCR product (545 bp) from 6 different premenopausal female RNA samples; top lanes 7 through 12 show AR PCR product from 6 different male RNA samples; bottom lanes 1 through 12 display ß-actin controls from same subjects (164 bp). B, These sex differences in AR expression were confirmed by competitive (quantitative) RT-PCR (P<0.001). indicates premenopausal women; , postmenopausal women; and •, men.

Effects of Androgen Exposure on Macrophage Lipid Loading
The functional effects of this sex difference in AR expression were then explored. Androgen exposure was associated with a dose-dependent increase in intracellular CE content in male-donor MDMs (n=9) (CE as a percentage of control, 109±10%, 117±3%, and 120±4% for DHT 4, 40, and 400 nmol/L, respectively; P=0.002 by ANOVA; Figure 2). This androgen-associated increase in CE accumulation was abrogated by coincubation with the nonsteroidal AR antagonist HF (CE content 95±8% for DHT 40 nmol/L and HF 400 nmol/L versus controls, P=0.58). HF 400 nmol/L alone did not alter CE content significantly (110±7%, P=0.1). Free cholesterol (FC) content in male-donor macrophages was not altered significantly by androgen exposure (P=0.40). Absolute lipid-loading values in macrophages from 1 male donor (but comparable in nature to the others) were as follows: for control cells, CE 62±4 nmol/mg cell protein, FC 139±16 nmol/mg; and for DHT 40 nmol/L–treated cells, CE 78±2 nmol/mg cell protein, FC 132±11 nmol/mg cell protein.

View larger version (53K): [in this window] [in a new window]   Figure 2. Androgen exposure (DHT) is associated with a dose-dependent increase in CE accumulation in human MDMs from male donors (n=9). This effect can be blocked by coincubation with specific androgen-receptor antagonist HF.

In contrast to the male-donor cells, CE content in female-donor macrophages (n=4) did not increase with androgen exposure (CE 86±11% and 75±15% for DHT 40 nmol/L and 400 nmol/L, respectively, P>0.2 versus controls). Similarly, FC accumulation was unchanged by androgen treatment.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although the male predisposition to atherosclerosis is well documented, there are few studies assessing the effects of androgens on atherogenic processes. In this study, we have demonstrated a marked sex difference in the AR mRNA status of mature MDMs from healthy human subjects. Because MDMs play a key role in the early development of atherosclerosis,^{6 9} with macrophage lipid uptake leading to foam cell and fatty streak formation, we postulated that a greater expression of AR in these cells might contribute to the male predisposition to atherosclerosis. Indeed, our data indicate that the androgen dihydrotestosterone caused a dose-related and receptor-mediated increase in human macrophage lipid accumulation in male- but not female-donor cells. This is probably due to higher levels of AR in the male MDMs; however, it is possible that androgens may also influence lipoprotein metabolism via other pathways.

Sex differences have previously been documented in the levels of AR message found in macrophage-like synoviocytes,¹³ and in rat aortic smooth muscle cells,¹⁴ androgen-receptor protein levels are higher in male than in female animals. In these experimental studies, however, no functional correlates of these observations were investigated. In this first study of AR expression in human MDMs, the observed sex difference in AR message was associated with enhanced lipid loading by male- but not female-donor cells, consistent with a proatherogenic effect.

Dihydrotestosterone was used in these experiments because it is a potent, nonaromatizable physiological androgen that is specific for the AR. This avoids the potentially confounding effects of estrogenic metabolites that arise from the aromatization of testosterone,¹⁵ noting that estrogens may also influence macrophage lipid loading.⁹ An acetylated form of LDL was used in these experiments, because acetylated LDL is the standard type of modified LDL used in lipoprotein uptake experiments and a specific ligand for the scavenger receptor, which may play a critical role in foam cell formation (although additional pathways of modified LDL uptake also occur in vivo).¹⁶

In summary, the androgen dihydrotestosterone increases CE accumulation in MDMs from male but not female subjects. Because this effect is receptor-mediated, the most likely reason for its sex specificity relates to the significantly higher levels of AR mRNA found in male- than in female-donor macrophages. These data are consistent with a significant effect of androgen exposure on foam cell formation in men.

	Acknowledgments

 
Dr McCrohon is supported by the University of Sydney and Department of Cardiology, Royal Prince Alfred Hospital; Dr Jessup by the National Health and Medical Research Council; and Dr Celermajer by the Medical Foundation, University of Sydney.

Received September 23, 1999; revision received November 4, 1999; accepted November 5, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Wingard DL, Suarez L, Barrett-Connor E. The sex differential in mortality from all causes and ischaemic heart disease. Am J Epidemiol. 1983;117:165–172.[Abstract/Free Full Text]
   
2. Toda T, Toda Y, Cho BH, Kummerow FA. Ultrastructural changes in the comb and aorta of chicks fed excess testosterone. Atherosclerosis. 1984;51:47–57.[Medline] [Order article via Infotrieve]
   
3. Adams MR, Williams JK, Kaplan JR. Effects of androgens on coronary artery atherosclerosis and atherosclerosis-related impairment of vascular responsiveness. Arterioscler Thromb Vasc Biol. 1995;15:562–570.[Abstract/Free Full Text]
   
4. McCrohon JA, Jessup W, Handelsman DJ, Celermajer DS. Androgen exposure increases human monocyte adhesion to vascular endothelium and endothelial cell expression of vascular cell adhesion molecule-1. Circulation.. 1999;99:2317–2322.[Abstract/Free Full Text]
   
5. Herman SM, Robinson JTC, McCredie RJ, Adams MR, Boyer MJ, Celermajer DS. Androgen deprivation is associated with enhanced endothelium-dependent dilatation in adult men. Arterioscler Thromb Vasc Biol. 1997;17:2004–2009.[Abstract/Free Full Text]
   
6. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990’s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
   
7. Brown MS, Goldstein JL, Krieger M, Ho YK, Anderson RGW. Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins. J Cell Biol. 1979;82:597–613.[Abstract/Free Full Text]
   
8. Garner B, Dean R, Jessup W. Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independent of superoxide production. Biochem J. 1994;301:421–428.
   
9. McCrohon JA, Jessup W, Nakhla S, Celermajer DS. Estrogen and progesterone reduce lipid accumulation in human monocyte-derived macrophages: a sex-specific effect. Circulation. 1999;100:2319–2325.[Abstract/Free Full Text]
   
10. Schmidt DM, Ernst JD. A fluorometric assay for the quantification of RNA in solution with nanogram sensitivity. Anal Biochem. 1995;232:144–146.[Medline] [Order article via Infotrieve]
    
11. Malucelli A, Sauerwein H, Pfaffl MW, Meyer HHD. Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in reverse transcription-polymerase chain reaction. J Steroid Biochem Mol Biol. 1996;58:563–568.[Medline] [Order article via Infotrieve]
    
12. Death AK, Yue DK, Turtle JR. Use of competitive RT-PCR for measuring metalloproteinase gene expression in mesangial cells exposed to a hyperglycaemic environment. Biotechniques. 1999;27:512–516.[Medline] [Order article via Infotrieve]
    
13. Cutolo M, Accardo S, Villaggio B, Barone A, Sulli A, Coviello DA, Carabbio C, Felli L, Miceli D, Farruggio R, Carruba G, Castagnetta L. Androgen and estrogen receptors are present in human synovial macrophages. J Clin Endocrinol Metab. 1996;81:820–827.[Abstract]
    
14. Higashiura K, Mathur RS, Halushka PV. Gender-related differences in androgen regulation of thromboxane A2 receptors in rat aortic smooth muscle cells. J Cardiovasc Pharmacol. 1997;29:311–315.[Medline] [Order article via Infotrieve]
    
15. Simpson ER, Zhao Y, Agarwal VR, Michael MD, Bulun SE, Hinshelwood MM, Graham-Lorence S, Sun T, Fisher CR, Qin K, Mendelson CR. Aromatase expression in health and disease. Recent Prog Horm Res. 1997;52:185–213.
    
16. Freeman M, Ekkel Y, Rohrer L, Penman M, Freedman NJ, Chisolm GM, Krieger M. Expression of type I and type II bovine scavenger receptors in Chinese hamster ovary cells: lipid droplet accumulation and non-reciprocal cross competition by acetylated and oxidized low density lipoprotein. Proc Natl Acad Sci U S A. 1991;88:4931–4935.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				R. Ray, C. M. Herring, T. A. Markel, P. R. Crisostomo, M. Wang, B. Weil, T. Lahm, and D. R. Meldrum Deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1498 - R1503. [Abstract] [Full Text] [PDF]

				M. K. C. Ng, S. Nakhla, A. Baoutina, W. Jessup, D. J. Handelsman, and D. S. Celermajer Dehydroepiandrosterone, an adrenalandrogen, increases human foam cell formation: a potentially pro-atherogenic effect J. Am. Coll. Cardiol., December 3, 2003; 42(11): 1967 - 1974. [Abstract] [Full Text] [PDF]

				M. K. C. Ng, C. M. Quinn, J. A. McCrohon, S. Nakhla, W. Jessup, D. J. Handelsman, D. S. Celermajer, and A. K. Death Androgens Up-Regulate Atherosclerosis-Related Genes in Macrophages From Males But Not Females: Molecular Insights Into Gender Differences in Atherosclerosis J. Am. Coll. Cardiol., October 1, 2003; 42(7): 1306 - 1313. [Abstract] [Full Text] [PDF]

				K. A. Griffiths, M. A. Sader, M. R. Skilton, J. A. Harmer, and D. S. Celermajer Effects of raloxifene on endothelium-dependent dilation, lipoproteins, and markers of vascular function in postmenopausal women with coronary artery disease J. Am. Coll. Cardiol., August 20, 2003; 42(4): 698 - 704. [Abstract] [Full Text] [PDF]

				C. P. Schneider, M. G. Schwacha, T. S. A. Samy, K. I. Bland, and I. H. Chaudry Androgen-mediated modulation of macrophage function after trauma-hemorrhage: central role of 5{alpha}-dihydrotestosterone J Appl Physiol, July 1, 2003; 95(1): 104 - 112. [Abstract] [Full Text] [PDF]

				P. Y. Liu, A. K. Death, and D. J. Handelsman Androgens and Cardiovascular Disease Endocr. Rev., June 1, 2003; 24(3): 313 - 340. [Abstract] [Full Text] [PDF]

				F. C. W. Wu and A. von Eckardstein Androgens and Coronary Artery Disease Endocr. Rev., April 1, 2003; 24(2): 183 - 217. [Abstract] [Full Text] [PDF]

				B. D. Sullivan, J. E. Evans, J. M. Cermak, K. L. Krenzer, M. R. Dana, and D. A. Sullivan Complete Androgen Insensitivity Syndrome: Effect on Human Meibomian Gland Secretions Arch Ophthalmol, December 1, 2002; 120(12): 1689 - 1699. [Abstract] [Full Text] [PDF]

				Y. Lukyanenko, J.-J. Chen, and J. C. Hutson Testosterone Regulates 25-Hydroxycholesterol Production in Testicular Macrophages Biol Reprod, November 1, 2002; 67(5): 1435 - 1438. [Abstract] [Full Text] [PDF]

				A. M. Kenny, K. M. Prestwood, C. A. Gruman, G. Fabregas, B. Biskup, and G. Mansoor Effects of Transdermal Testosterone on Lipids and Vascular Reactivity in Older Men With Low Bioavailable Testosterone Levels J. Gerontol. A Biol. Sci. Med. Sci., July 1, 2002; 57(7): M460 - 465. [Abstract] [Full Text]

				M. K.C. Ng, P. Y. Liu, A. J. Williams, S. Nakhla, L. P. Ly, D. J. Handelsman, and D. S. Celermajer Prospective Study of Effect of Androgens on Serum Inflammatory Markers in Men Arterioscler. Thromb. Vasc. Biol., July 1, 2002; 22(7): 1136 - 1141. [Abstract] [Full Text] [PDF]

				J. E Rossouw Hormones, genetic factors, and gender differences in cardiovascular disease Cardiovasc Res, February 15, 2002; 53(3): 550 - 557. [Full Text] [PDF]

				M. A. Sader and D. S. Celermajer Endothelial function, vascular reactivity and gender differences in the cardiovascular system Cardiovasc Res, February 15, 2002; 53(3): 597 - 604. [Full Text] [PDF]

				H. Igarashi, T. Kouro, T. Yokota, P. C. Comp, and P. W. Kincade Age and stage dependency of estrogen receptor expression by lymphocyte precursors PNAS, December 18, 2001; 98(26): 15131 - 15136. [Abstract] [Full Text] [PDF]

				H. Hanke, C. Lenz, B. Hess, K.-D. Spindler, and W. Weidemann Effect of Testosterone on Plaque Development and Androgen Receptor Expression in the Arterial Vessel Wall Circulation, March 13, 2001; 103(10): 1382 - 1385. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McCrohon, J. A. Articles by Celermajer, D. S. Search for Related Content PubMed PubMed Citation Articles by McCrohon, J. A. Articles by Celermajer, D. S. Related Collections Mechanism of atherosclerosis/growth factors