Histochem Cell Biol (2008) 130:399–405 DOI 10.1007/s00418-008-0426-7
ORIGINAL PAPER
Human umbilical vascular endothelial cells express estrogen receptor beta (ER) and progesterone receptor A (PR-A), but not ER and PR-B Bettina Toth · Gitti Saadat · Alrun Geller · Christoph Scholz · Sandra Schulze · Klaus Friese · Udo Jeschke
Accepted: 31 March 2008 / Published online: 18 April 2008 © Springer-Verlag 2008
Abstract Several reports deal with possible eVects of female sex hormones on human umbilical vein endothelial cells (HUVEC) including elasticity, activation of plasma membrane Na+/H+ exchange, VEGF receptor Flk-1/KDR and many others. In contrast to those Wndings, some publications pointed out that HUVEC lack expression of both the estrogen receptor (ER) and/or the progesterone receptor (PR). Because the majority of these investigations were carried out at a time period, when only one ER and one PR was known, the aim of this study was the systematic analysis of ER and ER as well as PR-A and PR-B expression in HUVEC with speciWc monoclonal antibodies by immunocytochemistry and quantitative RT-PCR (TaqMan). As a result, we could show that HUVEC lack ER but express ER. The expression of ER could be signiWcantly upregulated with 17-estradiol on mRNA and protein level. In addition, HUVEC express PR-A but not PR-B. PR-A expression could be signiWcantly upregulated with progesterone, again on mRNA and protein level. We conclude that estrogenic eVects on HUVEC are mediated via the ER and gestagens act via the PR-A pathway. Keywords Human umbilical vein endothelial cells · Estrogen receptor · Progesterone receptor B. Toth · G. Saadat · A. Geller · K. Friese Department of Obstetrics and Gynecology, Großhadern, Ludwig-Maximilians University, 81377 Munich, Germany e-mail:
[email protected] G. Saadat · A. Geller · C. Scholz · S. Schulze · K. Friese · U. Jeschke (&) Department of Obstetrics and Gynecology, Innenstadt, Maistrasse, Ludwig-Maximilians University, Maistrasse 11, 80337 Munich, Germany e-mail:
[email protected]
Introduction Endothelial cells are involved in a diversity of (patho-) physiologic processes including hemostasis, inXammation and angiogenesis. Human umbilical vein endothelial cells (HUVEC) serve as a widely used in vitro model to study eVects of natural and synthetic sex hormones on endothelial cells. The female sex hormones estradiol and progesterone exert their actions on target cells through the binding and activation of the estrogen receptor (ER) and progesterone receptor (PR), respectively. ER and PR are members of the steroid hormone superfamily of ligand-dependent transcription factors and bind to the control regions (promoters) of speciWc gene elements, where they recruit co-activators or co-repressors and control gene expression (Brosens et al. 2004). Although a variety of studies described estrogenic eVects in HUVEC, conXicting information is given on the expression of all four known ERs and PRs. Simoncini et al. (2005) investigated estrogen signaling pathways upon pulsed or continuous administration. They postulated that pulsed estrogen treatment of HUVEC lead to the activation of non-genomic pathways, particularly in the Wrst hour of administration. Other investigators highlighted estrogenic eVects on volume, growth and elasticity of HUVEC (Hillebrand et al. 2006; Morales et al. 1995) as well as the release of activation markers like prostacyclin (Hermenegildo et al. 2005a; Oviedo et al. 2005), nitric oxide (Florian et al. 2004), interleukins (Keck et al. 1998), vascular cell adhesion molecule-1 (Mukherjee et al. 2002; Tatsumi et al. 2002) and others (Chen et al. 2002; Akarasereenont et al. 2000). Although Jensen et al. (1998) and Tatsumi et al. (2002) showed that HUVEC do not express the classical ER,
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other investigators including Herve et al. (2006) and Evans et al. (2002a) demonstrated weak expression of both ER and ER mRNA in HUVEC. Focusing PR, only few studies on the expression of PR in HUVEC exist. Tatsumi et al. (2002) described PR-A and PR-B mRNA expression in HUVEC. However, the relative importance of ER, ER, PR-A and PR-B in mediating the cellular response to estrogen and progesterone is not well deWned, particularly the steroid receptor protein expression in HUVEC investigated by immunocytochemistry. Therefore, the aim of this study was to analyze the expression of both estrogen (ER and ER) and progesterone receptors (PR-A and PR-B) in HUVEC after stimulation with estradiol (E2) and progesterone and in unstimulated controls with speciWc monoclonal antibodies by immunocytochemistry and RT-PCR (TaqMan).
Materials and methods Cell culture HUVEC were obtained from Promocell (Heidelberg, Germany) at passage two and three. The cells were cultivated in phenol red free endothelial cell growth medium (ECGM) (customer formulation, Promocell, Heidelberg, Germany). ECGM contained 10% fetal bovine serum, 1.0 mg/ml hydrocortisone, 0.1 ng/ml endothelial cell growth factor, 1.0 ng/ml bFGE and 2 ml endothelial cell growth substrate as well as 5 ml streptomycin (Biochrom AG, Berlin, Germany) and 5 ml amphotericinB (Biochrom AG, Berlin, Germany) in 500 ml medium. HUVEC were used for experiments between passages two and three. All cell cultures were maintained in a humidiWed 5% CO2 atmosphere at 37°C. HUVEC were incubated with dextrane encapsulated water-soluble 17-estradiol (E2) and progesterone (Sigma, Taufkirchen, Germany) in diVerent concentrations (0–500 nmol/ml) and cultivated for exactly 72 h on chamber slides (Nunc, Wiesbaden, Germany). Cells were Wxed with methanol/ethanol (50%/50%) (Merck, Darmstadt, Germany). We analyzed nine cultures for each concentration, Wve diVerent spots on each cell culture slide were investigated. The Human Investigation Review Board
of the Ludwig-Maximilians-University Munich approved the study. Immunocytochemistry Expression of ER, ER, PR-A and PR-B on HUVEC was analyzed by using speciWc monoclonal antibodies (Table 1) and the ABC staining method (Vectastain Elite mouse-IgGKit, Vector, Burlingame, Canada). Staining intensity was investigated by using the semiquantitative Remmele score (Remmele et al. 1986). BrieXy, the IRS score was calculated by multiplication of optical staining intensity (graded as 0 = none, 1 = weak, 2 = moderate and 3 = strong staining) and the percentage of positive staining cells (0 = no staining, 1 · 10% of the cells, 2 = 11–50% of the cells, 3 = 51–80% of the cells and 4 ¸ 81% of the cells). The intensity and distribution pattern of the speciWc immunocytochemical staining reaction was evaluated by two blinded, independent observers without knowing the pathological evaluation of each specimen. HUVEC were cultivated under sterile conditions in chamber slide cultures Quadriperm (Nunc, Wiesbaden, Germany) for up to 72 h, dried, wrapped and stored at ¡80°C. After thawing, cells were brieXy Wxed with methanol/ethanol (Merck, Darmstadt, Germany; 5% in PBS, 5 min). Slides were incubated in methanol/H2O2 (30 min) to inhibit endogenous peroxidase activity, washed in PBS (5 min) and treated with goat serum (20 min, RT) to reduce non-speciWc background staining. Incubation with the primary antibody (Table 1) was done overnight at 4°C. Sections were then incubated with the biotinylated secondary anti-mouse antibody (1 h, RT) and avidin-biotinylated peroxidase (45 min, RT). Between each step, the sections were washed with PBS (phosphate buVered saline, pH 7.4), three times. Peroxidase staining reaction was done with diaminobenzidine/H2O2 (1 mg/ml; 5 min) and stopped in tap water (10 min). Sections were counterstained in hematoxylin (1 min) and then coverslipped. In controls, the primary antibody was replaced with pre-immune mouse serum. Positive and negative controls were always included. The slides were Wnally embedded in mounting buVer and examined with a Zeiss (Jena, Germany) Axiophot photomicroscope. The level of ER/PR expression was determined in a blinded
Table 1 Antibodies used for immunocytochemisty Antibody
Clone
Isotype
Dilution
Source
ER
ER1D5
Mouse IgG1
1:50
Immunotech, Prague, Czech Republic Serotec, Düsseldorf, Germany
ER
PPG5/10
Mouse IgG2a
1:600
PR-A
1A6
Mouse IgG1
1:50
Immunotech, Prague, Czech Republic
PR-B
SAN27
Mouse IgG1
1:50
Novocastra, Newcastle, UK
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fashion by two independent observers in one run with identical staV, equipment, and chemicals. From each section, Wve digital pictures were taken at random of diVerent places of stained HUVEC cells (200fold magniWcation; 3CCD color camera; Axiocam, and examined with a Zeiss Axiophot photomicroscope, Carl Zeiss, Jena, Germany).
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60°C. The ABI PRISM 7500 Fast (Applied Biosystems, Weiterstadt, Germany) was used to perform the PCR assays. QuantiWcation was carried out by the Ct-method using glyceraldehyde phosphate dehydrogenase (GAPDH) as housekeeping gene (Hs99999905_m1 assay for GAPDH mRNA detection, Applied Biosystems, Weiterstadt, Germany). Statistics
RNA extraction from HUVECs A total amount of 1 £ 106 HUVEC stimulated with either 10 nmol/ml E2 or 320 nmol/ml Progesterone and in addition 1 £ 106 normal control HUVEC were used for extraction of mRNA. Total RNA was extracted by NucleoSpin®RNAII Kit (Macherey-Nagel, Düren, Germany), according to the manufacturer⬘s protocol. PuriWed RNA was quantiWed and evaluated for purity by UV spectrometry.
The SPSS/PC software package version 15.0 was used for collection, processing, and statistical data analysis. Statistical analysis was performed using the non-parametrical Wilcoxon test for comparison of paired samples. P < 0.05 values were considered statistically signiWcant.
Results
Reverse transcription
Estrogen receptor alpha (ER)
Reverse transcription (RT) was carried out with the “High Capacity cDNA Reverse Transcription Kit” (Applied Biosystems, Weiterstadt, Germany) according to the protocol in a mastercycler gradient (Eppendorf, Hamburg, Germany). RT conditions were: 10 min 25°C, 2 h 37°C, 5 s 85°C and 4°C on hold.
HUVEC showed no expression of ER after cultivation for up to 72 h (Fig. 1a). In addition, cells were stimulated with 1, 10, and 100 pmol/ml and 1, 10, and 100 nmol/ml E2, respectively. However, after cultivation for 72 h, again no expression of ER on HUVEC was detected. Breast cancer cells (MCF7) served as positive controls for ER staining (Fig. 1b).
Quantitative RT-PCR (TaqMan®) Estrogen receptor beta (ER) RT-PCR reactions were performed in quadruplicate in optical 96-well reaction microtiter plates covered with optical caps, in a volume of 20 l containing 1 l TaqMan® Gene Expression Assay 20£ (Hs00230957_m1 for ER- mRNA detection and Hs00172183_m1 for PR-A mRNA detection both Applied Biosystems, Weiterstadt, Germany), 10 l TaqMan® Fast Universal PCR Master Mix 2£ (Applied Biosystems, Weiterstadt, Germany), 1 l template and 8 l H2O (DEPC treated DI water, Sigma, Taufkirchen, Germany). Thermical cycling conditions were: 20 s at 95°C, followed by 40 cycles of ampliWcation with 3 s at 95°C and 30 s at
In contrast to ER, HUVEC did express ER while being cultured for 72 h (Fig. 2a). Furthermore, expression of ER was signiWcantly upregulated after administration of 1, 10, and 100 pmol/ml E2 (Fig. 2b–d) and in addition 1 and 10 nmol/ml E2 (P < 0.044) (Fig. 2e, f), respectively. Unstimulated controls exhibited ER expression in up to 38% of the cultured cells. HUVEC stimulated with 1 and 10 pmol/ml E2 showed ER expression in up to 70% of the cultivated cells whereas higher levels of E2
Fig. 1 HUVEC cells do not express ER in vitro without (a) or with supplementation of E2. MCF-7 breast cancer cells served as positive controls for ER staining (b), both £25 lens
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Fig. 2 HUVEC showed a low expression of ER in unstimulated cells in vitro (a). In cells stimulated with 0.001 and 0.01 nmol/ml E2 a moderate elevated expression of ER (b, c) was seen, whereas HUVEC stimulated with higher concentrations of E2 (0.1, 1 and 10 nmol/ml, d–f showed a strong expression of ER; all pictures £25 lens
(0.1–10 nmol/ml) lead to strong ER expression in over 90% of the investigated cells. Progesterone receptor A (PR-A) In contrast to ER, HUVEC did express PR-A (Fig. 3a). Expression of PR-A was found in 50–60% of unstimulated HUVEC after cultivation for 72 h. Addition of progesterone in concentrations of 0.1 and 1 nmol/ml lead to an increase of PR-A expression up to 65% (Fig. 3b, c), whereas concentrations of 10 and 100 nmol/ml increased PR-A expression in more than 90% of the cells (Fig. 3d, e).
RT-PCR ER and PR-A mRNA expression were analyzed in either E2 or progesterone stimulated HUVEC and normal control HUVEC by quantitative RT-PCR. ER mRNA expression was increased 2.64-fold (P = 0.043) in E2 stimulated HUVEC compared to normal controls and PR-A mRNA was 3.44 (P = 0.028) fold higher in progesterone stimulated HUVEC compared to normal controls (Fig. 5).
Discussion Progesterone receptor B (PR-B) HUVEC did not express PR-B at all (Fig. 4a). Neither in cultured nor in progesterone-stimulated HUVEC expression of PR-B could be detected. Endometrial cancer cells (Ishikawa) served as positive controls for PR-B staining (Fig. 4b).
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Within our study, we were able to demonstrate that HUVEC express ER and PR-A, whereas no expression of ER and PR-B could be detected by immunocytochemistry. Focusing investigations on the eVects of female sex hormones on HUVEC, there are methodical diVerences in the
Histochem Cell Biol (2008) 130:399–405
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Fig. 3 HUVEC showed a mediate expression of PR-A in unstimulated cells in vitro (a). Stimulation with 0.1 nmol/ml progesterone lead to a elevated expression of PR-A (b), whereas HUVEC stimulated with higher concentrations of progesterone 1 nmol/ml (c), 10 nmol/ml (d) and 100 nmol/ml (e) showed a strong expression of PR-A; all pictures £25 lens
Fig. 4 HUVEC cells do not express PR-B in vitro without (a) or with supplementation of progesterone. Ishikawa endometrial cancer cells served as positive controls for PR-B staining (b), both £25 lens
conWrmation of ER and PR expression. In 1998, Jensen et al. performed immunocytochemistry and real time PCR to demonstrate that HUVEC lack expression of the classical ER (Jensen et al. 1998). After the appearance of the non classical ER was published, Evans et al. demonstrated weak mRNA expression of ER and ER in HUVEC in the year 2002 (Evans et al. 2002b).
In the same year, Tatsumi et al. showed PR-A and PR-B mRNA expression on HUVEC by real time PCR (Tatsumi et al. 2002). Furthermore, Herve et al. 2006 found no ER mRNA expression and only weak ER mRNA expression on HUVEC (Herve et al. 2006). However, since speciWc monoclonal antibodies for the detection of ER and ER have been available, no
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Fig. 5 Expression of ER and PR-A mRNA in HUVEC stimulated with either E2 or progesterone analyzed with quantitative RT-PCR (TaqMan®)
systematic immunocytochemical studies on the expression of these receptors in HUVEC were published. Lei et al. (2001) investigated the regulation of growthregulated oncogene (GRO ) expression by E2 in HUVEC. Tamoxifen as ER inhibitor abrogated the downregulation of GRO induced by E2. The authors concluded that GRO is regulated by ER, as Tamoxifen abolished the E2 eVect in more than 50%. Beside Tamoxifen, also other ER antagonists (i.e., ICI 182.780) were investigated to antagonize possible eVects of E2 and other estrogens on HUVEC (Oviedo et al. 2005; Florian et al. 2004; Hayashi et al. 1995). Nevertheless, a clear correlation between estrogenic eVects and receptor expression was not accomplished. Christian et al. (2006) investigated ER expression in the coronary arteries of pre- and postmenopausal women (autopsy) and the correlation to coronary calciWcation. They were able to show that intimal ER, but not ER expression was correlated with coronary calciWcation and artherosclerosis. Therefore, we conclude that the known estrogenic eVects in HUVEC were mediated by the nonclassical ER. In addition, little is known on the eVects of gestagens in HUVEC. Keck et al. (1998) studied the eVects of steroids on interleukin-6 (IL-6) production and on proliferation of HUVEC. Progesterone had no measurable eVects on IL-6 expression and did not aVect the proliferation rate of endothelial cells. Tatsumi et al. (2002) showed that in HUVEC mRNA expression of both isoforms of PR, (PR-A and PR-B) as well as androgen receptor could be detected. Addition of progesterone or dienogest did not aVect IL-1-stimulated ICAM-1 or VCAM-1 expression. In contrast, medroxyprogesterone acetate (Chardonnens et al. 1999), norethindrone acetate and levonorgestrel dose-dependently increased cell adhesion molecules. In addition, Hermenegildo et al.
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(2005b) showed that progesterone and MPA increased HUVEC prostacyclin production in a PR-dependent manner, by enhancing COX-1 and COX-2 expression and activities. Taken together, progesterone action on HUVEC seems to be mediated predominantly via PR although only investigations on mRNA expression of both isoforms of PR (PRA and PR-B) were performed. Within our study we could clearly show that addition of progesterone leads to an upregulation of PR-A in HUVEC whereas PR-B is not expressed in these cells. In summary, our study showed that eVects of female sex hormones on cell cycle, activation and apoptosis of HUVEC are mediated via the ER and not the ER, whereas progesterone acts via PR-A. Acknowledgments This work is part of the doctoral thesis of Gitti Saadat. Bettina Toth was supported by “Friedrich Baur-Stiftung”, “Förderung für Forschung und Lehre” (FöFoLe), “Hochschul-Wissenschafts Programm” and “LMUexcellent Mentoring Programm”, Ludwig-Maximilians-University, Munich, Germany. We gratefully acknowledge the following colleagues: (1) from the Department of Obstetrics and Gynecology- Maistrasse, Ludwig-Maximilians-University: Susanne Kunze and Christina Kuhn for technical assistance, (2) from the Department of Internal Medicine III, Großhadern, LudwigMaximilians-University: Verena Pihusch and Marius Penovici for helpful discussion concerning HUVEC cell culture
References Akarasereenont P, Techatraisak K, Thaworn A, Chotewuttakorn S (2000) The induction of cyclooxygenase-2 by 17beta-estradiol in endothelial cells is mediated through protein kinase C. InXamm Res 49:460–465 Brosens JJ, Tullet J, Varshochi R, Lam EW (2004) Steroid receptor action. Best Pract Res Clin Obstet Gynaecol 18:265–283 Chardonnens D, Cameo P, Aubert ML, Pralong FP, Islami D, Campana A, Gaillard RC, Bischof P (1999) Modulation of human cytotrophoblastic leptin secretion by interleukin-1alpha and 17beta-oestradiol and its eVect on HCG secretion. Mol Hum Reprod 5:1077– 1082 Chen W, Lee JY, Hsieh WC (2002) EVects of dexamethasone and sex hormones on cytokine-induced cellular adhesion molecule expression in human endothelial cells. Eur J Dermatol 12:445– 448 Christian RC, Liu PY, Harrington S, Ruan M, Miller VM, Fitzpatrick LA (2006) Intimal estrogen receptor (ER) beta, but not ERalpha expression, is correlated with coronary calciWcation and atherosclerosis in pre- and postmenopausal women. J Clin Endocrinol Metab 91:2713–2720 Evans MJ, Lai K, Shaw LJ, Harnish DC, Chadwick CC (2002a) Estrogen receptor alpha inhibits IL-1beta induction of gene expression in the mouse liver. Endocrinology 143:2559–2570 Evans MJ, Harris HA, Miller CP, Karathanasis SK, Adelman SJ (2002b) Estrogen receptors alpha and beta have similar activities in multiple endothelial cell pathways. Endocrinology 143:3785– 3795 Florian M, Lu Y, Angle M, Magder S (2004) Estrogen induced changes in Akt-dependent activation of endothelial nitric oxide synthase and vasodilation. Steroids 69:637–645
Histochem Cell Biol (2008) 130:399–405 Hayashi T, Yamada K, Esaki T, Kuzuya M, Satake S, Ishikawa T, Hidaka H, Iguchi A (1995) Estrogen increases endothelial nitric oxide by a receptor-mediated system. Biochem Biophys Res Commun 214:847–855 Hermenegildo C, Oviedo PJ, Garcia-Perez MA, Tarin JJ, Cano A (2005a) EVects of phytoestrogens genistein and daidzein on prostacyclin production by human endothelial cells. J Pharmacol Exp Ther 315:722–728 Hermenegildo C, Oviedo PJ, Garcia-Martinez MC, Garcia-Perez MA, Tarin JJ, Cano A (2005b) Progestogens stimulate prostacyclin production by human endothelial cells. Hum Reprod 20:1554–1561 Herve MA, Meduri G, Petit FG, Domet TS, Lazennec G, Mourah S, Perrot-Applanat M (2006) Regulation of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR by estradiol through VEGF in uterus. J Endocrinol 188:91–99 Hillebrand U, Hausberg M, Stock C, Shahin V, Nikova D, Riethmuller C, Kliche K, Ludwig T, Schillers H, Schneider SW, Oberleithner H (2006) 17beta-estradiol increases volume, apical surface and elasticity of human endothelium mediated by Na+/H+ exchange. Cardiovasc Res 69:916–924 Jensen I, Rinaldo CH, Nordbo Berge L, Seternes OM, Moens U (1998) Human umbilical vein endothelial cells lack expression of the estrogen receptor. Endothelium 6:9–21 Keck C, Herchenbach D, PWsterer J, Breckwoldt M (1998) EVects of 17beta-estradiol and progesterone on interleukin–6 production and proliferation of human umbilical vein endothelial cells. Exp Clin Endocrinol Diabetes 106:334–339 Lei ZB, Li XY, Wang BC, Yang YF, You NZ, Sun J (2001) Regulation of growth-regulated oncogene alpha expression by estrogen in human endothelial cells. Acta Pharmacol Sin 22:1003–1006
405 Morales DE, McGowan KA, Grant DS, Maheshwari S, Bhartiya D, Cid MC, Kleinman HK, Schnaper HW (1995) Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model. Circulation 91:755–763 Mukherjee TK, Dinh H, Chaudhuri G, Nathan L (2002) Testosterone attenuates expression of vascular cell adhesion molecule-1 by conversion to estradiol by aromatase in endothelial cells: implications in atherosclerosis. Proc Natl Acad Sci USA 99:4055– 4060 Oviedo PJ, Hermenegildo C, Tarin JJ, Cano A (2005) Raloxifene promotes prostacyclin release in human endothelial cells through a mechanism that involves cyclooxygenase-1 and -2. Fertil Steril 83:1822–1829 Remmele W, Hildebrand U, Hienz HA, Klein PJ, Vierbuchen M, Behnken LJ, Heicke B, Scheidt E (1986) Comparative histological, histochemical, immunohistochemical and biochemical studies on oestrogen receptors, lectin receptors, and Barr bodies in human breast cancer. Virchows Arch A Pathol Anat Histopathol 409:127–147 Simoncini T, Fornari L, Mannella P, Varone G, Caruso A, Garibaldi S, Genazzani AR (2005) DiVerential estrogen signaling in endothelial cells upon pulsed or continuous administration. Maturitas 50:247–258 Tatsumi H, Kitawaki J, Tanaka K, Hosoda T, Honjo H (2002) Lack of stimulatory eVect of dienogest on the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by endothelial cell as compared with other synthetic progestins. Maturitas 42:287–294
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