VEGF signaling is disrupted in the hearts of mice lacking estrogen receptor alpha

European Journal of Pharmacology 641 (2010) 168–178

Contents lists available at ScienceDirect

European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Cardiovascular Pharmacology

VEGF signaling is disrupted in the hearts of mice lacking estrogen receptor alpha Subrina Jesmin a,b,c,d,e,⁎, Chishimba N. Mowa a,b, Sayeeda Nusrat Sultana a,b, Nobutake Shimojo d, Hiroko Togashi d,e, Yoshio Iwashima f, Norihiro Kato a,b, Akira Sato d, Ichiro Sakuma d,e, Michiaki Hiroe a,b, Yuichi Hattori g, Naoto Yamaguchi h, Hiroyuki Kobayashi c a

Department of Gene Diagnostics and Therapeutics, Research Institute, International Medical Center of Japan, Tokyo, Japan Department of Cardiology, Research Institute, International Medical Center of Japan, Tokyo, Japan Program of Master of Clinical Biomedical Science, Tokai University, Isehara, Japan d Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo, Japan e Department of Pharmacology, Hokkaido University School of Medicine, Sapporo, Japan f Department of Geriatirc Medicine, Osaka University Graduate School of Medicine, Osaka, Japan g Department of Pharmacology, School of Medicine, University of Toyama, Toyama, Japan h Center for Medical Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan b c

a r t i c l e

i n f o

Article history: Received 28 August 2009 Received in revised form 26 March 2010 Accepted 27 May 2010 Available online 4 June 2010 Keywords: Heart Estrogen Estrogen receptors VEGF and its signaling cascade Coronary capillary density Apoptosis

a b s t r a c t Estrogen has widely been credited for cardioprotection in women. However, the exact mechanisms that underlie these beneficial estrogenic effects are not completely understood. Here, we sought to: 1) elucidate estrogen's influence on levels of vascular endothelial growth factor (VEGF), a key regulator of cardiovascular processes, and components of its basic signaling machinery (VEGF receptors, Akt, and eNOS) in the heart, and 2) delineate the specific estrogen receptor signaling pathway that mediates its beneficial effects using mice lacking either estrogen receptor alpha or estrogen receptor beta. We analyzed pattern of VEGF signaling and the associated coronary capillary density in the hearts of wild-type (WT), estrogen receptor alpha knockout (ERα-KO), and estrogen receptor beta knockout (ERβ-KO) female mice. Deletion of estrogen receptor alpha causes a marked decrease in coronary capillary density compared to wild-type (WT) mice, while that of estrogen receptor beta had a minimal effect. Consistent with reduced coronary capillary density, cardiac expression levels of VEGF and its signaling molecules (two receptors, phosphorylated Akt, and eNOS) in ERα-KO mice were reduced to half of WT, in contrast to ERβ-KO mice that only showed a slight decrease. Moreover, activity of eNOS was greatly lowered in ERα-KO mice. These data suggest that estrogen acts largely via estrogen receptor alpha to regulate VEGF transcription and possibly components of its basic signaling and ultimately, the development of coronary microvasculature in the heart. This molecular and histological data, in part, sheds some insights into potential mechanisms that may likely underlie estrogen's cardioprotective effects. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Estrogen is a predominant circulating sex steroid hormone in females and has been widely credited for cardioprotection in women (Ballard and Edelberg, 2005). This conclusion is supported by a body of evidence that includes: 1) a clear association between decreased levels of circulating estrogen (induced by natural or surgical menopause) with greater likelihood of developing cardiovascular diseases (Nakamura et al., 2005), which appears to be reversed by estrogen replacement therapy (Sumino et al., 2005); 2) greater incidence of

⁎ Corresponding author. Division of Gene Therapeutics, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. Tel.: + 81 3 3202 7181x2897; fax: + 81 3 3202 7364. E-mail address: [email protected] (S. Jesmin). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.05.020

cardiovascular diseases in men, who have a much lower level of circulating estrogen, than in women (Arnlov et al., 2006); and 3) localization of estrogen receptors in cardiovascular tissues (Haynes et al., 2002). Overall, the biological effects of estrogen are mediated by two estrogen receptor molecules, the classical receptor estrogen receptor alpha and the more recently discovered estrogen receptor beta (Kuiper et al., 1996; Walter et al., 1985), whose expression varies in different tissues and species (Grandien, 1996; Kuiper et al., 1997; Saunders et al., 1997). In murine, as well as in human myocardial tissues, estrogen receptor alpha is better characterized and established than estrogen receptor beta (Saunders et al., 1997; Mercier et al., 2002; Nuedling et al., 1999). To date, the exact mechanisms that underlie the cardioprotective effects of estrogen are not completely understood and recently, key clinical studies, namely the Women's Health Initiative (WHI) study and the Heart and Estrogen/Progestin Replacement Study (HERS), did not find any significant benefits of estrogen

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

replacement therapy on cardioprotection (Colditz et al., 1987; Kannel et al., 1976; Langer et al., 2005; Rossouw et al., 2002; Stevenson, 2004; Wenger, 2005; Writing Group for the Postmenopausal Estrogen/ Progestin Interventions (PEPI) Trial, 1995). Thus, there is a need for a more detailed understanding of the basic mechanisms underlying the estrogenic effects on the heart. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine and mitogen for endothelial cells (Ferrara, 2002; Yoon et al., 2004). The angiogenic actions of VEGF are thought to be mediated primarily by two cell surface receptors, namely fetal liver kinase 1 [Flk-1/kinase domain region (KDR)] and fms-like tyrosine kinase (Flt1), which are tyrosine protein kinases expressed primarily, but not exclusively, on endothelial cells (Matsumoto and Claesson-Welsh, 2001; Matsumoto and Mugishima, 2006). At present, the mitogenic actions of VEGF are thought to be mediated largely by KDR (Matsumoto and Claesson-Welsh, 2001; Matsumoto and Mugishima, 2006). It is possible that estrogen may exert its cardioprotective effects via VEGF, because: 1) the regulatory sequences of VEGF gene contain estrogen responsive elements (ERE) and 2) estrogen has been shown to induce VEGF mRNA transcription in different tissue types (Buteau-Lozano et al., 2002). For instance, we recently demonstrated significant reductions in levels of VEGF (and KDR) and total coronary capillary density in the hearts of middle-aged female rats subjected to ovariectomy (Jesmin et al., 2002). Importantly, estrogen replacement therapy nearly completely restored both reduced VEGF expression and total capillary density to pre-ovariectomy (intact) levels (Jesmin et al., 2002). Collectively, these data suggest that VEGF stimulates angiogenesis (i.e., formation of new capillaries from existing coronary vessels) in middle-aged females, thus prompting us to propose that estrogen deficiency-induced reduction in VEGF and KDR levels in coronary vessels may underlie the mechanism or pathogenesis of coronary heart disease in postmenopausal women. Here, we build on our recent findings (Jesmin et al., 2002), and sought to delineate the specific pathway that mediate VEGF signaling and associated expansion of myocardial capillary density in the heart using female mice lacking either estrogen receptor alpha or estrogen receptor beta. Our results show that estrogen receptor alpha, but not estrogen receptor beta, is the predominant receptor that mediates the effects of estradiol in female mouse hearts. These effects possibly include cardioprotection and its stimulatory effects on transcription of VEGF and, likely, components of VEGF's basic signaling machinery. Since it has been claimed that estrogen can block apoptosis of cardiomyocytes in ovariectomized mice with myocardial infarction, which consequently improves the prognosis of heart failure (Patten et al., 2004), we were also interested in delineating possible involvement of estrogen receptor subtypes in the apoptotic events in female mouse hearts. 2. Materials and methods

169

1993). Resultant chimeras were backcrossed to C57BL/6 for ten generations (N10). Taconic received stock from the NIEHS in 1998, and animals were transferred to Taconic for commercial production in 2000. Homozygotes and heterozygotes were generated by mating male and female heterozygous ERα-KO mice. ERα-KO mice used in the present study completely lack functional estrogen receptor alpha protein, and survive into adulthood, but are infertile, indicating the necessity of estrogen for proper gonadal development in both sexes (data sheet, Taconic). The ERβ-KO mouse was developed by Oliver Smithies and John Kreg of the University of North Carolina, Kenneth Korach of NIEHS and Jan-Ake Gustafsson of the Karolinska Institute (Krege et al., 1998). The model was created by targeting the Esr2 gene in ES cells of the BK4 subline of E14TG2a (129P2/OlaHsd) and injecting the targeted ES cells into C57BL/6 blastocysts. Resultant chimeras were backcrossed to C57BL/6 for eight generations (N8). Heterozygotes were intercrossed to generate homozygous ERβ-KO mice. Taconic received stock from NIEHS in January 2001 and animals were transferred to Taconic for commercial production in September 2001. The mice were derived by embryo transfer through backcrossing to C57BL/6NTac (N9). The colony is maintained by mating of homozygous males to heterozygous females. According to data sheet from Taconic, male and female homozygotes (ERβ-KO) completely lack functional estrogen receptor beta proteins, develop normally and survive into adulthood. The heart tissues were harvested under an overdose of pentobarbital (80 mg/kg), and immediately frozen in liquid nitrogen and stored at −80 °C. Some tissues were analyzed using either enzyme-linked immunosorbent assay (ELISA), immunoblotting, or real-time polymerase chain reaction (PCR), and the others were fixed overnight in 4% paraformaldehyde and routinely embedded in paraffin wax for analyses of capillary morphology or immunohistochemistry. For ELISA and immunoblotting, the extracted heart samples [left ventricular tissues] were minced and homogenized in PBS on ice, then centrifuged at 10,000 g for 15 min at 4 °C, and the resulting supernatants were stored at −80 °C. It should be noted that according to the heart weight, the required PBS was added to the heart sample (left ventricular sample) before homogenization. Then the total protein concentration of supernatant was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL) and based on the total protein concentration, data generated from the present experiments were normalized for each sample. 2.2. ELISA Levels of VEGF, KDR (VEGF R2), caspase-3 (R&D Systems, Minneapolis, MN, USA), and phospho-Akt (Thr 308 and Ser 473) (BioSource International, Inc., Camarillo, California, USA) were determined in supernatant of left ventricular extracts using the quantitative sandwich enzyme immunoassay technique according to the manufacturer's instructions. The values obtained from ELISA were normalized by total protein concentration for each sample and then used for statistical analysis.

2.1. Animals 2.3. Western blot analysis This study was conducted in accordance with the National Institutes of Health (NIH) guidelines on the use of laboratory animals and with approval of the Hokkaido University Graduate School of Medicine Animal Care and Use Committee. Estrogen receptor alpha knockout (ERα-KO) (001480-M-F c5BL/6-EstrA(tm) N10 Female, homozygous), estrogen receptor beta knockout (ERβ-KO) (001954-M-F B6.129Esr2btm1N Female, homogzygous), and age-matched wild-type (WT) female mice (B6-F c57BL/6NTac Female) were purchased from Taconic (New York, NY, USA). The mice were used at 16 weeks of age (n = 12 for each group). The ERα-KO mouse was developed by Kenneth S. Korach of the National Institute of Environmental Health Sciences (NIEHS) and colleagues in 1993 by targeting the Esr1 gene in E12TG2a ES cells and injecting the targeted cells into C57BL/6J blastocysts (Lubahn et al.,

The following commercially available antibodies were used: a) antihuman VEGF rabbit polyclonal antibody (Immuno-Biological Laboratories, Fujioka, Japan), b) anti-human Flt-1 rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), c) anti-human KDR rabbit polyclonal antibody (Santa Cruz Biotechnology), d) anti-mouse Ser473-phospho-Akt rabbit polyclonal (Cell Signaling, Beverly, MA, USA), e) anti-human endothelial nitric oxide synthase (eNOS) rabbit polyclonal antibody (Affinity BioReagents, Golden, CO, USA), f) antibovine basic fibroblast growth factor (bFGF) mouse monoclonal antibody (Upstate Biotechnology, Lake Placid, NY, USA), g) antihuman Bcl-2 rabbit polyclonal antibody (Santa Cruz Biotechnology), h) rabbit polyclonal Akt antibody (Cell Signaling), i) mouse estrogen

170

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

receptor alpha monoclonal antibody (Novocastra Laboratories, Benton Lane, Newcastle upon Tyne, UK), j) rabbit antihuman estrogen receptor beta polyclonal antibody (Affinity BioReagents), and k) anti-Xenopus laevis beta-actin mouse monoclonal antibody (Abcam, Cambridge, MA, USA). The protein concentration of supernatant was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Immunoblotting was performed as described in our previous report (Jesmin et al., 2004). Samples of left ventricular tissue homogenates were run on a 7.5%–10% SDS-polyacrylamide gel and electrotransferred to a polyvinylidene diflouride filter membrane. The membrane was then blotted with the indicated antibody and processed via chemiluminescence. 2.4. Immunofluorescence staining For immunohistochemical determination of target molecules, frozen 8-μm thick sections were exposed to the fluorescent secondary antibody after overnight incubation with each primary antibody, as fully described in our previous reports (Jesmin et al., 2002, 2004). Immunofluorescent images were observed under laser scanning confocal imaging system (Jesmin et al., 2002, 2004). 2.5. Nitric oxide (NO) assay NO was indirectly detected in supernatant of left ventricular tissues as nitrite using a NO (NO2−/NO3−) Assay kit (R&D Systems). In this method, at first 200 μl left ventricular supernatant and 200 μl of reaction buffer (1×) were taken in an ultrafilter (e.g. CENTRISART CUTT-OFF 10000 from SARTORIUS, Cat. No. 13 239-E) and centrifuged at 20 °C for 45 min (1250 × g respectively 4000 rpm, r = 7 cm) and then ultrafiltrate was collected and used in the test. The nitrate present in the sample was reduced to nitrite by reduced nicotinamide adenine dinucleotide phosphate in the presence of the enzyme nitrate reductase. The nitrite formed reacted with sulfanilamide and N-(1naphthyl)-ethylenediamine dihydrochloride to give a red-violet diazo dye. The diazo dye was measured at 550 nm, on the basis of its absorbance within the visible range. The generated value was normalized by total protein concentration for each sample. 2.6. Measurement of 17ß-estradiol in plasma and left ventricular tissues by enzyme immunoassay (EIA) For determining tissue estrogen levels, the left ventricular tissues were washed in cold PBS just after tissue harvest and snap frozen on dry ice. The tissues were homogenized in 0.1 molar (M) Tris–HCl (pH 7.2) buffer by a high speed homogenizer, at a consistent ice-cold temperature, centrifuged at 1000 × g for 15 min to pellet any insoluble material, and thereafter the protein concentrations were measured. Finally, the local tissue concentrations of 17ß-estradiol were quantified using an enzyme immunoassay kit (ESTRADIOL EIA, PANTEX, Santa Monica, CA, USA), according to the manufacturer's instructions. The specificity of this kit has previously been confirmed in our laboratory using various types of rat tissue including serum, plasma, ovary, brain and penis. The values obtained were compared with those of the Rodent Estradiol ELISA Test Kit (Endocrine Technologies, Inc., Newark, CA, USA). The lowest level of 17ß-estradiol detectable by the kit used in this study is 4.6 pg/ml. Intra-assay and interassay coefficients of variation (%CV) were 5.9 and 5.2 respectively. 2.7. RNA preparation and real-time quantitative PCR Total RNA from left ventricular tissues was isolated using RNeasy (Qiagen, Tokyo, Japan). After the RNA was isolated, treated with DNase I, and quantified, it was reverse transcribed to cDNA by omniscript

reverse transcriptase using a first-strand cDNA synthesis kit (Qiagen). The reaction was performed at 37 °C for 60 min. The levels of mRNA of target genes were analyzed by real-time quantitative PCR with TaqMan probe using an ABI Prism 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster, CA, USA). The gene-specific primers and TaqMan probes were synthesized from Primer Express v.1.5 software (Perkin-Elmer), according to the published cDNA sequences for each gene. The PCR mixture (25 μl total volume) consisted of 450 nM of both forward and reverse primers for each gene (Perkin-Elmer), 200 nM of FAM-labeled primer probes (Perkin-Elmer), and TaqMan Universal PCR Master Mix (Perkin-Elmer). Each PCR amplification was performed in triplicate, using the following profile: 1 cycle of 95 °C 10 min, and 40 cycles of 94 °C for 15 s, and 60 °C for 1 min. The quantitative values of target mRNAs were normalized by β-actin mRNA. VEGF forward: 5′-TGAGACCCTGGTGGACATCTT-3′ VEGF reverse: 5′-CACACAGGACGGCTTGAAGA-3′ VEGF probe: 5′-CCCCGATGAGATAGAGTAT-3′ KDR forward: 5′-GAAACTGAATGGCACCGTGTT-3′ KDR reverse: 5′-GCAGGGAGGCATTCTGGAAT-3′ KDR probe: 5′-CTAACAGCACAAACGACATCT-3′ Flt-1 forward: 5′-AGGAAACCACAGCAGGAAGAC-3′ Flt-1 reverse: 5′-GCGAGCCATCTTTTAACCATACGAT-3′ Flt-1 probe: 5′-CCATGAAAGTGAAGGCCTT-3′ eNOS forward: 5′-GATCCTAACTTGCCTTGCATCCT-3′ eNOS reverse: 5′-TGTAATCGGTCTTGCCAGAATCC-3′ eNOS probe: 5′-CTGGTATTGCACCCTTCC-3′ Bcl-2 forward: 5′-TGCGCTCAGCCCTGTG-3′ Bcl-2 reverse: 5′-GGTAGCGACGAGAGAAGTCATC-3′ Bcl-2 probe: 5′-CCACCTGTGGTCCACCTG-3′ β-actin forward: 5′-GGCCGGGACCTGACA-3′ β-actin reverse: 5′-GCTGTGGTGGTGAAGCTGTAG-3′ β-actin probe: 5′-ACTACCTCATGAAGATCC-3′ 2.8. Cell death detection ELISA Apoptosis was studied by measuring the cytoplasmic histoneassociated DNA fragments (mono- and oligo-nucleasomes) in left ventricular tissues by photometric enzyme immunoassay (cell death detection ELISA; Roche Diagnostics Division, Basel, Switzerland). 2.9. TUNEL assay Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was performed using the Apoptosis in situ Detection Kit (Wako, Osaka, Japan). 2.10. Capillary morphology Our previous study has shown that histochemical staining with the lectin Griffonia simplicifolia (GSA-B4) is a sensitive and reliable method to visualize capillary vasculature (Jesmin et al., 2002). Eight-micrometer-thick serial frozen coronal sections of left ventricular tissues were stained with GSA-B4 (Sigma Chemical, St. Louis, MO, USA) and then incubated in diaminobenzidine enhancing solution (Vector Laboratories, Burlingame, CA, USA). Vascular endothelium was stained with lectin, which stained capillaries as black/dark brown dots. Sections were examined using a microscope (Olympus, Tokyo, Japan), and counts were carefully made of stained capillaries as previously described (Jesmin et al., 2002). Capillary density was also assessed microscopically on 5-μm-thick deparaffinized tissue sections that were immunostained by anti-von

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

Willebrand factor (vWF) antibody (Dako, Hamburg, Germany) or CD34 (Santa Cruz Biotechnology). The antibodies were made visible by a secondary exposure of the sections to Cy3-conjugated AffiniPure donkey anti-rabbit IgG (Jackson Immuno Research Laboratories, West Grove, PA, USA).

2.11. Statistical analysis Results were expressed as mean ± S.D. Data were compared using one-way ANOVA. Post hoc comparisons were made with Fisher's protected least significance t-test for multiple comparisons. Differences were considered significant at P b 0.05.

3. Results 3.1. General features of experimental animals At 16 weeks of age, the body weights of ERα-KO (24.2 ± 1.6 g) and ERβ-KO mice (26.4 ± 1.8 g) were significantly (P b 0.01) greater than that of WT mice (21.8 ± 1.1 g). The heart weight was also significantly (P b 0.01) larger in ERα-KO (111.25 ± 12.3 mg) and ERβ-KO (115.5 ± 8.2 mg) compared to WT mice (99.07 ± 4.2 mg), but when we calculated heart weight-to-body weight ratio, notably there was no significant difference among the study groups (WT:ERα-KO:ERβ-KO: 0.22 ± 0.013:0.219 ± 0.014:0.230 ± 0.016). The plasma estrogen level was significantly (P b 0.01) higher in ERα-KO (2.5 ± 2.0 pg/ml) than in WT (0.8 ± 0.2 pg/ml) and ERβ-KO mice (0.9 ± 0.4 pg/ml). The estrogen levels in left ventricular tissues were 4.1 ± 0.8, 4.8 ± 0.2, and 5.1 ± 0.5 pg/mg in WT, ERα-KO, and

171

ERβ-KO mice, respectively, i.e., the difference between groups were statistically insignificant. 3.2. Expression of VEGF VEGF in plasma was significantly down-regulated in both ERα-KO and ERβ-KO mice (Fig. 1A). Left ventricular VEGF expression was remarkably down-regulated in ERα-KO mice, but was only slightly decreased in ERβ-KO compared to WT mice at both protein and mRNA levels, as determined by ELISA, immunoblotting and real-time PCR (Fig. 1B–D). 3.3. Expression of VEGF receptors KDR and Flt-1 Levels of KDR, an angiogenic receptor of VEGF, were in parallel to those of VEGF in the hearts of both ERα-KO and ERβ-KO mice. Thus, left ventricular expression levels of KDR protein in ERα-KO went down by 50% compared to WT mice, as demonstrated by ELISA (Fig. 2A), while ERβ-KO mice showed a minimal decrease (Fig. 2A–C). These changes in KDR protein were consistent with levels of its mRNA (Fig. 2C). Left ventricular levels of Flt-1 protein, a vascular permeability receptor of VEGF, also decreased by 60% and 40% in ERα-KO and ERβ-KO, respectively, in comparison with WT mice, when analyzed by immunoblotting (Fig. 2D). However, the Flt-1 mRNA levels were higher in both ERα-KO and ERβ-KO than in WT mice (data not shown). We do not have a clear understanding of this paradox, but it may be related to the presence of endogenous inhibitions at various stages of translation (Mallat et al., 2001) and/or rapid proteolytic degradation of Flt-1 protein by protease-like pigment epithelium-derived factor (Perry and Meyuhas, 1990).

Fig. 1. Plasma VEGF level (A) and protein and mRNA expression levels of VEGF in left ventricular tissues (B–D) from WT, ERα-KO, and ERβ-KO mice. ELISA (A, B) and Western blot analysis (C) were performed for protein expression. In immunoblotting, β-actin served as loading control. Total RNA isolated from left ventricular tissues was analyzed by real-time quantitative PCR (D). Values are mean ± S.D. *P b 0.001 vs. WT; #P b 0.001 vs. ERα-KO; n = 12.

172

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

Fig. 2. Protein and mRNA expression levels of KDR (A–C) and protein expression level of Flt-1 (D) in left ventricular tissues from WT, ERα-KO, and ERβ-KO mice. ELISA (A) and Western blot analysis (B, D) were performed for protein expression, and immunoreactive KDR (B) and Flt-1 (D) were detected as a single band with a molecular mass of 200 and 180 kDa, respectively. In immunoblotting, β-actin served as loading control. Total RNA isolated from left ventricle was analyzed by real-time quantitative PCR (C). Values are mean ± S.D. *P b 0.05 vs. WT; #P b 0.05 vs. ERα-KO; n = 12.

3.4. Changes in Akt phosphorylation, eNOS expression, and NO level

3.6. Coronary capillary morphology

The serine/threonine kinase Akt is an important downstream molecule of VEGF angiogenic signaling (Nuedling et al., 1999; Matsumoto and Claesson-Welsh, 2001). Left ventricular expression of Akt, dually phosphorylated at Thr308 and Ser473, was significantly reduced in ERα-KO, compared to ERβ-KO and WT mice (Fig. 3A–C). No apparent difference in total Akt level among groups was noted. Activation of Akt phosphorylation activates eNOS, which in turn induces NO production (Nuedling et al., 1999; Matsumoto and Claesson-Welsh, 2001). Left ventricular expression of eNOS was significantly less in ERα-KO than in WT mice both at protein (Fig. 3D) and mRNA (Fig. 3E) levels. NO levels in left ventricular tissues were also evidently lower in ERα-KO mice (Fig. 3F). The reduced levels of eNOS expression and NO were observed in ERβ-KO mice, but the reduction in left ventricular NO level was significantly less pronounced than that seen in ERα-KO mice (Fig. 3F).

Coronary capillary expansion was analyzed using specific markers for endothelia, vWF (brown color) and lectin (brick red). Lectin- or vWFstained endothelial cells in left ventricular tissues were less in ERα-KO than in WT and ERβ-KO mice (Fig. 5B). The graphical presentation of coronary capillary density was shown in Fig. 5C with the level of significance. Coronary capillary density calculated from lectin staining was significantly lower in ERα-KO than in WT and ERβ-KO mice. 3.7. Left ventricular expression of estrogen receptor alpha and estrogen receptor beta in female mice Both estrogen receptor subtypes were detected in left ventricular tissue of WT female mice by immunoblotting, with levels of estrogen receptor α being greater than those of estrogen receptor beta (Fig. 5A). 3.8. Apoptosis-related markers

3.5. Immunohistochemical detection of VEGF and its signaling molecules Immunohistochemistry revealed that the down-regulation of VEGF, KDR, eNOS, and phosphorylated Akt in left ventricular tissues was more marked in ERα-KO compared to ERβ-KO mice (Fig. 4). It should be noted that these molecules were expressed not only in coronary blood vessels, but also in cardiomyocytes.

To assess whether deletion of estrogen receptors causes or is associated with apoptotic cell death and delineate the estrogenic pathway mediating this effect in the heart, we performed cell death detection ELISA. The percentage of left ventricular cell death remained unchanged, regardless of whether estrogen receptor was deleted or not (Fig. 6A). The same results were obtained from TUNEL staining

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

173

Fig. 3. Analysis of phosphorylated Akt (pAkt), eNOS and NO levels in left ventricular tissues from WT, ERα-KO, and ERβ-KO mice. Levels of Akt phosphorylated at Ser473 (A, B) and Thr308 (C) were determined by ELISA (A, C) and immunoblotting (B). Protein (D) and mRNA expression (E) levels of eNOS from left ventricular tissues were determined by immunoblotting and real-time quantitative PCR, respectively. Left ventricular NO level (F) was determined by colorimetric assay. In immunoblotting, β-actin served as loading control. Values are mean ± S.D. *P b 0.05 vs. WT; #P b 0.001 vs. ERα-KO; n = 12.

(data not shown). Moreover, left ventricular expression levels of two important pro-apoptotic molecules, caspase-3 and Bax, were unaffected by deletion of estrogen receptors (Fig. 6B, C). In contrast, mRNA and protein expression of Bcl-2, which suppresses activation of caspase-3 protein through regression of cytochrome c release from mitochondria (Cai et al., 2006; Martinou et al., 2000), were significantly decreased in left ventricular tissues from ERα-KO and ERβ-KO compared with WT mice, with striking reductions in ERα-KO (Fig. 6D, E). 3.9. Expression of FGF-2 Finally, we investigated whether left ventricular expression of FGF-2, another important angiogenic growth factor, is altered by

deletion of estrogen receptors. Interestingly, it was found that FGF-2 expression in left ventricular tissues was also significantly diminished in ERα-KO compared to WT and ERβ-KO mice (Fig. 6F). 4. Discussion Here, we attempted to elucidate the molecular and histological characteristics associated with mechanisms likely to underlie the cardioprotective effects of estrogen and the specific estrogenic estrogen receptor pathway that mediates this effect. Expression and/or activity of VEGF and components of its basic signaling machinery (KDR, Flt-1, Akt, and eNOS) were down-regulated in the hearts of mice lacking estrogen receptor subtypes, estrogen receptor alpha and estrogen receptor beta. However, the reductions in the

174

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

Fig. 4. Representative immunofluorescent images for VEGF, KDR, pAkt and eNOS in left ventricular cross sections from WT, ERα-KO, and ERβ-KO mice. Positive staining was focused on coronary vessels. Magnification × 200.

angiogenic molecules were more pronounced in ERα-KO mice, suggesting that estrogen receptor alpha may be the primary subtype that mediates the beneficial cardiovascular effects of estrogen in females. These data corroborate with another set of data that showed a significant decrease in total coronary capillary density in ERα-KO mice compared to WT and ERβ-KO mice. Finally, we showed that deletion of either estrogen receptor alpha or estrogen receptor beta does not affect cell death in left ventricular tissues, as well as expression of key pro-apoptotic markers, such as caspase-3 and Bax. However, to our surprise, cardiac levels of Bcl-2, an important antiapoptotic marker, were significantly down-regulated in ERα-KO when compared to WT and ERβ-KO mice. Numerous animal studies have demonstrated the beneficial effects of estrogen on the vasculature (Nuedling et al., 1999; Lubahn et al., 1993; Reckelhoff et al., 2000; Williams and Suparto, 2004). For example, it has been shown that estrogen inhibits vascular remodeling in response to mechanical injury (Reckelhoff et al., 2000; Bar et al., 1993; Beldekas et al., 1981; Orshal and Khalil, 2004) and hyperlipidemia (Rifici and Khachadurian, 1992). However, the impact of estrogen on the heart is more complex and is influenced by multiple factors, and its cardioprotective effects are poorly defined. In animal models, we know that: 1) estrogen attenuates the development of cardiac hypertrophy in response to pressure overload (van Eickels et al., 2001), 2) estrogen decreases the inflammatory signaling and cardiac dysfunction following acute ischemia (Wang et al., 2006a,b), and 3)

ovariectomy induces significant reduction in total coronary capillary density and expression levels of VEGF and its receptor KDR in the hearts of middle-aged female rats (Jesmin et al., 2002), an effect reversed by estrogen replacement therapy (Jesmin et al., 2004). In women, although epidemiological reports have consistently supported the beneficial cardiovascular effects of estrogen, data from very recent randomized studies contradict the findings of these earlier reports (Colditz et al., 1987; Kannel et al., 1976; Langer et al., 2005; Rossouw et al., 2002; Stevenson, 2004; Wenger, 2005). Specifically, the idea that exogenous estrogen is cardioprotective in postmenopausal women has recently been challenged by the unexpected data generated by the Heart and Estrogen/Progestin Replacement Study (HERS) (Langer et al., 2005; Stevenson, 2004; Writing Group for the PEPI Trial, 1995). According to the HERS report, that hormone replacement therapy fails to demonstrate an overall cardioprotective action, and, to the contrary, increases the incidence of thromboembolism in postmenopausal women (Stevenson, 2004; Wenger, 2005). Another study, with similar outcomes, the Women's Health Initiative (WHI) study, indicates an increased risk of pulmonary embolism, coronary artery disease, and invasive breast cancer in postmenopausal women receiving hormone replacement therapy (Stevenson, 2004). Nonetheless, most investigators have noted that while these results are vital, their relevance to the traditional use of hormone replacement therapy is limited (Colditz et al., 1987; Kannel et al., 1976; Rossouw et al., 2002; Hulley et al., 1998). These discrepancies could be effectively resolved by improved knowledge and

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

Fig. 5. (A) Representative immunoblots for estrogen receptor alpha and estrogen receptor beta in left ventricular tissues from study mice. (B) Morphology of coronary capillaries in left ventricular sections from WT, ERα-KO, and ERβ-KO mice. Photomicrographs of left ventricular sections stained with vWF antibody appearing as brown dot (left panels) and stained with lectin appearing as black/dark brown dots (right panels) are shown. Magnification × 400. (C) Bar graph summarizing the total capillary density, obtained by the lectin staining method. Values are means ± S.D. (30 fields × 12 samples).*P b 0.05 vs. WT.

understanding of the basic science, precisely, the exact mechanisms underlying estrogen's effects on the physiology of the heart, both in vitro and in vivo. The animal models lacking estrogen receptors, as used in the present study, are ideal for a more precise dissection of estrogen's estrogen receptor mediated role in cardioprotection. The reductions in levels of VEGF and its signaling molecules in ERα-KO mouse hearts, as revealed here, suggest a role for estrogen in the cardiac physiology of females and that this effect is likely mediated by estrogen receptor alpha. This finding is consistent with reports of several studies using other body tissues (Kazi et al., 2005; Lee et al., 2004; Mueller et al., 2000) and plasma (Christodoulakos et al., 2004) from postmenopausal human subjects and animal models. For example, estrogen replacement therapy of ovariectomized rats induces VEGF expression in the brain (Jesmin et al., 2003). Estrogen is

175

also capable of inducing VEGF expression in rodent uterus in a rapid and dose-dependent manner (Mueller et al., 2000). Estrogen has also been shown to directly regulate VEGF gene transcription in endometrial cells and in Ishikawa adenocarcinoma cells (Mueller et al., 2000). As stated earlier, estrogen-induced gene transcription is estrogen receptor dependent, and is likely activated through a variant ERE localized 1.5 kb upstream from the VEGF transcription start site (Mueller et al., 2000). Site-directed mutagenesis of this ERE fully abrogates estrogen-induced VEGF mRNA expression, confirming the functional significance of this single motif (Mueller et al., 2000). More recent studies have utilized estrogen receptor knockout mice to show that estrogen receptor alpha but not estrogen receptor beta, mediates transcription of VEGF in endometrial tissue (Kazi et al., 2005). Interestingly, in our recent study, coronary VEGF levels were diminished in ovariectomized female middle-aged rat hearts, with a parallel change in expression of estrogen receptor beta subtype, but not estrogen receptor alpha (Jesmin et al., 2002). Although this appears contradictory to our present data, the apparent difference may be explained by the differential cellular distribution of the estrogen receptor subtypes in the cardiovascular system. Whereas, estrogen receptor beta appears to be the predominant subtype in blood vessels, estrogen receptor alpha appears to be the main subtype in cardiac muscles (Wang et al., 2006a,b). Also differences in the reproductive age of the animals used could also account for the discrepancy, i.e., middle-aged rats versus young estrogen receptor knockout mice or, alternatively, the absence of cross-talk/interaction between the two estrogen receptor subtypes in estrogen receptor knockout mice, which is known to influence the function of estrogen via each estrogen receptor in a tissue-dependent manner (Kuiper et al., 1997). It is possible that other factors other than those cited here, could account for this discrepancy. Finally, the fact that differences in VEGF concentration is more pronounced at the protein, and not mRNA, in all the three models, used in the present study suggests that post-translational regulation may be more important than transcriptional regulation. For instance, in lung epithelial cells, hypoxia increased stability and release of VEGF protein, while diminishing levels of mRNA (Shenberger et al., 2007). The fact that levels of VEGF and its signaling molecules were not completely down-regulated by deletion of estrogen receptors implies existence of a compensatory mechanism, redundancy or other regulatory factors. It is well known that VEGF production is stimulated by numerous factors, including interleukin-1, endothelin-1, calcium ions, phorbol esters, cytokines, heavy metals, hypoxia, and other sex steroid hormones (Mueller et al., 2000; Gu and Adair, 1997; Toi et al., 1995), most of which are expressed in the heart (Gu and Adair, 1997; Bastard et al., 2006; Mogan and Larson, 2004). Stimulation of VEGF with these factors may account for the “residual” levels of VEGF in the hearts following estrogen receptor deletion. Furthermore, the fact that levels of other growth factors, such as FGF-2, also decreased with VEGF suggests that the reduction in capillary density may not necessarily be solely caused by decrease in VEGF levels. It is intriguing to note that levels of both KDR and FGF-2, an apparently estrogen receptor independent pro-angiogenic factor, were also down-regulated in the same way as VEGF. The precise explanation for these observations is at present unclear. However, it is reasonable to speculate that the expression of FGF-2 mRNA is down-regulated in an indirect way, i.e., since VEGF has been reported to affect levels of FGF-2 via endogenous PIGF expression (Fujii et al., 2008), which in turn impacts VEGF levels, and reduces levels of VEGF in estrogen receptor knockout, likely leading to diminished FGF-2 levels in estrogen receptor knockout mice. As for decrease in KDR mRNA, we have previously reported a spatial and temporal relationship in levels of KDR and VEGF in the cervix, i.e., they increase over the course of pregnancy and diminish immediately after labor (Mowa et al., 2004). This pattern of expression is consistent with changes in the serum levels of 17β estradiol in rodents. Another study by O'Neil et al. (2008) reported that estrogen

176

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

Fig. 6. Percentage of cardiac cell death (A) and expression of the molecules related to apoptosis (B–E) and of FGF-2 (F) in left ventricular tissue specimens from WT, ERα-KO, and ERβ-KO mice. Activity of caspase-3 (B) and protein expression of BAX (C) were determined by ELISA, protein and mRNA expression levels of Bcl-2 (E, F) by immunoblotting and realtime quantitative PCR, respectively, and expression of FGF-2 by immunoblot analysis. In immunoblotting, β-actin served as loading control. Values are mean ± S.D. *P b 0.01 vs. WT; #P b 0.01 vs. ERα-KO; n = 12.

up-regulates KDR in the jejunum (O'Neil et al., 2008). It is therefore, not surprising that levels of KDR, as reported here, decrease in estrogen receptor knockout mice. Recently, the role of anti-apoptotic effect, as one of the mechanisms underlying estrogen's cardioprotective effects, has gained appreciation (Patten et al., 2004; Pelzer et al., 2001; Wang et al., 2006a,b). Autopsy studies have shown that women tend to have less cardiomyocyte apoptosis in normal and failing hearts compared to men (Cai et al., 2006). This gender difference in cardiomyocyte survival provides a plausible explanation for the beneficial effect of the female gender on the heart failure progression. More recently, it has been shown that physiological levels of estrogen replacement in ovariectomized female mice can reduce infarct size, both in the early and late stages after left coronary ligation, suggesting that physiolog-

ical estrogen replacement may be associated with diminished cardiomyocyte apoptosis in the infarct zone and peri-infarct zone 24 h after coronary ligation (van Eickels et al., 2003). However, the estrogen receptor subtype that mediates this effect, i.e., a decrease in occurrence of cardiomyocyte apoptosis after estrogen replacement, is unknown. Here, we showed that neither deletion of estrogen receptor alpha nor estrogen receptor beta directly affects cardiac cell death in female hearts. We found that expression of important pro-apoptotic molecules, caspase-3 and Bax, was unaltered. A large body of evidence suggests that VEGF/Akt signaling is crucial in endothelial cell survival (Kliche and Waltenberger, 2001; Zachary, 2003). In addition, VEGF has been shown to induce Bcl-2 expression (Liu et al., 2000). We speculate that the diminished signaling of VEGF/Akt cascade in the estrogen receptor knockout hearts, especially in ERα-KO, may contribute to the

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

diminished levels of cardiac Bcl-2, which could in turn account for the pre-disposition of the female hearts to the onset of apoptotic events. Future studies should subject animals to some endurance exercise, in order to tease out the specific effects of estrogen receptor absence vs. endurance stress on apoptosis. Moreover, although we believe that the ERα-KO mice used in the present study completely lack the functional estrogen receptor alpha protein (Taconic data sheet), there is a need in the future to tease out the effects of estrogen receptor–estrogen independent pathway (other non-estrogenic ligands act on estrogen receptor, such as growth factors) on cardioprotection (Cenni et al., 1999; Kato et al., 2000; Sinkevicius et al., 2008). Furthermore, since the plasma estrogen level was greater in ERα-KO mice, we cannot completely rule out whether some of the effects seen in the present study could be brought about through increased estrogen receptor beta signaling. In conclusion, the present study reveals that the absence or deficiency of functional estrogen receptor disrupts levels of VEGF and components of its signaling machinery (KDR, Flt-1, eNOS, and Akt), as well as those of FGF-2 and Bcl-2 in female mouse hearts. Such disrupting effects, in addition to reduced total coronary capillary density, were more profound in ERα-KO compared with WT and ERβKO mice. We interpret these findings to suggest that estrogen, largely mediated via estrogen receptor alpha has a cardioprotective effect through the regulation of the VEGF-mediated coronary microvascular development. Acknowledgments This work was supported in part by a grant-in-aid for Scientific Research and for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18300215, 18650186, 1705488, 21249086) and Japan Society for the Promotion of Science (21300234). A part of this work has also been supported by a research grant from Kanzawa Medical Research Foundation (Japan). The authors thank Sohel Zaedi, Dr. Seiji Maeda, Dr. Motoyuki Iemitsu and Dr. Shamsul Haque Prodhan for their technical assistances. References Arnlov, J., Pencina, M.J., Amin, S., Nam, B.H., Benjamin, E.J., Murabito, J.M., Wang, T.J., Knapp, P.E., D'Agostino Sr., R.B., Bhasin, S., Vasan, R.S., 2006. Endogenous sex hormones and cardiovascular disease incidence in men. Ann. Intern. Med. 145, 176–184. Ballard, V.L., Edelberg, J.M., 2005. Harnessing hormonal signaling for cardioprotection. Sci. Aging Knowl. Environ. 2005 (51), re6. Bar, J., Tepper, R., Fuchs, J., Pardo, Y., Goldberger, S., Ovadia, J., 1993. The effect of estrogen replacement therapy on platelet aggregation and adenosine triphosphate release in postmenopausal women. Obstet. Gynecol. 81, 261–264. Bastard, J.P., Maachi, M., Lagathu, C., Kim, M.J., Caron, M., Vidal, H., Capeau, J., Feve, B., 2006. Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur. Cytokine Netw. 17, 4–12. Beldekas, J.C., Smith, B., Gerstenfeld, L.C., Sonenshein, G.E., Franzblau, C., 1981. Effects of 17β-estradiol on the biosynthesis of collagen in cultured bovine aortic smooth muscle cells. Biochemistry 20, 2162–2167. Buteau-Lozano, H., Ancelin, M., Lardeux, B., Milanini, J., Perrot-Applanat, M., 2002. Transcriptional regulation of vascular endothelial growth factor by estradiol and tamoxifen in breast cancer cells: a complex interplay between estrogen receptors alpha and beta. Cancer Res. 62, 4977–4984. Cai, J., Jiang, W.G., Grant, M.B., Boulton, M., 2006. Pigment epithelium-derived factor inhibits angiogenesis via regulated intracellular proteolysis of vascular endothelial growth factor receptor 1. J. Biol. Chem. 281, 3604–3613. Christodoulakos, G., Lambrinoudaki, I., Panoulis, C., Papadias, C., Sarandakou, A., Liakakos, T., Alexandrou, A., Creatsas, G., 2004. Effect of hormone therapy, tibolone and raloxifene on circulating vascular endothelial growth factor in Greek postmenopausal women. Eur. J. Endocrinol. 151, 187–1920. Colditz, G.A., Willett, W.C., Stampfer, M.J., Rosner, B., Speizer, F.E., Hennekens, C.H., 1987. Menopause and the risk of coronary heart disease in women. N. Engl. J. Med. 316, 1105–1110. Ferrara, N., 2002. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin. Oncol. 29, 10–14. Fujii, T., Yonemitsu, Y., Onimaru, M., Inoue, M., Hasegawa, M., Kuwano, H., Sueishi, K., 2008. VEGF function for upregulation of endogenous PlGF expression during FGF-2-mediated therapeutic angiogenesis. Atherosclerosis 200, 51–57.

177

Grandien, K., 1996. Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor mRNA isoform. Mol. Cell. Endocrinol. 116, 207–212. Gu, J.W., Adair, T.H., 1997. Hypoxia-induced expression of VEGF is reversible in myocardial vascular smooth muscle cells. Am. J. Physiol. 273, H628–H633. Haynes, M.P., Li, L., Russell, K.S., Bender, J.R., 2002. Rapid vascular cell responses to estrogen and membrane receptors. Vascul. Pharmacol. 38, 99–108. Hulley, S., Grady, D., Bush, T., Furberg, C., Herrington, D., Riggs, B., Vittinghoff, E., 1998. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/Progestin Replacement Study (HERS) research group. JAMA 280, 605–613. Jesmin, S., Sakuma, I., Hattori, Y., Kitabatake, A., 2002. In vivo estrogen manipulations on coronary capillary network and angiogenic molecule expression in middle-aged female rats. Arterioscler. Thromb. Vasc. Biol. 22, 1591–1597. Jesmin, S., Hattori, Y., Sakuma, I., Liu, M.Y., Mowa, C.N., Kitabatake, A., 2003. Estrogen deprivation and replacement modulate cerebral capillary density with vascular expression of angiogenic molecules in middle-aged female rats. J. Cereb. Blood Flow Metab. 23, 181–189. Jesmin, S., Sakuma, I., Hattori, Y., Kitabatake, A., 2004. Regulatory molecules for coronary expressions of VEGF and its angiogenic receptor KDR in hypoestrogenic middle-aged female rats. Mol. Cell. Biochem. 259, 189–196. Kannel, W.B., Hjortland, M.C., McNamara, P.M., Gordon, T., 1976. Menopause and risk of cardiovascular disease the Framingham study. Ann. Intern. Med. 85, 447–452. Kato, S., Masuhiro, Y., Watanabe, M., Kobayashi, Y., Takeyama, K.I., Endoh, H., Yanagisawa, J., 2000. Molecular mechanism of a cross-talk between oestrogen and growth factor signalling pathways. Genes Cells 5, 593–601. Kazi, A.A., Jones, J.M., Koos, R.D., 2005. Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogen-induced recruitment of both estrogen receptor alpha and hypoxia-inducible factor 1 to the vascular endothelial growth factor promoter. Mol. Endocrinol. l19, 2006–2019. Kliche, S., Waltenberger, J., 2001. VEGF receptor signaling and endothelial function. IUBMB Life 52, 61–66. Krege, J.H., Hodgin, J.B., Couse, J.F., Enmark, E., Warner, M., Mahler, J.F., Madhabananda, S., Korach, K.S., Gustaffson, J.A., Smithies, O., 1998. Generation and reproductive phenotypes of mice lacking estrogen receptor b. Proc. Natl. Acad. Sci. U.S.A. 95, 15677–15682. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., Gustafsson, J.A., 1996. Cloning of a novel receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. U. S. A. 93, 5925–5930. Kuiper, G.G., Carlsson, B., Grandien, K., Enmark, E., Häggblad, J., Nilsson, S., Gustafsson, J.A., 1997. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138, 863–870. Langer, R.D., Pradhan, A.D., Lewis, C.E., Manson, J.E., Rossouw, J.E., Hendrix, S.L., LaCroix, A.Z., Ridker, P.M., 2005. Baseline associations between postmenopausal hormone therapy and inflammatory, haemostatic, and lipid biomarkers of coronary heart disease. The Women's Health Initiative Observational Study. Thromb. Haemost. 93, 1108–1116. Lee, J.E., Chung, K.W., Han, W., Kim, S.W., Kim, S.W., Shin, H.J., Bae, J.Y., Noh, D.Y., 2004. Effect of estrogen, tamoxifen and epidermal growth factor on the transcriptional regulation of vascular endothelial growth factor in breast cancer cells. Anticancer Res. 24, 3961–3964. Liu, W., Ahmad, S.A., Reinmuth, N., Shaheen, R.M., Jung, Y.D., Fan, F., Ellis, L.M., 2000. Endothelial cell survival and apoptosis in the tumor vasculature. Apoptosis 5, 323–328. Lubahn, D.B., Moyer, J.S., Golding, T.S., Couse, J.F., Korach, K.S., Smithies, O., 1993. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc. Natl. Acad. Sci. U. S. A. 90, 11162–11166. Mallat, Z., Fornes, P., Costagliola, R., Esposito, B., Belmin, J., Lecomte, D., Tedgni, A., 2001. Age and gender effects on cardiomyocyte apoptosis in the normal human heart. J. Gerontol. A Biol. Sci. Med. Sci. 56, M719–M723. Martinou, J.C., Desagher, S., Antonsson, B., 2000. Cytochrome c release from mitochondria: all or nothing. Nat. Cell Biol. 2, E41–E43. Matsumoto, T., Claesson-Welsh, L., 2001. VEGF receptor signal transduction. Sci. STKE 112, RE21. Matsumoto, T., Mugishima, H., 2006. Signal transduction via vascular endothelial growth factor (VEGF) receptors and their roles in atherogenesis. J. Atheroscler. Thromb. 13, 130–135. Mercier, I., Colombo, F., Mader, S., Calderone, A., 2002. Ovarian hormones induce TGF-beta (3) and fibronectin mRNAs but exhibit a disparate action on cardiac fibroblast proliferation. Cardiovasc. Res. 53, 728–739. Mogan, C., Larson, D.F., 2004. Rationale of platelet gel to augment adaptive remodeling of the injured heart. J. Extra-Corpor. Technol. l36, 191–196. Mowa, C.N., Jesmin, S., Sakuma, I., Usip, S., Togashi, H., Yoshioka, M., Hattori, Y., Papka, R., 2004. Characterization of vascular endothelial growth factor (VEGF) in the uterine cervix over pregnancy: effects of denervation and implications for cervical ripening. J. Histochem. Cytochem. 52, 1665–1674. Mueller, M.D., Vigne, J.L., Minchenko, A., Lebovic, D.I., Leitman, D.C., Taylor, R.N., 2000. Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta. Proc. Natl. Acad. Sci. U. S. A. 97, 10972–10977. Nakamura, Y., Suzuki, T., Sasano, H., 2005. Estrogen actions and in situ synthesis in human vascular smooth muscle cells and their correlation with atherosclerosis. J. Steroid Biochem. Mol. Biol. 93, 263–268. Nuedling, S., Kahlert, S., Loebbert, K., Doevendans, P.A., Meyer, R., Vetter, H., Grohe, C., 1999. 17 Beta-estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo. Cardiovasc. Res. 43, 666–674. O'Neil, M.R., Lardy, G.P., Reynolds, L.P., Caton, J.S., Vonnahme, K.A., 2008. Impacts of linseed meal and estradiol-17 beta on mass, cellularity, angiogenic factors, and vascularity of the jejunum. J. Anim. Sci. 86, 3014–3022.

178

S. Jesmin et al. / European Journal of Pharmacology 641 (2010) 168–178

Orshal, J.M., Khalil, R.A., 2004. Gender, sex hormones, and vascular tone. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R233–R249. Patten, R.D., Pourati, I., Aronovitz, M.J., Baur, J., Celestin, F., Chen, X., Michael, A., Haq, S., Nuedling, S., Grohe, C., Force, T., Mendelsohn, M.E., Karas, R.H., 2004. 17 Betaestradiol reduces cardiomyocyte apoptosis in vivo and in vitro via activation of phospho-inositide-3 kinase/Akt signaling. Circ. Res. 95, 692–699. Pelzer, T., Neumann, M., de Jager, T., Jazbutyte, V., Neyses, L., 2001. Estrogen effects in the myocardium: inhibition of NF-kappa B DNA binding by estrogen receptor-alpha and -beta. Biochem. Biophys. Res. Commun. 286, 1153–1157. Perry, R.P., Meyuhas, O., 1990. Translational control of ribosomal protein production in mammalian cells. Enzyme 44, 83–92. Reckelhoff, J.F., Zhang, H., Srivastava, K., 2000. Gender differences in development of hypertension in spontaneously hypertensive rats: role of the renin–angiotensin system. Hypertension 35, 480–483. Rifici, V.A., Khachadurian, A.K., 1992. The inhibition of low-density lipoprotein oxidation by 17-estradiol. Metabolism 41, 1110–1114. Rossouw, J.E., Anderson, G.L., Prentice, R.L., LaCroix, A.Z., Kooperberg, C., Stefanick, M.L., Jackson, R.D., Beresford, S.A., Howard, B.V., Johnson, K.C., Kotchen, J.M., Ockene, J., 2002. Risks and benefits of estrogen plus progestin in healthy postmenopausal women principal results from the Women's Health Initiative randomized controlled trial. JAMA 288, 321–333. Saunders, P.T., Maguire, S.M., Gaughan, J., Millar, M.R., 1997. Expression of oestrogen receptor beta in multiple rat tissues visualised by immunohistochemistry. J. Endocrinol. 154, R13–R16. Shenberger, J.S., Zhang, L., Powell, R.J., Barchowsky, A., 2007. Hyperoxia enhances VEGF release from A549 cells via posttranscriptional processes. Free Radic. Biol. Med. 43, 844–852. Sinkevicius, K.W., Burdette, J.E., Woloszyn, K., Hewitt, S.C., Hamilton, K., Sugg, S.L., Temple, K.A., Wondisford, F.E., Korach, K.S., Woodruff, T.K., Greene, G.L., 2008. An estrogen receptor-alpha knock-in mutation provides evidence of ligand-independent signaling and allows modulation of ligand-induced pathways in vivo. Endocrinology 149, 2970–2979. Stevenson, J.C., 2004. Hormone replacement therapy: review, update, and remaining questions after the Women's Health Initiative Study. Curr. Osteoporos. Rep. 2, 12–16.

Sumino, H., Ichikawa, S., Kasama, S., Kumakura, H., Takayama, Y., Sakamaki, T., Kurabayashi, M., 2005. Effect of transdermal hormone replacement therapy on carotid artery wall thickness and levels of vascular inflammatory markers in postmenopausal women. Hypertens. Res. 28, 579–584. Toi, M., Inada, K., Hoshina, S., Suzuki, H., Kondo, S., Tominaga, T., 1995. Vascular endothelial growth factor and platelet-derived endothelial cell growth factor are frequently coexpressed in highly vascularized human breast cancer. Clin. Cancer Res. 1, 961–964. van Eickels, M., Grohe, C., Cleutjens, J.P., Janssen, B.J., Wellens, H.J., Doevendans, P.A., 2001. 17-Beta-estradiol attenuates the development of pressure-overload hypertrophy. Circulation 104, 1419–1423. van Eickels, M., Patten, R.D., Aronovitz, M.J., Alsheikh-Ali, A., Gostyla, K., Celestin, F., Grohe, C., Mendelsohn, M.E., Karas, R.H., 2003. 17-beta-estradiol increases cardiac remodeling and mortality in mice with myocardial infarction. J. Am. Coll. Cardiol. 41, 2084–2092 (Erratum in: J. Am. Coll. Cardiol. 42, 183). Walter, P., Green, S., Green, G., Krust, A., Bornert, J.M., Jeltsch, J.M., Staub, A., Jensen, E., Scrace, G., Waterfield, M., 1985. Cloning of the human estrogen receptor cDNA. Proc. Natl. Acad. Sci. U. S. A. 82, 7889–7893. Wang, M., Crisostomo, P., Wairiuko, G.M., Meldrum, D.R., 2006a. Estrogen receptor-alpha mediates acute myocardial protection in females. Am. J. Physiol. 290, H2204–H2209. Wang, M., Tsai, B.M., Reiger, K.M., Brown, J.W., Meldrum, D.R., 2006b. 17-Beta-estradiol decreases p38 MAPK-mediated myocardial inflammation and dysfunction following acute ischemia. J. Mol. Cell. Cardiol. 40, 205–212. Wenger, N.K., 2005. Menopausal hormone therapy: currently no evidence for cardiac protection. Pediatr. Blood Cancer 44, 625–629. Williams, J.K., Suparto, I., 2004. Hormone replacement therapy and cardiovascular disease: lessons from a monkey model of postmenopausal women. ILAR J. 45, 139–146. Writing Group for the PEPI Trial, 1995. Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. The Postmenopausal Estrogen/Progestin Interventions (PEPI) trial. JAMA 273, 199–208. Yoon, Y.S., Johnson, I.A., Park, J.S., Diaz, L., Losordo, D.W., 2004. Therapeutic myocardial angiogenesis with vascular endothelial growth factors. Mol. Cell. Biochem. 264, 63–74. Zachary, I., 2003. VEGF signalling: integration and multi-tasking in endothelial cell biology. Biochem. Soc. Trans. 31, 1171–1177.