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Circulation. 2006;114:2261-2270
Published online before print November 6, 2006, doi: 10.1161/CIRCULATIONAHA.106.631465
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(Circulation. 2006;114:2261-2270.)
© 2006 American Heart Association, Inc.

Molecular Cardiology

Estrogen Receptors {alpha} and ß Mediate Contribution of Bone Marrow–Derived Endothelial Progenitor Cells to Functional Recovery After Myocardial Infarction

Hiromichi Hamada, MD, PhD; Myeong Kon Kim, MD; Atsushi Iwakura, MD, PhD; Masaaki Ii, MD, PhD; Tina Thorne, MS; Gangjian Qin, MD; Jun Asai, MD; Yoshiaki Tsutsumi, MD, PhD; Haruki Sekiguchi, MD; Marcy Silver, MS; Andrea Wecker, BS; Evelyn Bord, BS; Yan Zhu, PhD; Raj Kishore, PhD; Douglas W. Losordo, MD

From the Division of Cardiovascular Research, St Elizabeth Medical Center of Boston, Tufts University School of Medicine, Boston, Mass (H.H., M.K.K., A.I., M.I., T.T., G.Q., J.A., Y.T., H.S., M.S., A.W., E.B., Y.Z., R.K., D.W.L.), and Stem Cell Translational Research, Kobe Institute of Biomedical Research and Innovation/RIKEN Center for Developmental Biology, Kobe, Japan (M.I.).

Correspondence to Douglas W. Losordo, MD, Cardiovascular Research, Caritas St. Elizabeth Medical Center of Boston, 736 Cambridge St, Boston, MA 02135. E-mail douglas.losordo{at}tufts.edu

Received April 3, 2006; revision received July 6, 2006; accepted August 11, 2006.


*    Abstract
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Background— Estradiol (E2) modulates the kinetics of circulating endothelial progenitor cells (EPCs) and favorably affects neovascularization after ischemic injury. However, the roles of estrogen receptors {alpha} (ER{alpha}) and ß (ERß) in EPC biology are largely unknown.

Methods and Results— In response to E2, migration, tube formation, adhesion, and estrogen-responsive element–dependent gene transcription activities were severely impaired in EPCs obtained from ER{alpha}-knockout mice (ER{alpha}KO) and moderately impaired in ERßKO EPCs. The number of ER{alpha}KO EPCs (42.4±1.5; P<0.001) and ERßKO EPCs (55.4±1.8; P=0.03) incorporated into the ischemic border zone was reduced as compared with wild-type (WT) EPCs (72.5±1.3). In bone marrow transplantation (BMT) models, the number of mobilized endogenous EPCs in E2-treated mice was significantly reduced in ER{alpha}KO BMT (WT mice transplanted with ER{alpha}KO bone marrow) (2.03±0.18%; P=0.004 versus WT BMT) and ERßKO BMT (2.62±0.07%; P=0.02 versus WT) compared with WT BMT (2.87±0.13%) (WT to WT BMT as control) mice. Capillary density at the border zone of ischemic myocardium also was significantly reduced in ER{alpha}KO BMT and ERßKO BMT compared with WT mice (WT BMT, 1718±75/mm2; ER{alpha}KO BMT, 1107±48/mm2; ERßKO BMT, 1567±50/mm2). ER{alpha} mRNA was expressed more abundantly on EPCs compared with ERß. Moreover, vascular endothelial growth factor was significantly downregulated on ER{alpha}KO EPCs compared with WT EPCs both in vitro and in vivo.

Conclusions— Both ER{alpha} and ERß contribute to E2-mediated EPC activation and tissue incorporation and to preservation of cardiac function after myocardial infarction. ER{alpha} plays a more prominent role in this process. Moreover, ER{alpha} contributes to upregulation of vascular endothelial growth factor, revealing possible mechanisms of an effect of E2 on EPC biology. Finally, these data provide additional evidence of the importance of bone marrow–derived EPC phenotype in ischemic tissue repair.


Key Words: angiogenesis • bone marrow cells • hormones • myocardial infarction • receptors, estrogen


*    Introduction
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Clinical and experimental evidence suggests that estrogen plays important roles in physiological and pathological angiogenesis. In adult organisms, angiogenesis is virtually absent under normal conditions except in the female reproductive tract. The role of estrogen in uterine angiogenesis has been demonstrated by findings in the estrogen receptor {alpha} (ER{alpha})–knockout (KO) mouse, in which angiogenesis is impaired, and by the demonstration that ER antagonists can inhibit angiogenesis.1 The positive correlation between ER expression, angiogenic activity, and breast tumor invasiveness also supports the angiogenic effect of estrogen mediated by estrogen receptors,2–4 In addition, the estrogen metabolite 2-methoxyestradiol has been shown to be a potent antiangiogenic agent.5

Editorial p 2203

Clinical Perspective p 2270

Vascular endothelial cells express at least 2 estrogen receptors: ER{alpha}6,7 and ERß.8 Estrogen enhances endothelial cell activities in vitro1,9 and has favorable effects on ischemic neovascularization in vivo.10,11 In human studies, it was reported that postmenopausal hormone replacement therapy was associated with reduced risk of mortality after myocardial infarction (MI).12

Tissue ischemia induces upregulation of angiogenic growth factors and mobilization of circulating cellular elements that together enable development of an accessory vasculature for organ survival. Recently, endothelial progenitor cells (EPCs) isolated from peripheral blood have been shown to incorporate into foci of neovascularization in the adult, ie, postnatal vasculogenesis.13,14 These circulating EPCs are derived from bone marrow and mobilized endogenously in response to tissue ischemia or exogenously by cytokine stimulation.15–18

Previous findings have suggested that estrogen also could augment the recruitment of EPCs for vascular repair.11,19,20 Our laboratory showed that the effects of estrogen on EPC recruitment in vascular repair were endothelial nitric oxide synthase dependent.11,20 However, the potential role of estrogen receptor in EPC recruitment for myocardial microvascular repair has not been studied. In this study, we investigated the roles of ER{alpha} and ERß in estrogen-induced, EPC-mediated tissue repair in the setting of acute MI.


*    Methods
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Cell Culture
Mononuclear cells were isolated from mouse bone marrow and plated for EPCs as described previously21,22 and in the online Data Supplement. The rationale for using a culture method to enrich the EPC fraction of bone marrow cells is as follows: For phenotyping and assessing gene expression, circulating cells in the mouse are too scarce, thus mandating the use of a more abundant source of cells. A method has been developed and used repeatedly in our laboratory (and in multiple peer-reviewed publications15,16,23) that favors the growth of cells with endothelial lineage capability while diminishing the non-EPC population. Medium was replaced with phenol red–free medium (EBM, Camblex, Walkersville, Md) with 5% charcoal-dextran–treated fetal bovine serum (Biosource, Invitrogen, Carlsbad, Calif) at day 5 to remove the effects of estrogen-like activity of phenol red and estrogen derived from the serum. After 2 days in further culture, the cells were used as an EPC-rich cell population for cell function studies and real-time polymerase chain reaction (RT-PCR).

In Vitro Cell Function Assays (Proliferation, Migration, Tube Formation, and Adhesion Activity)
Cell proliferation was assessed by [3H]thymidine incorporation into DNA as described before.24 Migration was measured in a modified Boyden’s chamber. Tube formation assay was performed as described before using Matrigel-Matrix (BD Biosciences, San Jose, Calif).25 The adhesion assay is described in the Data Supplement.

Animals
All mice used in this study were handled in accordance with the guidelines of the Animal Care and Use Committee at St Elizabeth’s Medical Center of Boston (Mass). Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, Maine) were used as WT mice. Full details about ER{alpha}-null mutant mice and ERß-null mutant mice were given recently.26 In ER{alpha}-null mutant mice, exon 3 of ER{alpha} was deleted, and no ER{alpha} proteins were found in this strain. In ERß-null mutant mice, exon 3 of ERß was deleted, and no ERß proteins were found in this strain. These mice are of C57BL/6 background.

Bone Marrow Transplantation Model
Female C57BL/6J mice 9 to 10 weeks old were studied. Mice underwent ovariectomy on day –35, followed by bone marrow transplantation (BMT) at day –28. WT, ER{alpha}KO, or ERßKO mice 8 to 12 weeks old were used as donors of the bone marrow. The BMT procedure was performed as described previously.16,21 At day –7, by which time the bone marrow of the recipient mice was reconstituted, BMT mice received either 17b-estradiol (E2) pellets (Innovative Research of America, Sarasota, Fla) or placebo-containing pellets implanted subcutaneously into the dorsal neck region of the animals. To achieve typical E2 levels found at mid-cycle, a 90-day release pellet containing 1.7 mg E2 was used. Circulating E2 levels in mice with the E2 pellets and in mice with the placebo pellets were previously reported.20 Seven days later (day 0), animals underwent MI surgery.

Surgical Procedure
MI was induced by permanent left anterior descending coronary artery ligation as described previously11 using intraperitoneal injection of avertin 0.015 mg/kg and assisted ventilation (Harvard Apparatus, Holliston, Mass).

In Vivo Tissue Homing Assay
Female C57BL/6J mice 9 to 10 weeks old underwent ovariectomy at day –28, followed by either 1.7 mg E2 pellet or placebo-containing pellet implantation, together with splenectomy at day –7. Seven days later (day 0), animals underwent MI surgery. Cultured EPCs were coincubated with 2 µg/mL DiI-acLDL (Biomedical Technologies, Stoughton, Mass) for 1 hour, and 5x105 EPCs were injected intravenously immediately after MI surgery. Hearts of these mice were harvested at the indicated time after MI surgery for histology.

Fluorescence-Activated Cell Sorting Analysis
To evaluate the number of circulating EPCs, 1 mL blood was taken at days –1, 7, and 28, and mononuclear cells were isolated by density centrifugation with Histopaque-1083 (Sigma-Aldrich, St Louis, Mo) for fluorescence-activated cell sorting analysis (Becton Dickinson, Franklin Lakes, NJ). The viable mononuclear cell population (2 to 4x106 cells were available from 1 mL blood) was analyzed for the expression of Sca-1–FITC (BD PharMingen, San Diego, Calif) and Flk-1–PE (BD PharMingen). Isotype-identical antibodies served as negative controls (Jackson ImmunoResearch, West Grove, Pa).

Echocardiography
Left ventricular function was assessed by transthoracic echocardiography (SONOS 5500, Hewlett Packard, Palo Alto, Calif) at days –1, 7, 14, 21, and 28. Left ventricular end-diastolic dimension, left ventricular end-systolic dimension, and fractional shortening at the papillary muscle level of the left ventricle were measured, and the mean value of 3 measurements was determined for each sample.

Histological Analysis
In BMT models, hearts were harvested at day 28 for histological analysis. The explanted hearts were sliced in a bread-loaf fashion into transverse sections from apex to base and fixed with 4% paraformaldehyde. Tissues were stained for Masson’s trichrome staining to measure the average ratio of fibrosis area to total left ventricular area.

Immunohistochemistry
The hearts of treated mice were harvested at predetermined times after surgery and frozen in optical coherence tomography compound (Sakura Finetek USA, Inc, Torrance, Calif). For capillary detection, sections were stained with mouse anti-CD31 antibody (BD PharMingen). For detection of vascular endothelial growth factor (VEGF), sections were incubated with mouse anti-VEGF antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif). Details are described in the Data Supplement.

Quantitative RT-PCR
RNA was collected from 8x105 cells per sample with RNA STAT-60 (TEL-TEST, Inc, Friendswood, Texas). Total RNA was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, Calif), and amplification was performed on the Taqman 7300 (Applied Biosystems, Foster City, Calif). Primer and probe sequences are described in the Data Supplement.

Determination of E2-Surface Binding With E2-Conjugated BSA-FITC
E2-ER binding study was performed as previously described27 with modification, as described in the Data Supplement.

Reporter Gene Luciferase Assay for Estrogen-Responsive Element–Dependent Transcription
EPCs were transiently transfected with estrogen-responsive element (ERE)–luciferase reporter construct using Fusene 6 transfection reagent (Roche, Palo Alto, Calif) according to the manufacturer’s instructions, and luciferase activity was determined as described before.28

Statistical Analysis
All values are expressed as mean±SE. Statistical significance was evaluated through the use of an unpaired t test for comparisons between 2 groups. For comparison among 3 or 4 groups, 1-factor analysis of variance was used, followed by an unpaired t test to compare 2 groups within them. When multiple time-point measurements were taken, repeated-measures analysis was done, followed by an unpaired t test. A value of P<0.05 was considered statistically significant.

The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.


*    Results
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 up arrowMethods
 *Results
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E2 Effects on EPC Cell Function Are Abolished in ER-Knockout EPCs
First, we evaluated the role of ER expression on EPC cell function in vitro. In migration assays (Figure 1a), EPC migration was significantly increased in WT cells by physiological concentrations of E2 but was absent in ER{alpha}KO EPCs and reduced in ERßKO EPCs (WT EPCs: 8.89±0.17%/E2 0 mol/L, 14.1±1.50%/E2 10–9 mol/L, P<0.01 versus without E2; 14.8±0.81%/E2 10–8 mol/L, P<0.001 versus without E2; ERßKO EPCs: 10.6±0.20%/E2 0 mol/L, 13.4±1.74%/E2 10–8 mol/L, P<0.01 versus without E2). In proliferation assays (Figure 1b), E2-induced proliferation of in vitro expanded EPCs obtained from both ER{alpha}KO and ERßKO mice was significantly reduced compared with EPCs obtained from WT littermates (WT EPCs: 8540±420 cpm/E2 10–8 mol/L versus ER{alpha}KO EPCs: 4320±50 cpm/E2 10–8 mol/L, P<0.001; WT EPCs: 8540±420 cpm/E2 10–8 mol/L versus ERßKO EPCs: 4650±240 cpm/E2 10–8 mol/L, P<0.001). Similarly, adhesion activity to vitronectin, which is increased in a dose-dependent manner by E2 in WT EPCs, was absent in ER{alpha}KO EPCs and reduced in ERßKO EPCs (Figure 1c) (WT EPCs: 1.00±0.03/E2 0 mol/L, 1.27±0.04/E2 10–9 mol/L, P<0.05 versus without E2, 1.57±0.06/E2 10–8 mol/L, P<0.001 versus without E2; ER{alpha}KO EPCs: E2 0 mol/L, 1.00±0.19; E2 10–9 mol/L: 0.89±0.03 (P=NS versus 0 mol/L), E2 10–8 mol/L: 1.02±0.08 (P=NS versus 0 mol/L); ERßKO EPCs: 1.00±0.06/E2 0 mol/L, 1.23±0.04/E2 10–9 mol/L, P<0.001 versus without E2, 1.24±0.10/E2 10–8 mol/L, P<0.01 versus without E2). Finally, we evaluated tube formation, an established method to assess functional angiogenic activity in vitro. Capillary-like tube formation requires several biological activities such as endothelial cell proliferation, cell migration, protease secretion, and cell-to-cell interaction. As shown in Figure 1d and 1e, WT EPCs showed an E2 concentration–dependent response in capillary network formation (21.8±0.8 mm/E2 0 mol/L, 114±4.2 mm/E2 10–9 mol/L, P<0.001 versus without E2, 219±8.4 mm/E2 10–8 mol/L, P<0.001 versus without E2). Under similar culture condition, ERßKO EPCs (97.8±5.9 mm/E2 10–8 mol/L) made fewer networks compared with WT EPCs (P<0.001 versus WT EPCs), and tube formation by ER{alpha}KO EPCs (18.0±0.8 mm/E2 10–8 mol/L) was severely impaired (P<0.001 versus WT EPCs).


Figure 1179366
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  		Figure 1. In vitro cell function in WT, ER{alpha}KO, and ERßKO EPCs. a, Migration toward E2 in EPCs from WT, ER{alpha}KO, and ERßKO mice. EPCs were allowed to migrate toward different molar concentrations of E2 as indicated. SDF-1 200 ng/mL is used as a positive control. n=4. b, Proliferation activity of EPCs from WT, ER{alpha}KO, and ERßKO mice under different E2 molar concentration is assessed by thymidine uptake assay. n=3. c, Adhesion activity to rat vitronectin-coated plates of EPCs from WT, ER{alpha}KO, and ERßKO mice under different E2 molar concentration. The number of adhered cells without E2 is considered 1.0; the number of adhered cells with different E2 molar concentrations is indicated as relative adhesion ratio. n=6. d, Tube formation assay in Matrigel matrix of EPCs from WT, ER{alpha}KO, and ERßKO mice under different E2 molar concentrations. e, Total tube length are measured and indicated. n=4. Similar results were obtained at least 3 times in these in vitro experiments. Bars represent mean±SE. V indicates VEGF 50 ng/mL. *P<0.001, +P<0.01, **P<0.05.

E2 Contribution to EPC Tissue Homing Is Impaired in ER-Knockout EPCs
From the results of the in vitro cell function assays, we hypothesized that E2 could modulate chemotactic activity in EPCs via both ERs. Accordingly, we evaluated EPC homing in vivo in a tissue homing assay using mouse acute MI models. Incorporation of DiI-labeled WT EPCs, injected just after MI induction, was observed in the border zone of the ischemic myocardium (Figure 2a) at day 3 (36.7±3.4 cells/x200 field). At day 5, the number of EPCs at the site increased (63.6±2.5 cells/x200 field) and persisted until at least day 10 (day 7, 65.7±4.0 cells; day 10, 61.5±4.0 cells; Figure 2b). Next, we compared WT, ER{alpha}KO, and ERßKO EPC incorporation at the border zone of the ischemic myocardium at day 7, choosing this time point as the approximate peak on the basis of pilot studies. Incorporation of WT EPCs in ovariectomized mice with placebo pellets was used as a negative control (40.2±3.7 cells). As shown in Figure 2c and 2d, the number of incorporated cells per x200 magnification field was significantly higher in WT EPCs (72.5±1.3) compared with ER{alpha}KO (42.4±1.5; P<0.001) and ERßKO EPCs (55.4±1.8; P=0.03).


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  		Figure 2. In vivo EPC tissue homing assay. a, Histology at the border zone of ischemic myocardium on indicated days after MI surgery when WT EPCs were injected into an E2 pellet–implanted WT mouse. Injected EPCs are labeled with DiI-acLDL (red). Nuclear is stained with DAPI (blue). Scale bar represents 100 µm. b, Number of DiI-positive cells at the border zone was counted and shown as mean±SE n=5. c, Histology at the border zone of ischemic myocardium at day 7 after MI surgery when WT, ER{alpha}KO, and ERßKO EPCs were injected into an E2 pellet–implanted WT mouse. Samples of WT EPC injection into WT mice with placebo pellets are used as negative controls. d, Quantification of injected, labeled EPCs in the MI border zone shown as mean±SE. n=5. WT E2+ indicates WT EPC injection into WT mouse with E2 pellet; WT E2–, WT EPC injection into WT mouse with placebo pellet; ER{alpha}KO E2+, ER{alpha}KO EPC injection into WT mouse with E2 pellet; ERßKO E2+, ERßKO EPC injection into WT mouse with E2 pellet. *P<0.001, +P=0.03 vs WT E2+, respectively.

E2 Contribution to EPC Mobilization From Bone Marrow Was Impaired From ER-Knockout Bone Marrow
To evaluate the effect of ER-mediated effects on circulating EPC kinetics in vivo, peripheral blood was collected at serial time points after MI in WT, ER{alpha}KO, and ERßKO BMT models and prepared for fluorescence-activated cell sorting analysis. The light-scatter pattern of mononuclear cells was similar in WT, ER{alpha}KO, and ERßKO cells (Figure 3a). As shown in Figure 3b, a significantly greater number of circulating Sca-1+/Flk-1+ cells were observed in WT BMT mice 1 week after MI (2.87±0.13% of total mononuclear cells) compared with ER{alpha}KO (2.03±0.18%; P=0.004 versus WT BMT with E2 pellet) and ERßKO (2.62±0.07%; P=0.02 versus WT BMT with E2 pellet) BMT mice.


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  		Figure 3. EPC mobilization from bone marrow in response to MI in WT, ER{alpha}KO, and ERßKO BMT to WT mice. a, Forward scatter (FSC-H) and side scatter (SSC-H) plot for circulating blood mononuclear cells in steady-state WT, ER{alpha}KO, and ERßKO mice. Numbers show percent of data-collected cells per whole cells (circled area). b, Percentage of Sca-1+/Flk-1+ mononuclear cells in circulating blood before and 1 and 4 weeks after MI is indicated as mean±SE. WT E2+ indicates WT BMT to WT mouse with E2 pellet (n=12); WT E2–, WT BMT to WT mouse with placebo pellet (n=12); ER{alpha}KO E2+, ER{alpha}KO BMT to WT mouse with E2 pellet (n=8); ERßKO E2+, ERßKO BMT to WT mouse with E2 pellet (n=8). *P=0.004, +P=0.02.

Protective Effect of E2 in MI Is Reduced in ER-Knockout BMT Model
Physiological and histological assessments were then performed after MI in WT, ER{alpha}KO, and ERßKO BMT mice. WT mice with WT BMT plus placebo pellets were evaluated as negative controls. Left ventricular end-diastolic dimensions and systolic function were not significantly different between WT BMT and ER mutant BMT mice early after MI (Figure 4a). However, beginning 3 weeks after MI, echocardiography revealed better preservation of fractional shortening in the WT BMT mice compared with ER{alpha}KO BMT mice (WT BMT versus ER{alpha}KO BMT: P=0.02 at 3 weeks after MI, P=0.007 at 4 weeks after MI; Figure 4a, left).


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  		Figure 4. Cardiac phenotype in WT, ER{alpha}KO, and ERßKO BMT to WT mice with MI surgery. WT E2+ indicates WT BMT to WT mouse with E2 pellet (n=12); WT E2–, WT BMT to WT mouse with placebo pellet (n=12); ER{alpha}KO E2+, ER{alpha}KO BMT to WT mouse with E2 pellet (n=8); ERßKO E2+, ERßKO BMT to WT mouse with E2 pellet (n=8). a, Left, Fractional shortening before and after MI is shown as mean±SE. *P<0.001 among 4 groups; P=0.02, ER{alpha}KO vs WT+E2; P=NS, ERßKO vs WT+E2. +P=0.01 among 4 groups; P=0.007, ER{alpha}KO vs WT+E2; P=NS, ERßKO vs WT+E2. Right, Left ventricular end-diastolic dimension before and after MI is shown as mean±SE. *P=0.004 among 4 groups; P=NS, ER{alpha}KO vs WT+E2; P=NS, ERßKO vs WT+E2. +P=0.005 among 4 groups; P=NS, ER{alpha}KO vs WT+E2; P=NS, ERßKO vs WT+E2. b, Representative cross sections of left ventricle at the papillary muscle level 4 weeks after MI stained with Masson’s trichrome. Scale bar represents 2 mm. c, Percent of fibrosis area in cross sections of left ventricle at the papillary muscle level in each BMT mice 4 weeks after MI. Bars represent mean±SE. n=5. P<0.001 among 4 groups. *P=0.03, ER{alpha}KO vs WT+E2. +P=0.04, ERßKO vs WT-E2. e, Cross sections at the border zone of ischemic myocardium in each BMT mice 4 weeks after MI stained with CD31 are shown. Scale bar represents 100 µm. f, Capillary density at the border zone of ischemic myocardium in each BMT mice 4 weeks after MI. Bars represent mean±SE. n=4. P<0.001 among 4 groups. *P<0.001, ER{alpha}KO E2+ vs WT E2+.

Masson’s trichrome–stained tissues in ER{alpha}KO and ERßKO BMT mice indicated marked dilation of the left ventricular cavity consistent with the echocardiographic measurements (Figure 4b). The area of fibrosis was significantly less in WT BMT mice than in ER{alpha}KO and ERßKO BMT mice (WT BMT, 13.5±1.1%; ER{alpha}KO BMT, 19.4±2.4%; ERßKO BMT, 17.9±1.1%; Figure 4c). WT BMT mice with placebo pellets were analyzed as negative controls (WT BMT with placebo, 21.7±0.8%). Capillary density at the border zone of ischemic myocardium 4 weeks after MI was significantly greater in WT BMT mice with E2 compared with ER{alpha}KO BMT and ERßKO BMT mice with E2 (WT BMT, 1718±75/mm2; ER{alpha}KO BMT, 1107±48/mm2; ERßKO BMT, 1567±50/mm2; Figure 4d and 4e). In WT BMT mice with placebo pellets, capillary density was 1136±83/mm2.

ER{alpha}/ERß Expression and Binding Activity to E2 on Mouse EPCs
In the above series of experiments, ER{alpha}KO EPCs appeared to have a more prominent phenotype than ERßKO EPCs. To better understand the potential mechanisms for this, we first evaluated ER{alpha} and ERß mRNA expression in WT mouse EPCs by RT-PCR. Both receptors were expressed on EPCs from WT mice cultured for 7 days (Figure 5a). Next, we used quantitative RT-PCR to evaluate the relative expression of individual receptors in EPCs and showed that ER{alpha} mRNA was expressed more abundantly on WT EPCs compared with ERß mRNA (relative expression versus 18S: ER{alpha}, 1.88±0.18, ERß, 0.01±0.01; Figure 5b and 5c; note that the y-axis scale is 10x between Figure 5b and Figure 5c). Because ERß expression was low by RT-PCR, we reevaluated it using other primer sets that were within exon 5 of the ERß sequence (Figure 5c, right). In WT EPCs, the relative expression of ERß versus 18S was again much lower than ER{alpha} expression (0.12±0.03). In ERßKO mice, ERß exon 5 gene transcription was accelerated (13.2±0.27), which indicates that the gene was actually present in ERßKO mice except exon 3, where the ERß gene was disrupted. In addition, E2 10–8 mol/L induces ER{alpha} mRNA upregulation in WT EPCs (P=0.004) and ERßKO EPCs (P=0.015; Figure 5d).


Figure 5179366
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  		Figure 5. ER expression on mouse EPCs. a, ER{alpha} and ERß expression by RT-PCR in WT EPCs cultured for 4 and 7 days. b, ER{alpha} expression on EPCs from WT, ER{alpha}KO, and ERßKO mice by quantitative RT-PCR. Uterus tissues of WT mice are used as positive control (ER{alpha} relative expression vs 18S is 116; lane 4). n=4. c, ERß expression on EPCs from WT, ER{alpha}KO, and ERßKO mice using primers within exon 3 or 5 by quantitative RT-PCR. Spleen tissues of WT mice are used as positive controls. n=4. Exon 5 ERß-relative expression vs 18S is 13.2 in ERßKO EPCs (lane 7) and 48.0 in ERßKO spleen (lane 8). D, ER{alpha} expression on EPCs from WT and ERßKO mice is upregulated with 10–8 mol/L E2 treatment. n=3. *P=0.004, **P=0.015. e, Representative images of E2-surface binding with E2-conjugated BSA-FITC. WT+E2 10–6 mol/L. EPCs were pretreated with non–FITC-conjugated E2 10–6 mol/L for 15 minutes. f, Percentage of FITC-positive cells is shown. n=5. *P<0.001, ER{alpha}KO vs WT EPCs. g, ERE-dependent transcription of reporter gene luciferase is shown in EPCs from each genotype. n=3. **P<0.01, ER{alpha}KO vs WT EPCs with E2. *P<0.03, ERßKO vs WT EPCs with E2. Similar results were obtained at least 3 times in these in vitro experiments. Bars represent mean±SE.

Bovine serum albumin–FITC–tagged estradiol (E2coBSA-FITC) was used to investigate estrogen binding in WT, ER{alpha}KO, and ERßKO EPCs (Figure 5e and 5f). After 4 hours of incubation, 64.5±4.1% of cells were positive for FITC in WT EPCs, whereas 11.4±2.9% of cells were positive in ER{alpha}KO EPCs (P<0.001). There were no significant differences in binding activity between WT and ERßKO EPCs (58.8±3.0%). Preincubation with unlabeled E2 10–6 mol/L diminished E2coBSA binding (20.8±2.5%), indicating ligand specificity. As an additional measure of individual ER function, ERE-dependent transcription of the reporter gene luciferase was evaluated in EPCs from each genotype. As shown in Figure 5g, E2 treatment (10–8 mol/L) of WT EPCs led to a 35-fold increase in reporter activity over untreated cells. In contrast to WT EPCs, reporter activity was significantly decreased in EPCs from both ER{alpha}KO and ERßKO mice (P<0.01 and P<0.03, respectively), indicating that E2-induced, ERE-dependent gene transcription requires both ER{alpha} and ERß.

E2 Upregulates VEGF Through ER{alpha} in EPCs
To investigate ER{alpha}-associated gene expression, we evaluated the repertoire of angiogenic molecules expressed in EPCs by analyzing RNA extracted from WT and ER{alpha}KO EPCs. Of the several angiogenic molecules differentially expressed in WT and ER{alpha}KO EPCs, VEGF-A was the most consistent gene expressed differentially, which was confirmed by quantitative RT-PCR (Figure 6a). When WT EPCs were treated with 10–8 mol/L E2, the abundance of VEGF transcripts increased 5-fold within 1 hour of E2 exposure to WT EPCs (Figure 6b) and persisted for 8 hours (relative expression versus 18S: 26.2±2.4/0 h, 123±12/1 h, 128±21/3 h, 114±14/5 h, and 84±10/8 h), returning to basal levels at 24 hours of E2 treatment (39.2±1.9/12 h and 28.4±2.0/24 h). In contrast, in ER{alpha}KO EPCs, E2 exposure resulted in a brief, modest increase in VEGF mRNA (relative expression versus 18S: 10.6±0.4/0 h, 24.0±0.2/1 h, 11.9±0.8/3 h, 9.4±0.9/5 h, and 11.0±0.3/8 h). In the mouse MI model, after intravenous injection of EPCs just after induction of myocardial injury, we observed that the incorporated WT EPCs, but not ER{alpha}KO EPCs, expressed VEGF (Figure 6c), corroborating the in vitro findings and providing further evidence that ER{alpha}-mediated VEGF expression might represent a key feature of the E2-mediated EPC-derived effect on MI recovery.


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  		Figure 6. E2 upregulates VEGF through ER{alpha} in EPCs. a, VEGF expression on EPCs from WT, ER{alpha}KO, and ERßKO mice cultured in normal endothelial cell medium (EBM-2). Relative VEGF expression vs 18S is indicated. n=3. Bars represent mean±SE. *P<0.001, ER{alpha}KO vs WT EPCs. b, VEGF expression on EPCs from WT and ER{alpha}KO mice with 10–8 mol/L E2 treatment at indicated times. EPCs are cultured in E2-free medium for 48 hours and starved for 12 hours before the experiment. Relative VEGF expression vs 18S is indicated. n=3. Bars represent mean±SE. *P<0.001. Similar results were obtained at least 3 times in these in vitro experiments. c, VEGF is expressed on injected WT EPCs, not on ER{alpha}KO, at the border zone of ischemic myocardium 7 days after MI. Just after MI surgery, 5x105 EPCs labeled with DiI-acLDL (red) are injected. Scale bar represents 100 µm.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Previous studies performed in our laboratory indicated that E2 could enhance the recruitment of EPCs and had favorable effects on neovascularization in ischemic tissues.11 The focus of the present study was the role of ERs in activation of EPCs during ischemic neovascularization.

These data, from in vitro EPC functional assays, assessment of in vivo EPC homing to ischemic myocardium, and EPC mobilization from bone marrow after MI in mice in which the bone marrow had been replaced with ER{alpha}KO or ERßKO mutant marrow, indicate that both ER{alpha} and ERß are functional in EPC-mediated ischemic neovascularization.

The present findings further reveal that the effects of E2 on EPC activation are mediated more prominently via ER{alpha} than ERß. Prior studies of the roles of ER{alpha} and ERß in mediating the macrovascular protective effects of estrogen acting via endothelial cells.8,29–31 including studies in transgenic mice in which either ER{alpha} or ERß expression had been disrupted, have revealed that ER{alpha}, not ERß, mediates the protective effects of estrogen after vascular injury.30,31 Our data indicate that certain microvascular effects of E2 involving bone marrow–derived EPCs are mediated predominantly via ER{alpha}, but the data also provide evidence of a clear function for ERß in this setting.

Our data also provide several clues to explain the apparently disproportionate role of ER{alpha} in EPC-mediated ischemic neovascularization. First, we found that ER{alpha} mRNA expression was >10 times higher than ERß. In agreement with previous evidence in mature endothelial cells,32 physiological levels of E2 induce ER{alpha} mRNA upregulation in EPCs, indicating that the ligand has potent effects on the expression of its own receptor. Moreover, FITC-conjugated E2-ER binding assay and reporter gene luciferase assay for ERE-dependent transcription support that ER{alpha} is a main functional receptor for E2 in EPCs.

Further potential mechanisms of the ER{alpha}-specific effects on EPCs were provided by gene expression profiling, which indicated that VEGF was markedly upregulated by E2 in WT but not ER{alpha}KO EPCs. The in vitro findings in isolated EPCs were corroborated in vivo, showing VEGF expression in incorporated WT EPCs in the mouse MI model compared with minimal expression by ER{alpha}KO EPCs. Previous studies have shown that estrogens increase VEGF expression in uterine tissue,33,34 endometrial cells,35,36 endometrial adenocarcinomas,37,38 breast cancer cells,39,40 and vascular smooth muscle cells.41 In cancer cells, E2 was shown to increase VEGF transcriptional activity through both ER{alpha} and ERß in VEGF promoter luciferase assays42,43 via E2-ER complexes binding variant ERE in the promoter region of VEGF gene.42,44 Our data indicate that this mechanism is not at play in EPCs.

The actual mechanism by which E2 influences EPC kinetics through ERß remains unknown. Among 128 angiogenic genes investigated by gene array analysis, we could not find any candidates with consistent and specific expression differences between WT and ERßKO EPCs. A more comprehensive search to identify previously unsuspected candidate genes is under way.

The findings of this report could be of interest for a variety of scientific reasons; however, the impact of our data resides to a large degree on the significant overall effect of estradiol on post-MI outcome. In the present investigation, we concentrated our studies on the mobilization, recruitment, and incorporation of bone marrow–derived cells into vascular structures; however, this paradigm alone may not offer the full explanation for the effect of E2. For example, we did not investigate the possibility that E2 augmented other pathways of tissue repair such as mobilization and homing of cells that can differentiate into cardiomyocytes45–47 and stimulation of local cardiac progenitors.48 In the setting of acute myocardial injury, improved outcome may result via protection (eg, antiapoptosis) and via the full paradigm of tissue repair (ie, replacement of damaged structures). Tissue repair thus requires neovascularization, as documented in the present investigation; however, our studies do not exclude additional E2-mediated effects. Cardiac myocytes and fibroblasts are known to express ER,49,50 providing a mechanism for direct effects of E2 on these components of the tissue response to MI.

Finally, beyond the specific findings of this report, these data have important implications because they provide additional evidence of the critical importance of the phenotype of bone marrow–derived EPCs in regulating ischemic tissue repair and suggest that modulation of EPC phenotype may have important therapeutic implications.


*    Acknowledgments
 
We thank Mickey Neely for secretarial assistance.

Sources of Funding

This study was supported in part by NIH grants (HL-53354, HL-57516, HL-63414, HL-77428, HL-80137, HL-P01-66957).

Disclosures

Dr Losordo has significant relationships as a principal investigator, collaborator, or consultant on research grants with the following companies: Baxter, Inc, Corautus, Cordis, Curis, Anormed, and Boston Scientific Corp. The other authors report no conflicts.


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 up arrowDiscussion
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CLINICAL PERSPECTIVE

Despite the negative results of recent randomized trials of hormone replacement therapy for the prevention of cardiovascular events, abundant clinical and preclinical data provide evidence of a protective effect of estrogen. For example, premenopausal women are significantly less likely to experience cardiovascular events than are age-matched men, and premature (eg, surgical) menopause is associated with an increased incidence of coronary disease. Most recently, mutations in certain estrogen receptors in humans have been associated with increased incidence of cardiac disease. The present study examined the role of estrogen receptors (ERs) {alpha} and ß in the improved recovery from myocardial infarction resulting from estradiol (E2) administration. The data reveal that although both ER{alpha} and ERß mediate the effects of E2 on myocardial infarction recovery, ER{alpha} appears to be more critical. These data may lead to the identification of new therapeutic targets for post–myocardial infarction recovery by dissecting the effects of ER{alpha} and ERß. With the knowledge that ER{alpha} is more important in the overall benefit of E2, specific downstream signaling pathways that directly mediate the benefit of E2 may be targeted pharmacologically, thereby permitting development of a therapy that mimics the benefits of E2 without the side effects (thrombosis, gynecomastia in men, etc) of the natural or synthetic hormone.


*    Footnotes
 
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.631465/DC1.




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