Estradiol Accelerates Reendothelialization in Mouse Carotid Artery Through Estrogen Receptor-α but Not Estrogen Receptor-β
Background—The atheroprotective effect of 17β-estradiol (E2) has been suggested in women and clearly demonstrated in animals through both an effect on lipid metabolism and a direct effect on the cells of the arterial wall. It has been shown, for example, that E2 promotes endothelium-dependent relaxation and accelerates reendothelialization in rats. Similar studies have been undertaken in mice to appreciate the molecular mechanism of this process.
Methods and Results—We report here a model of electric carotid injury adapted from that described by Carmeliet et al (1997) that allows us to precisely evaluate the reendothelialization process. We demonstrate that E2 accelerates endothelial regeneration in castrated female wild-type mice. In ovariectomized transgenic mice in which either the estrogen receptor (ER)-α or ERβ gene has been disrupted, E2 accelerated reendothelialization in female ERβ knockout mice, whereas this effect was abolished in female ERα knockout mice.
Conclusions—This study demonstrates that ERα but not ERβ mediates the beneficial effect of E2 on reendothelialization and potentially the prevention of atherosclerosis.
Epidemiological and observational studies1 2 support a vasculoprotective effect of estrogens. This is traditionally thought to be due to potentially favorable changes in blood lipids and lipoproteins,3 but a number of experimental studies4 5 6 7 8 9 strongly suggest a direct effect on the vascular system. Among the cell types present in the normal and atherosclerotic vessel wall, endothelium represents both a key actor in the pathophysiology of atherosclerosis and an important target for 17β-estradiol (E2).
Twenty years of intensive experimental work has confirmed the crucial role of endothelium in the physiology of circulation. The endothelium is uniquely positioned at the interface between the blood and the vessel wall. As such, it performs multiple functions: it is involved in the regulation of coagulation, leukocyte adhesion in inflammation, vessel tone, and vascular smooth muscle growth and also acts as a barrier to transvascular flux of liquids and solutes. In addition, endothelial abnormalities appear to be central to the pathogenesis of atherosclerosis.10 11 The importance of endothelial integrity was initially demonstrated by the facilitation of atherosclerotic lesion development in hypercholesterolemic animal models after removal of endothelium. Although subsequent studies demonstrated the presence of endothelium overlying the lesions, repeated mechanical, hemodynamic, and/or immunological injury probably contributes to morphological and functional alterations of the endothelium as well as its senescence. Telomere length is decreased in cells of the arterial tree subject to hemodynamic stress and prone to atherosclerosis, demonstrating an accelerated cell turnover at these sites.12 Recent experimental work has demonstrated that low shear stress, which in some respects mimics turbulent blood flow, favors apoptosis of cultured endothelial cells.13 14 Altogether, these observations suggest that the capacity of endothelium to maintain a cell monolayer could be of crucial importance in the prevention of atherosclerosis and its complications.
Several models of arterial injury have been developed to study endothelial regrowth. Injury of an artery by passage of a balloon catheter causes endothelial denudation and medial damage and produces both an endothelial and a smooth muscle cell proliferative response in nonhuman primates, swine, dogs, rabbits, and rats.15 In such models, E2 was reported to promote endothelial regrowth in castrated female rats.16 The effect of E2 has long been thought to be mediated through activation of the estrogen receptor (ER)-α. ERα is a ligand-dependent transcriptional activator17 that modulates gene expression in target cells not only in reproductive tissues but also in bone and vessels.18 19 Moreover, ERβ, recently discovered and cloned from rat prostate,20 was found to be expressed in many other tissues, including injured arteries.21 22
Evaluation of the respective roles of ERα and ERβ in the reendothelialization process requires a reliable model of mouse arterial injury in which the extent of endothelial regrowth can be quantified. Removal of the endothelium in the mouse carotid artery with a flexible wire was described in 199323 and allowed the arterial smooth muscle proliferation and/or neointima formation in various transgenic mice to be evaluated. This model of endovascular carotid injury suggested that ERα was not involved in one aspect of the protective effect of E2, ie, the prevention of medial smooth muscle proliferation.24 However, the specific evaluation of arterial reendothelialization appears to be difficult with this technique.23 Recently, Carmeliet et al25 described a novel model of electric injury to the mouse femoral artery that consisted of destroying endothelial, smooth muscle, and adventitial cells and allowing subsequent quantification of both arterial neointima formation and reendothelialization.25 We have adapted this model of electric arterial injury to allow the precise study of arterial reendothelialization and to determine which ER subtype was involved in this process. Our results indicate that the beneficial effect of estrogen on reendothelialization is mediated by ERα in female mice.
All procedures involving experimental animals were performed in accordance with the recommendations of the French Accreditation of Laboratory Animal Care. They were housed in stainless steel cages in groups of 5, kept in a temperature-controlled facility on a 12-hour light-dark cycle, and fed normal laboratory mouse chow diet.
Targeted disruption (knockout) of mouse ERα and ERβ genes was generated by homologous recombination, resulting in ERα- and ERβ-null mice.25A Six backcrosses with C57Bl/6 mice were performed.
For all surgical procedures, mice were anesthetized by injection of 150 mg/kg ketamine IP and allowed to recover on a 37°C heat pack. Mice were ovariectomized at 4 weeks of age and given either 60-day time-release E2 pellets (0.1 mg E2, Innovative Research of America, ie, releasing 80 μg · kg−1 · d−1) or placebo-containing pellets implanted subcutaneously into the backs of the animals with a sterile trochar. Electric carotid artery injury was performed 2 weeks later, ie, in 6-week-old mice. Three days after injury, the animals were killed and the vessels harvested.
Electric Injury Model
We adapted the electric injury model described by Carmeliet et al25 on the femoral artery to the common carotid artery, the latter being easier to dissect. Surgery was carried out with a dissection microscope (Nikon SMZ-2B) in 6-week-old female mice weighing 20 g on average. Because the proximal part of the carotid artery is intrathoracic, the injury could not be applied to the whole common carotid artery. The left common carotid artery was exposed via an anterior incision of the neck. The electric injury (in fact primarily thermal) was applied to the distal part of the common carotid artery. The carotid artery was injured by electric current with a bipolar microregulator. To standardize the temperature increase in the vessel wall, we used forceps with large tips (1 mm) instead of microsurgical forceps (200 μm) and a bipolar microregulator Force FX (Valleylab). The “precise” mode of this apparatus allowed delivery of electric energy within a narrow range of resistance, because the generator microprocessor disrupted the electric current when the resistance increased as a consequence of temperature increase. This allowed the increase in tissue temperature to be controlled and avoided the risk of desiccation and coagulation of the arterial wall. The optimal conditions were determined as follows: electric current of 2 W applied for 2 seconds to each millimeter of carotid artery over a total length of 4 mm with the help of a size marker placed parallel to the long axis of the carotid. Despite optimization of the technique, coagulation and thrombosis of the carotid artery occurred in ≈10% of the cases, which were then excluded from the study.
One to 7 days later, the endothelial regeneration process was evaluated by staining the denuded areas with Evans blue dye as previously described.23 Briefly, 50 μL of solution containing 5% Evans blue diluted in saline was injected into the tail vein with a 30-gauge needle 10 minutes before euthanization, followed by fixation with a perfusion of 4% phosphate-buffered formalin (pH 7.0) for 5 minutes. Blood, saline, and fixative were removed through an incision in the right atrium. The left common carotid artery was dissected with an adjacent portion of the aortic arch and carotid bifurcation. The artery was then opened longitudinally and placed between slides with Fluoprep. After transparency scanning and numeration, the total and stained carotid artery areas were planimetered with an image analyzer (VISIOL@b2000). The ratio between the area stained in blue and the total carotid artery area was calculated. The surface of the area that remained deendothelialized was indexed to the total carotid artery area to take into account the changes in vessel area due to both the elasticity of the carotid artery and the flattening of the vessel between slides. The coefficients of variation of the endothelial regrowth were 2.6%, 4.5%, and 13.5% at days 1, 3, and 5, respectively (Figure 1C⇓).
Histology and Scanning Electron Microscopy
Arteries were also embedded in paraffin, and sections perpendicular to the long axis of the carotid were cut from the proximal, middle, and distal thirds of the injured carotid artery. Sections were subjected to standard hematoxylin and eosin staining.
To verify that the area stained with Evans blue corresponded to the deendothelialized area and that the unstained area corresponded to the reendothelialized area, 3 animals were killed 3 days after the electric injury procedure. Perfusion fixation was carried out as described above with phosphate-buffered 4% paraformaldehyde in vivo. The vessels were cut open longitudinally, pinned flat on a silicone-coated dish, and photographed. The vessels were then further fixed with 2% glutaraldehyde for 24 hours, rinsed with PBS, incubated with 2% osmium tetroxide, and then dehydrated through a series of ethanol dilutions. The tissue was dried to critical point and mounted on scanning electron microscopy stubs with colloidal silver paste. After having been sputter-coated with gold/palladium, the specimens were examined with a scanning electron microscope.
Serum Hormone Concentrations
Radioimmunoassay kits for E2 were used according to the manufacturer’s instructions (Sorin Biomedica). Hormone levels could not be measured in the same mice that were injected with Evans blue because it interfered with the assay. Thus, additional groups of mice were ovariectomized at 4 weeks of age, given either E2- or placebo-containing pellets as described above, and killed 17 days later. Hormone levels were assayed for each individual mouse in a similar series of assays. The intra-assay coefficient of variability was 4.5%. The assay sensitivity, defined as 15% displacement of labeled tracer, was 0.5 pg E2.
Results are expressed as mean±SEM. To test the respective roles of E2 treatment and of genotype on reendothelialization, a 2-factor ANOVA was performed (comparison of the 6 groups) in ERα and in ERβ mice. When an interaction was observed between the 2 factors, the effect of E2 treatment was studied in each genotype with a t test. A value of P<0.05 was considered statistically significant.
Generation of the ERα and ERβ Mutant Mice
Heterozygous ERα- and ERβ-null mice, originally generated as C57Bl/6J×129 hybrids, were crossbred into the C57Bl/6 background to generate the heterozygous ERα+/− and ERβ+/− lines, which constituted the population that served as the parental genotypes for all animals in the studies. The offspring of the parental ERα+/− strain were ERα+/+, ERα+/−, and ERα−/−, and those of the parental ERβ+/− strain were ERβ+/+, ERβ+/−, and ERβ−/− littermates, respectively, which served as the subjects of our studies.
Description of the Model of Carotid Artery Injury
We first determined the time course of endothelial regeneration (days 1, 3, 5, and 7 after injury) in 6-week-old female C57Bl/6 mice. As shown in Figure 1⇑, 50% of the endothelial regeneration was observed on days 3 and 4, and complete reendothelialization was observed on day 7. We chose to study reendothelialization on day 3 to evaluate the effect of E2. To further ensure that the area stained with Evans blue dye corresponded to a deendothelialized area, electron scanning microscopy studies were performed. These confirmed that the area stained colocalized with an area of endothelial cell denudation and a monolayer of aggregated platelets. A front of migrating endothelial cells was observed at the edges of the lesion (Figure 2A⇓, 2B⇓, and 2C⇓).
Paraffin sections (day 2) with standard staining (hematoxylin/eosin and Masson’s trichrome) also demonstrated the absence of endothelial cells (or rare pyknotic nuclei), the absence of smooth muscle cells in the media (or only necrotic fragments), and infiltration of the adventitia by inflammatory cells (Figure 3a⇓ and 3b⇓). Verhoeff staining for elastic lamina revealed that only the most internal lamina was in some cases ruptured (not shown). At day 15, ad integrum restoration of the intima and of the media was observed (Figure 3c⇓ and 3d⇓). However, no neointimal hyperplasia was observed after electric injury of the carotid, even on day 30 (Figure 3e⇓ and 3ff⇓). Adventitia was still infiltrated by inflammatory cells, which persisted at day 15 (Figure 3c⇓ and 3d⇓) and at day 30 (Figure 3e⇓ and 3f⇓).
Effect of Estradiol on Endothelial Regeneration
Ovariectomized mice with an implanted placebo pellet showed nondetectable (<5 pg/mL ie, 20×10−12 mol/L) circulating levels of E2, whereas those implanted with a pellet releasing 0.1 mg E2 for 60 days (ie, 80 μg · kg−1 · d−1) showed serum E2 concentrations averaging 0.5×10−9 mol/L, irrespective of genotype (Tables 1⇓ and 2⇓). Ovariectomized, placebo-treated mice showed atrophied uteri (<20 mg); E2-treated mice showed a significant increase in uterine weight, except ERα−/− mice, which had atrophied uteri even when treated with E2 (Tables 1⇓ and 2⇓).
Endothelial regeneration was similar in all categories of mice with an implanted placebo pellet, irrespective of genotype (Tables 1⇑ and 2⇑, Figure 4⇓). In ERα-deficient mice, an interaction (P=0.004) between E2 treatment and genotype on reendothelialization was revealed by the 2-factor ANOVA. In these animals, E2 treatment significantly accelerated reendothelialization in ERα+/+ mice (P<0.0001), tended to accelerate the reendothelialization in ERα+/− mice (P=0.14), but had no effect in ERα−/− mice (P=0.95) (Figure 4⇓ and Table 1⇑). In contrast, in ERβ-deficient animals, E2 treatment significantly accelerated the reendothelialization process in ERβ+/+, ERβ+/−, and ERβ−/− mice in a similar fashion (P<0.0001), without influence of genotype (P=0.27, NS). Although the acceleration of the reendothelialization tended to be greater in E2-treated ERβ−/− mice treated with E2 than in ERβ+/+ mice, the difference was not statistically significant, because no interaction between E2 treatment and the genotype was observed (P=0.73, NS) (Figure 4⇓ and Table 2⇑).
Endothelium is both a key actor in the pathophysiology of atherosclerosis and an important target of E2 in the vessel wall. Indeed, the modulation of several properties of endothelium in response to E2 could account for the vasculoprotective effect of the hormone. One major function of endothelium is the release of the vasorelaxant and antiaggregant messenger nitric oxide, which is enhanced in oophorectomized females treated with estrogens26 27 28 either directly by upregulation of the endothelial NO synthase gene expression29 or indirectly by a receptor-mediated antioxidant effect.30 E2 was also reported to promote reendothelialization in castrated female rats,16 but to the best of our knowledge, this effect has not been reported in mice. Moreover, the availability of genetically modified mice with ERα or ERβ gene disruption offers the possibility of studying the role of each of these ER subtypes in the effect of E2 on endothelial regeneration.
Preliminary experiments led us to modify the model of electric injury initially described by Carmeliet et al25 to study the reendothelialization process. For technical reasons, we elected to perform the electric injury in the carotid instead of the femoral artery. As reported for the femoral site,25 we found that the reendothelialization process started immediately after injury and proceeded very actively during the first week after injury. The 4 mm of injured carotid artery was completely reendothelialized within 7 days, ie, 300 μm/d for each edge. Taking into account that the average length of an endothelial cell is 50 μm (see previous reports31 32 and Figure 2⇑) and that the doubling time of this cell population is in the range of 24 hours, proliferation would contribute to 50 μm/d from each edge over the 7-day period. It would appear from these calculations that reendothelialization relies essentially on local cell migration or circulating endothelial progenitor cells, which have been shown to be incorporated into foci of neovascularization.33 34
We observed that E2 accelerated endothelial regrowth in wild-type mice by 25%, a value close to what had been measured in the rat species.16 We then sought to determine which ER gene promoted the reendothelialization process. ERα has long been considered to be the unique target of E2, because it has been characterized in reproductive tissues, in bone, and in vessels, particularly in the endothelial cells.35 36 37 Iafrati et al24 reported that the prevention of medial enlargement (ie, smooth muscle cell proliferation) by E2 is preserved in ERα-deficient mice in a model of endovascular carotid injury. Very recently, the same group reported that E2 also inhibits medial enlargement in the injured carotid of ERβ-deficient mice.38 It should be underlined that in these studies, vascular smooth muscle cell proliferation was evaluated, whereas in our studies, the effect of E2 on endothelial regeneration was appreciated. Altogether, these series of data led to the intriguing possibility that reendothelialization could be mediated specifically by the ERα gene, whereas arterial smooth muscle cell proliferation could be mediated by a third, as yet unidentified ER gene. Further work should clarify this situation, but taking into account the crucial role of endothelium in the maintenance of vascular integrity, ERα should be considered a prime target of pharmacological studies in the area of cardiovascular diseases.
The work at INSERM U397 was supported in part by INSERM, the Ministère de la Recherche et de la Technologie, the Fondation de France, the Fondation de l’Avenir, and the Conseil Régional Midi-Pyrénées. The work at the Institut de Génétique et de Biologie Moléculaire et Cellulaire was supported by a grant from the CNRS, INSERM, the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche Médicale, the Fondation pour la Recherche Médicale, and by GIP/HMR. Dr Dupont was supported by a fellowship from Hoechst Marion Roussel. We thank J.-L. Fontanilles, G.B.M., Hôpital Rangueil, for his valuable technical assistance.
- Received May 22, 2000.
- Revision received July 11, 2000.
- Accepted August 1, 2000.
- Copyright © 2001 by American Heart Association
Stampfer M, Grodstein F. Cardioprotective effect of hormone replacement therapy is not due to a selection bias. BMJ. 1994;309:808–809.
Grodstein F, Stampfer MJ, Colditz GA, et al. Postmenopausal hormone therapy and mortality [see comments]. N Engl J Med. 1997;336:1769–1775.
Lobo RA, Speroff L. International consensus conference on postmenopausal therapy and the cardiovascular system. Fertil Steril. 1994;61:592–595.
Gilligan DM, Quyyumi AA, Cannon RO, et al. Effects of physiological levels of estrogen on coronary vasomotor function in postmenopausal women. Circulation. 1994;89:2545–2551.
Hough IL, Zilversmit DB. Effect of 17β-estradiol on aortic cholesterol content and metabolism in cholesterol-fed rabbits. Arteriosclerosis. 1986;6:57–63.
Haarbo J, Leth-Espensen P, Stender S, et al. Estrogen monotherapy and combined estrogen-progestogen replacement therapy attenuate aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits. J Clin Invest. 1991;87:1274–1279.
Keaney JFJ, Gaziano JM, Xu Z, et al. Low-dose α-tocopherol improves and high-dose α-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest. 1994;93:844–851.
Bourassa P-A, Milos PM, Gaynor BJ, et al. Estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A. 1996;93:10022–10027.
Elhage R, Arnal JF, Pierragi M-T, et al. Estradiol-17β prevents fatty streak formation in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 1997;17:2679–2684.
Harrison D. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest. 1997;100:2153–2157.
Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;340:115–125.
Chang E, Harley CB. Telomere length and replicative aging in human vascular tissues. Proc Natl Acad Sci U S A. 1995;92:11190–11194.
Kaiser D, Freyberg M-A, Friedl P. Lack of hemodynamic forces triggers apoptosis in vascular endothelial cells. Biochem Biophys Res Commun. 1997;231:586–590.
Dimmeler S, Hermann C, Galle J, et al. Upregulation of superoxide dismutase and nitric oxide synthase mediates the apoptosis-suppressive effects of shear stress on endothelial cells. Arterioscler Thromb Vasc Biol. 1999;19:656–664.
Schwartz SM, Reidy MA, O’Brien ERM. Assessment of factors important in atherosclerotic occlusion and restenosis. Thromb Haemost. 1995;74:541–551.
Krasinski K, Spyridopoulos I, Asahara T, et al. Estradiol accelerates functional endothelial recovery after arterial injury. Circulation. 1997;95:1768–1772.
Green S, Walter P, Kumar V, et al. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature. 1986;320:134–139.
Mendelsohn M, Karas R. Estrogen and the blood vessel wall. Curr Opin Cardiol. 1994;9:619–628.
Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen. FASEB J. 1996;10:615–624.
Kuiper GGJM, Enmark E, Pelto-Huikko M, et al. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U S A. 1996;93:5925–5930.
Mosselman S, Polman J, Dijkema R. ERβ: identification and characterization of a novel human estrogen receptor. FEBS Lett. 1996;392:49–53.
Mäkelä S, Savolainen H, Aavik E, et al. Differentiation between vasculoprotective and uterotrophic effect with different binding affinities to estrogen receptors α and β. Proc Natl Acad Sci U S A. 1999;96:7077–7082.
Lindner V, Fingerle J, Reidy MA. Mouse model of arterial injury. Circ Res. 1993;73:792–796.
Iafrati M, Karas R, Aronovitz M, et al. Estrogen inhibits the vascular injury response in estrogen receptor α-deficient mice. Nat Med. 1997;3:545–548.
Carmeliet P, Moos L, Stassen J, et al. Vascular wound healing and neointima formation induced by perivascular electric injury in mice. Am J Pathol. 1997;150:761–776.
Dupont S, Krust A, Gansmuller A, et al. Effect of single and compound knockouts of estrogen receptors α (ERα) and β (ERβ) on mouse reproductive phenotypes. Development. 2000;19:4277–4291.
Gisclard V, Miller VM, Vanhoutte P. Effect of 17β-estradiol on endothelium-dependent responses in the rabbit. J Pharmacol Exp Ther. 1988;244:19–22.
Hayashi T, Fukuto JM, Ignarro LJ, et al. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci U S A. 1992;89:11259–11263.
Keaney JF, Shwaery GT, Xu AM, et al. 17β-Estradiol preserves endothelial vasodilator function and limits low-density lipoprotein oxidation in hypercholesterolemic swine. Circulation. 1994;89:2251–2259.
Weiner CP, Lizasoain I, Baylis SA, et al. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci U S A. 1994;91:5212–5216.
Arnal JF, Clamens S, Pechet C, et al. Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine aortic endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production. Proc Natl Acad Sci U S A. 1996;93:4108–4113.
Levesque M, Liepsch D, Moravec S, et al. Correlation of endothelial cell shape and wall shear stress in stenosed dog aorta. Arteriosclerosis. 1986;6:220–229.
Davies P. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519–558.
Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964–967.
Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularisation. Science. 1999;275:964–967.
Colburn P, Buonassissi V. Estrogen-binding sites in endothelial cell cultures. Science. 1978;201:817–819.
Bayard F, Clamens S, Delsol G, et al. Oestrogen synthesis, oestrogen metabolism and functional oestrogen receptors in bovine aortic endothelial cells. In: Non-Reproductive Actions of Sex Steroids. Chichester, UK: Ciba Symposium Foundation (Wiley); 1995:122–138.
Venkov C, Rankin A, Vaughan D. Identification of authentic estrogen receptor in cultured endothelial cells: a potential mechanism for steroid hormone regulation of endothelial function. Circulation. 1996;94:727–733.
Karas R, Hodgin J, Kwoun M, et al. Estrogen inhibits the vascular injury response in estrogen receptor β-deficient mice. Proc Natl Acad Sci U S A. 1999;96:15133–15136.