J.- F. Arnal1 , H. Laurell1, F. Lenfant1, V. Douin-Echinard1, L. Brouchet1 and P. Gourdy1
(1)
INSERM U589, INSTITUT L. BUGNARD, 1 avenue Jean Poulhès, 31403 Toulouse Cedex, France
J.- F. Arnal
Email: arnal@toulouse.inserm.fr
Abstract
Whereas hormonal replacement/menopause therapy (HRT) in postmenopausal women increases coronary artery disease risk, epidemiological studies (protection in premenopaused women) suggest and experimental studies (prevention of the development of fatty streaks in animals) demonstrate a major atheroprotective action of estradiol (E2). The understanding of the deleterious and beneficial effects of estrogens is thus required. The atheroprotective effect of E2 is absent in mice deficient in mature T and B lymphocytes, demonstrating the crucial role of the endothelium/immune system pair. The immunoinflammatory system appears to play a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Whereas E2 favors an anti-inflammatory effect in vitro (cultured cells), it elicits in vivo a pro-inflammation at the level of several subpopulations of the immunoinflammatory system, which could contribute to plaque destabilization. Endothelium appears to be an important target for E2, since it potentiates endothelial NO and prostacyclin production, thus promoting beneficial effects such as vasorelaxation and inhibition of platelet aggregation. Prostacyclin, but not NO, appear to be involved in the atheroprotective effect of E2, which also accelerates endothelial regrowth, thus favoring vascular healing. Finally, most of these E2 effects are mediated by estrogen receptor α and are independent of estrogen receptor β. In summary, a better understanding of the mechanisms of estrogens on the normal and atheromatous arteries is required and should help to optimize the prevention of cardiovascular disease after menopause. These mouse models should help to screen existing and future selective estrogen receptor modulators (SERMs).
Estrogens play a pivotal role in sexual development and reproduction and are also implicated in a number of physiological processes in various tissues, including the cardiovascular system. Numerous epidemiological studies suggest that estrogens protect women against cardiovascular diseases before the age of menopause. After menopause, women's cardiovascular risk becomes progressively closer to that of men, reinforcing the hypothesis of an atheroprotective effect of estrogens. However, the two controlled prospective and randomized studies published so far did not demonstrate a beneficial effect of hormone replacement therapy (HRT), neither in secondary prevention (Heart and Estrogen/Progestin Replacement Study, HERS) (Hulley et al. 1998) nor in primary prevention (Women's Health Initiative study, WHI) (Rossouw et al. 2002). This is in contrast to the large amount of data from experimental models of atherosclerosis, where estradiol (E2) treatment prevents the development of fatty streaks in comparison with castrated animals given a placebo (Hodgin and Maeda 2002).
Waters et al. (2004) recently defined five main priorities in the area of menopause treatment and cardiovascular (CV) risk including: 1. The determination of the mechanisms of the CV events during the first year of the HRT, 2. The understanding of the beneficial effects of endogenous estrogens.
1 The Atheromatous Process: Numerous Cellular Players for Several Scenarios
The first step of the atheromatous process is the penetration of atherogenic lipoproteins, in particular low-density lipoproteins (LDL) through the endothelial monolayer (Steinberg 2002). LDL oxidation occurs in the subendothelial space and probably represents a necessary modification to the subsequent steps, because oxidized LDL induces in turn an activation of the endothelium, consisting in particular of the increased expression of adhesion molecules, such as intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1). These molecules are required to slow down circulating monocytes, stop them, and allow their subsequent migration into the intima. In the subendothelial space, the activation of the monocytes induces their differentiation into macrophages, and this probably contributes to increasing the level of LDL oxidation. These modified LDLs can be recognized by scavenger receptors expressed by macrophages. Thus, macrophages attempt to clean the intima, thereby preventing the accumulation of oxidized LDL. As oxidized LDL accumulates intracellularly, macrophages progressively turn into foam cells, which make up the major component of fatty streaks.
Blood flow shear stress represents a crucial protective factor, where abnormal shear stress promotes endothelial activation and dysfunction, to date the best-recognized explanation for a focal location of atheroma functions (Gimbrone 1999; Shyy and Chien 2002; Tedgui and Mallat 2001). Classic risk factors (high blood pressure, hypercholesterolemia, smoking, diabetes) appear to favor and/or aggravate endothelial dysfunction. They can also favor the production of chemokines and cytokines by the different cellular actors (endothelium, monocytes-macrophages, smooth muscle cells, but also lymphocytes). Protective factors are less recognized, although high-density lipoproteins (HDL) appear to be of major importance.
Expansion of the fatty streak tends to be limited and circumvented by a scarring reaction of the smooth muscle cells migrating to the intima and secreting collagen. The balance between the inflammation level and the strength of fibromuscular cap determines the stability of the atheromatous plaque (Ross 1999). Plaque rupture exposes thrombogenic materials, leading to the formation of a thrombus, which threatens the viability of the tissue downstream from the occluded artery. Unfortunately, plaque rupture is not satisfactorily modeled in mouse models, and this is probably the greatest limitation of the current experimental approach.
Many groups have been working to describe the cellular and molecular mechanisms leading to the aggravation of or protection from atheroma (Ross 1999; Tedgui and Mallat 2001; Hansson et al. 2002; Libby 2002), leading to the generation of the two major models of hypercholesterolemic mice: mice deficient in apolipoprotein E (apoE-KO) and mice deficient in LDL-receptor. ApoE-KO mice are hypercholesterolemic (3–4 g cholesterol/l) under a chow diet and have very low levels of HDL cholesterol (Zhang et al. 1992). Accordingly, they spontaneously and rapidly (within a few weeks) develop fatty streak at the root of the aorta. Mice deficient in LDL-receptor must be given a Western diet (fat plus cholesterol) to develop fatty streaks, because their lipoprotein profile under chow diet is less severe than the apoE-KO mice.
So far, the cellular and molecular dissection of the pathophysiology of atheroma was explored by breeding hypercholesterolemic mice and mice deficient in another specific gene. For instance, hypercholesterolemic mice also deficient in either monocyte-macrophage (through a deficit of macrophage colony stimulating factor) (Smith et al. 1995) or mature B and T lymphocytes (RAG-2 gene deficient) (Dansky et al. 1997) respectively develop one-tenth and one-half the amount of fatty streaks than control hypercholesterolemic mice. Mice deficient in various cytokines in general demonstrated the aggravating role of pro-inflammatory cytokine (such as interferon γ (IFNγ), interleukins (IL)-1α and β, IL-12, IL-18) and the protective role of anti-inflammatory cytokines (mainly IL-10) in the development of the atherosclerotic process (see Libby 2002 and Tedgui and Mallat 2001 and references therein).
Platelets were recently found to participate to the constitution of fatty streak lesions at a very early stage, in particular at the level of the carotid bifurcation, a lesion-prone site, by interacting with the activated endothelium before any macrophage infiltration (Massberg et al. 2002). This process involves the platelet GPIbα (glycoprotein 1b, alpha polypeptide), and the adhesive proteins P-selectin and/or von Willebrand factor, which mediate the attachment of platelets to activated endothelial cells. Blocking these interactions completely (–100%) prevented fatty streak formation at the level of the carotid bifurcation in apoE-KO mice, but only partially (–30%) at the level of the aortic sinus (Massberg et al. 2002). While this points out that platelets can be a target for antiatherosclerotic therapies, this also suggests that modulation of the adhesive properties of the endothelium may be of pathophysiological relevance, especially at the level of the carotid bifurcation.
2 E2 Prevents Fatty Streak in Models of Atheroma
Studies in primates, mainly conducted by Clarkson et al., have provided convincing evidence for the primary prevention of coronary artery atherosclerosis when estrogens are administered soon after the development of estrogen deficiency (Clarkson and Appt 2005). Equally convincing are the data from monkey studies indicating the total loss of the beneficial effects of estrogens if the treatment is delayed for a period equal to 6 postmenopausal years for women. Moreover, in the monkey model, an attempt has been made to identify the most effective hormone treatment regimen in preventing the progression of coronary artery atherosclerosis. By far the most successful approach uses estrogen containing oral contraceptive during the perimenopausal transition, followed directly by hormone replacement therapy postmenopausally. However, the monkey model does not provide an understanding of the cellular or molecular mechanisms of E2 action.
Several groups, including ours, have been working to describe the vascular effects of E2 and elucidate the cellular and molecular mechanisms. Castration of ApoE-KO or LDL receptor (LDL-R) KO mice is followed by an increase in the fatty streak lesion area and, in castrated mice, E2 prevents this fatty streak deposit. However, serum E2 concentrations on the order of those encountered during gestation are necessary for maximal protection (Bourassa et al. 1996; Elhage et al. 1997a). Although E2 treatment induces a decrease in serum cholesterol concentrations, the decrease involving both LDL and HDL fractions is too minor to explain the hormone's atheroprotective effect (Elhage et al. 1997a; Hodgin and Maeda 2002), which seems to result mainly from a direct effect of E2 on the cells of the arterial wall (Arnal et al. 2004). A similar conclusion was previously obtained by other groups (Haarbo et al. 1991; Holm et al. 1999) using hypercholesterolemic rabbits. In addition, they elegantly showed the crucial role of intact endothelium, as the antiatherogenic effect of E2 was abolished, or even reversed, after balloon catheter injury (Holm et al. 1999).
3 Involvement of the Inflammatory–Immune System
A mentioned above, cell populations of the inflammatory–immune system (monocytes-macrophages, lymphocytes, etc.) play crucial roles in the pathophysiology of atherosclerosis (Binder et al. 2002; Hansson et al. 2002; Libby 2002; Tedgui and Mallat 2001). Indeed, we demonstrated that E2 prevents the deposit of fatty streaks in immunocompetent ApoE-KO mice, whereas it has no effect in mice deficient in both apoE and RAG-2 gene expression, since mature B and T lymphocytes are lacking (Elhage et al. 2000). One hypothesis resulting from these observations was that lymphocytes, or at least a subpopulation of them, were the mediators of the atheroprotective effect. After crossing ApoE-KO mice with mice deficient in either TCRαβ, CD4, CD8, or TCRδγT lymphocytes, we reported that TCRαβ T lymphocytes play a major role in fatty streak development (Elhage et al. 2004). However, the protective effect of E2 persisted in all these strains, showing that none of these lymphocyte subpopulations specifically mediated the atheroprotective effect of E2 (Elhage et al. 2005).
4 Does E2 Have a Pro- or Anti-inflammatory Effect?
At variance with macrophages in fatty streaks, peritoneal macrophages are a cell population that can be obtained in considerable amounts, and thus the chronic effect of E2 on the inflammatory–immune system can easily be studied in these cells. We first must mention again that a chronic in vivo treatment of mice by E2 led to a pro-inflammatory response in peritoneal macrophages, whereas an acute (only a few hours of E2) in vitro treatment of the macrophage cell line RAW 264.7 by E2 led to an anti-inflammatory effect (Ghisletti et al. 2005). This striking discrepancy demonstrates the importance of the in vivo approach to understand the pathophysiological effects of E2.
We also evaluated the possibility that E2 promotes the production of anti-inflammatory cytokines. In collaboration with two groups of immunologists, we demonstrated that the profile of cytokine secretion in CD4+ (Maret et al. 2003) as well as in NK T lymphocytes (Gourdy et al. 2005) is altered by E2. In these studies, an increase in IFNγ production and a decrease in anti-inflammatory cytokine production were observed, i.e., toward a Th1 profile. Similarly, we observed an increased production of IL-1 (α and β), IL-12, and IL-18 by macrophages obtained from E2-treated mice compared with those from castrated mice (B. Calippe et al., unpublished data).
According to current knowledge, the pro-inflammatory effect of E2 cannot account for its preventive effect of fatty streak accumulation (Binder et al. 2002; Hansson et al. 2002; Libby 2002; Tedgui and Mallat 2001). In contrast, it could contribute to the destabilization of atheromatous plaques, and thus represent a good candidate to explain the increase in cardiovascular events during the year following the onset of HRT (Hulley et al. 1998; Rossouw et al. 2002). Nonetheless, the non-natural progestin (medroxyprogesterone acetate) used in these clinical trials could indeed have undesirable, deleterious effects (Anderson et al. 2004).
5 Estrogen Receptor α Mediates Most of the Vascular Effects of E2
Endothelium is involved in the regulation of coagulation, leukocyte adhesion in inflammation, transvascular flux of cells, vascular smooth muscle growth, etc. and also is a major target for E2. Endothelial messengers, such as nitric oxide (NO) (Mendelsohn 2000 and references therein) and prostacyclin are increased by E2. Indeed, E2 can increase NO bioactivity acutely by stimulating endothelial NO synthase activity (Mendelsohn 2000) and chronically by decreasing the breakdown of NO, as a consequence of a decreased production of reactive oxygen species (Arnal et al. 1996; Wagner et al. 2001). Although the atheroprotective effect of E2 appears independent of NO production (Elhage et al. 1997b), induction of COX-2 and prostacyclin production was recently proposed to play an important role in the prevention of fatty streak at the level of the thoracoabdominal aorta (Egan et al. 2004).
E2 effects can be mediated by estrogen receptor alpha (ERα) and beta (ERβ), two members of the nuclear receptor superfamily that are encoded by two distinct genes (Couse and Korach 1999). A collaborative effort with the Krust and Chambon group led to the clearcut demonstration of a prominent role of ERα in vascular physiology in vivo. Full length ERα (66 kDa) is composed of six domains (named from A to F) and two independent transactivation functions AF1 and AF2 (Tora et al. 1989). The ERα and ERβ genes were previously disrupted by targeted mutagenesis (Couse and Korach 1999). Mice targeted for ERα through the insertion of the Neo gene in exon 1 (therefore named ER-αNeoKO) was first generated by Korach's group in 1993 (Lubahn et al. 1993). These mice were subsequently shown to present a leakage due to a non-natural alternative splicing of the ERα mRNA, resulting in the expression of a truncated 55-kD isoform (Couse et al. 1995; Kos et al. 2002; Pendaries et al. 2002). Such an ERα isoform, lacking most of the A/B domain and therefore the AF-1 transactivating function, was sufficient to mediate the E2 effect on the endothelial NO production (Pendaries et al. 2002) and postinjury medial hyperplasia (Iafrati et al. 1997; Pare et al. 2002).
In contrast, the generation and studies of mice that fully and unambiguously lack ERα (Dupont et al. 2000) showed that ERα is necessary in the response of E2 on NO production (Pendaries et al. 2002). Erβ-deficient mice had, however, a normal NO production (Darblade et al. 2002). Altogether, these data allow us to conclude that an ERα lacking the A/B domain (and therefore AF-1) is sufficient to mediate some of the vascular effects of estrogen. Interestingly, an ERα 46-kD isoform, lacking the N-terminal portion (domains A/B), can be physiologically expressed through an alternative splicing (Flouriot et al. 2000) in the uterus (Faye et al. 1986; Pendaries et al. 2002), in cultured endothelial cells (Li et al. 2003) and in macrophages (H. Laurell, unpublished data).
However, the prevention of fatty streaks appears to require the full length ERα (66 kD) (Hodgin et al. 2001). Indeed, in contrast to the E2 protection elicited in apoE-KO mice, E2 treatment of ovariectomized ERα-Neo/apoE double KO female mice caused a nonsignificant (p = 0.12) reduction in lesion size and no reduction in total plasma cholesterol (Hodgin et al. 2001). However, it should be mentioned that E2 treatment significantly reduced the complexity of plaques in ERα-Neo/apoE double KO female mice, although not to the same degree as in apoE-KO female mice. Although this could have been due to the existence of ERα-dependent atheroprotective effects of E2, the expression of the truncated 55kD ER-α isoform could also have been responsible for this E2 effect.
6 Effect on Artery Healing: Reendothelialization Mechanisms
As mentioned in the introduction, the loss of the integrity of the endothelial monolayer is another important aspect at early steps of atherosclerosis, but also after the destruction provoked by endoluminal angioplasty (often followed by stent implantation) (Bennett and O'Sullivan 2001). In this context, the acceleration of vascular healing, where reendothelialization plays a key role, is considered a major protective event against short- as well as long-term complications of endovascular therapy.
Endovascular de-endothelialization in mice is a complex and delicate manipulation as a consequence of the very small size of the carotid artery. Carmeliet et al. (1997) proposed destroying the endothelium using a perivascular electric injury approach, and an adaptation of this model was proposed by our group (Brouchet et al. 2001). Endovascular and electric perivascular injury are identical in their efficiency in destroying the endothelium, which will be temporarily replaced by a monolayer of platelets, but they differ in at least two major points. Firstly, whereas endovascular injury preserves most of the medial smooth muscle cells as well as adventitia cells (Lindner et al. 1993), electric injury destroys the cells of the three layers of the injured area, and in particular the smooth muscle cells, which do not recolonize the media even after several weeks. Secondly, immunoinflammatory cells (mainly macrophages) make up the main cell population in the perivascular electric injury model that may interact with regenerating endothelium. The accelerative effect of E2 on reendothelialization is mediated by ERα (Brouchet et al. 2001) and endothelial NO synthase appears absolutely required for this effect (Iwakura et al. 2003). We have recently explored the role of two molecules that also appear to be clearly involved in this process: fibroblast growth factor 2 (FGF2) and osteopontin (OPN).
FGF2 is one of the first growth factors to be characterized and remains one of the most potent. The expression of the five FGF2 isoforms of 18, 22, 22.5, 24, and 34 kDa in humans and the three isoforms of 18, 21, and 22 kDa in mice is particularly original, since they are synthesized not through alternative splicing of mRNA, but through an alternative use of translation initiation codons from a single mRNA (Prats et al. 1989). These isoforms differ only in their NH2 extremities, which confer a nuclear localization to the high molecular weight (HMW) CUG-initiated forms, whose function is for the most part unknown. In contrast, the low-molecular-weight (LMW) AUG-initiated FGF2 (18 kDa) is predominantly cytoplasmic and excreted, and known to activate the FGF receptors (FGFR), leading to stimulation of proliferation and migration. We recently reported that E2 stimulated migration in endothelial cells from Fgf2 +/+ , but not from Fgf2 –/– mice (deficient both in FGF2lmw and in FGF2hmw) (Garmy-Susini et al. 2004). More recently, we confirmed that E2 increased both the velocity of reendothelialization and the number of circulating EPCs (as previously described in Strehlow et al. 2003) in ovariectomized Fgf2 +/+ mice. However, both these effects of E2 were abolished in Fgf2 –/– mice. We then investigated the role of medullary FGF2 in these processes. In chimeric (i.e., bone-marrow [BM] transplanted) (Fgf2 –/– BM ⇒ Fgf2 +/+ ) mice, both effects of E2 on reendothelialization and on circulating EPC levels were abolished, whereas both were preserved in chimeric (Fgf2 +/+ BM ⇒ Fgf2 –/– ) mice, demonstrating that medullary, and not extramedullary, FGF2 is required for both effects of E2 (Fontaine et al. 2006). Similarly, ERα +/+ or ERα –/– BM graft experiments revealed that the effect of E2 on reendothelialization relies on medullary, but not on extramedullary ERα, emphasizing for the first time, to the best of our knowledge, a prominent role played by bone marrow in the E2 effect on reendothelialization.
OPN is an RGD-containing extracellular matrix phosphoprotein involved in cell adhesion and migration via a number of receptors, including several integrins and CD44 (Chaulet et al. 2001). OPN –/–mice are resistant to ovariectomy-induced bone resorption (Yoshitake et al. 1999). OPN is detected in vascular SMCs and macrophages of atherosclerotic plaques (Giachelli et al. 1993) and its expression is upregulated in neointimal hyperplasia and in regenerating endothelium (Liaw et al. 1995). However, the cellular source and target of OPN in accelerating endothelial regeneration remains to be demonstrated. In collaboration with Gadeau et al., we recently found that the effect de E2 on reendothelialization was abolished in OPN –/– mice in the two models of carotid injury (unpublished data).
7 Pathophysiological and Therapeutical Implications
In conclusion, E2 exerts an atheroprotective effect in all experimental models and most likely in women before menopause. Although serum cholesterol decreases, this influence on lipid metabolism is negligible. Similarly, although E2 induces an increase in endothelial NO production and/or bioavailability, this effect does not account for its protection of the constitution of fatty streak. The precise mechanisms of the atheroprotective effect of E2 at the level of the endothelium remains to be characterized. At the same time, E2 also induces an immunoinflammatory response toward a Th1 profile with increasing interferon γ production. This proinflammatory effect could have been prominent in advanced atheromatous plaques in postmenopausal women, favoring destabilization of the most unstable plaques. At present, this is the most likely explanation accounting for the increase in the frequency of cardiovascular events in postmenopausal women during the 1st year of HRT, as observed in the HERS and WHI studies. It should be noted that the women enrolled in these studies had been postmenopausal for several years (on average more than 10 years after the onset of menopause).
Although ERs are classically defined as ligand-activated transcription factors, it has become clear that extragenomic membrane short-term responses play an important role in cultured endothelial cells (Mendelsohn 2000), but also in osteoblasts (such as the activation of PI3kinases-AKT pathways as well as MAP kinase pathways) (Kousteni et al. 2001). An important challenge for the next years will be to describe the respective roles of these membrane effects and the classic effects.
These new acquisitions are a basis for new pharmacological developments that can prevent harmful effects and preserve the beneficial effects. The effects of selective estrogen receptor modulators (SERMs) on the different constituents in the atheroma plaque formation must now be analyzed on the basis of their specific regulation of the ERα but also of the ERβ. Various classes of estrogens and SERMs have been described according their molecular actions through ERα (Jordan 2001a,b; Katzenellenbogen and Katzenellenbogen 2002).
Due to the complexity of the mechanisms of action of estrogens and SERMs, their effect on each type of cell and tissue cannot be predicted from their structure. Indeed, only integrated models can screen the present and future SERMs in terms of the beneficial and deleterious effects. Theoretically, it is conceivable to design a SERM (or a combination of molecules) that retains most (if not all) of the desired effects of E2 (on the central nervous system to prevent hot flushes, on bone, endothelium, etc.), but which is devoid of the undesirable effects of E2 (mainly breast cancer and thromboembolism). Finally, though not within the scope of this review, it should be mentioned that the interaction with progestins is of major importance and should also be studied in parallel investigations.
Acknowledgements
We are grateful to Prof. F. Bayard for his input to our team's work over many years. We are grateful to P. Chambon, A. Krust, K. Korach, J.C. Guery, A.P. Gadeau, C. Filipe, A. Billon, and B. Calippe for helpful discussions over many years. We thank M.J. Fouque and A. Schambourg for their skillful technical assistance. The work described herein was supported in part by Université Paul Sabatier Toulouse III, INSERM, the European Vascular Genomics Network No. 503254, the Fondation de France, the Fondation de l'Avenir, and the Conseil Régional Midi-Pyrénées in France.
References
Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, Bonds D, Brunner R, Brzyski R, Caan B et al (2004) Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women's Health Initiative randomized controlled trial. JAMA 291:1701–1712PubMedCrossRef
Arnal JF, Clamens S, Pechet C, Nègre-Salvayre A, Allera C, Girolami JP, Salvayre R, Bayard F (1996) 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 USA 93:4108–4113PubMedCrossRef
Arnal JF, Gourdy P, Elhage R, Garmy-Susini B, Delmas E, Brouchet L, Castano C, Barreira Y, Couloumiers JC, Prats H et al (2004) Estrogens and atherosclerosis. Eur J Endocrinol 150:113–117PubMedCrossRef
Bennett MR, O'Sullivan M (2001) Mechanisms of angioplasty and stent restenosis: implications for design of rational therapy. Pharmacol Ther 91:149–166PubMedCrossRef
Binder CJ, Chang MK, Shaw PX, Miller YI, Hartvigsen K, Dewan A, Witztum JL (2002) Innate and acquired immunity in atherogenesis. Nat Med 8:1218–1226PubMedCrossRef
Bourassa PA, Milos PM, Gaynor BJ, Breslow JL, Aiello RJ (1996) Estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice. Proc Natl Acad Sci USA 93:10022–10027PubMedCrossRef
Brouchet L, Krust A, Dupont S, Chambon P, Bayard F, Arnal JF (2001) Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta. Circulation 103:423–428PubMedCrossRef
Carmeliet P, Moos L, Stassen J, De Mol M, Bouché A, van den Oord J, Kocks M, Collen D (1997) Vascular wound healing and neointima formation induced by perivascular electric injury in mice. Am J Pathol 150:761–776PubMed
Chaulet H, Desgranges C, Renault MA, Dupuch F, Ezan G, Peiretti F, Loirand G, Pacaud P, Gadeau AP (2001) Extracellular nucleotides induce arterial smooth muscle cell migration via osteopontin. Circ Res 89:772–778PubMedCrossRef
Clarkson TB, Appt SE (2005) Controversies about HRT: lessons from monkey models. Maturitas 51:64–74PubMedCrossRef
Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB, Smithies O, Korach KS (1995) Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol 9:1441–1454PubMedCrossRef
Couse JF, Korach KS (1999) Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 20:358–417PubMedCrossRef
Dansky H, Charlton S, McGee Harper M, Smith J (1997) T and B lymphocytes play a minor role in atherosclerotic plaque formation in the apolipoprotein E-deficient mouse. Proc Natl Acad Sci USA 94:4642–4646PubMedCrossRef
Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF (2002) Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor. Circ Res 90:413–419PubMedCrossRef
Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M (2000) Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes. Development 127:4277–4291PubMed
Egan KM, Lawson JA, Fries S, Koller B, Rader DJ, Smyth EM, Fitzgerald GA (2004) COX-2-derived prostacyclin confers atheroprotection on female mice. Science 306:1954–1957PubMedCrossRef
Elhage R, Arnal JF, Pierragi M-T, Duverger N, Fiévet C, Faye JC, Bayard F (1997a) Estradiol-17b prevents fatty streak formation in apolipoprotein E-deficient mice. Arterioscl Thromb Vasc Biol 17:2679–2684PubMedCrossRef
Elhage R, Bayard F, Richard V, Holvoet P, Duverger N, Fievet C, Arnal JF (1997b) Prevention of fatty streak formation of 17beta-estradiol is not mediated by the production of nitric oxide in apolipoprotein E-deficient mice. Circulation 96:3048–3052PubMedCrossRef
Elhage R, Clamens S, Reardon-Alulis C, Getz GS, Fievet C, Maret A, Arnal JF, Bayard F (2000) Loss of atheroprotective effect of estradiol in immunodeficient mice. Endocrinology 141:462–465PubMedCrossRef
Elhage R, Gourdy P, Brouchet L, Jawien J, Fouque MJ, Fievet C, Huc X, Barreira Y, Couloumiers JC, Arnal JF, Bayard F (2004) Deleting TCR alpha beta+ or CD4+ T lymphocytes leads to opposite effects on site-specific atherosclerosis in female apolipoprotein E-deficient mice. Am J Pathol 165:2013–2018PubMedCrossRef
Elhage R, Gourdy P, Huc X, Brouchet L, Castano C, Barreira Y, Couloumiers JC, Fievet C, Hansson G, Arnal J-F et al (2005) The atheroprotective effect of 17-estradiol depends on complex interactions in adaptive immunity. Am J Pathol 167:267–274PubMedCrossRef
Faye JC, Fargin A, Bayard F (1986) Dissimilarities between the uterine estrogen receptor in cytosol of castrated and estradiol-treated rats. Endocrinology 118:2276–2283PubMedCrossRef
Flouriot G, Brand H, Denger S, Metivier R, Kos M, Reid G, Sonntag-Buck V, Gannon F (2000) Identification of a new isoform of the human estrogen receptor-alpha (hER-alpha) that is encoded by distinct transcripts and that is able to repress hER-alpha activation function 1. EMBO J 19:4688–4700PubMedCrossRef
Fontaine V, Filipe C, Werner N, Gourdy P, Billon A, Garmy-Susini B, Bouchet L, Bayard F, Prats H, Doetschman T, Nickenig G, Arnal JF (2006) Essential role of bone marrow fibroblast growth factor-2 in the effect of estradiol on reendothelialization and endothelial progenitor cell mobilization. Am J Pathol 169:1855–1862PubMedCrossRef
Garmy-Susini B, Delmas E, Gourdy P, Zhou M, Bossard C, Bugler B, Bayard F, Krust A, Prats AC, Doetschman T et al (2004) Role of fibroblast growth factor-2 isoforms in the effect of estradiol on endothelial cell migration and proliferation. Circ Res 94:1301–1309PubMedCrossRef
Ghisletti S, Meda C, Maggi A, Vegeto E (2005) 17{beta}-Estradiol inhibits inflammatory gene expression by controlling NF-{kappa}B intracellular localization. Mol Cell Biol 25:2957–2968PubMedCrossRef
Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM (1993) Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest 92:1686–1696PubMedCrossRef
Gimbrone MA Jr (1999) Vascular endothelium, hemodynamic forces, and atherogenesis. Am J Pathol 155:1–5PubMedCrossRef
Gourdy P, Araujo LM, Zhu R, Garmy-Susini B, Diem S, Laurell H, Leite-de-Moraes M, Dy M, Arnal JF, Bayard F, Herbelin A (2005) Relevance of sexual dimorphism to regulatory T cells: estradiol promotes IFN-{gamma} production by invariant natural killer T cells. Blood 105:2415–2420PubMedCrossRef
Haarbo J, Leth-Espensen P, Stender S, Christiansen C (1991) Estrogen monotherapy and combined estrogen-progestogen replacement therapy attenuate aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits. J Clin Invest 87:1274–1279PubMedCrossRef
Hansson GK, Libby P, Schonbeck U, Yan ZQ (2002) Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res 91:281–291PubMedCrossRef
Hodgin J, Maeda N (2002) Estrogen and mouse models of atherosclerosis. Endocrinology 143:4495–4501PubMedCrossRef
Hodgin JB, Krege JH, Reddick RL, Korach KS, Smithies O, Maeda N (2001) Estrogen receptor alpha is a major mediator of 17beta-estradiol's atheroprotective effects on lesion size in Apoe–/– mice. J Clin Invest 107:333–340PubMedCrossRef
Holm P, Andersen HL, Andersen MR, Erhardtsen E, Stender S (1999) The direct antiatherogenic effect of estrogen is present, absent, or reversed, depending on the state of the arterial endothelium. A time course study in cholesterol-clamped rabbits. Circulation 100:1727–1733PubMedCrossRef
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 280:605–613PubMedCrossRef
Iafrati MD, Karas RH, Aronovitz M, Kim S, Sullivan TR Jr, Lubahn DB, O'Donnell TF Jr, Korach KS, Mendelsohn ME (1997) Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice. Nat Med 3:545–548PubMedCrossRef
Iwakura A, Luedemann C, Shastry S, Hanley A, Kearney M, Aikawa R, Isner JM, Asahara T, Losordo DW (2003) Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury. Circulation 108:3115–3121PubMedCrossRef
Jordan VC (2001a) The past, present, and future of selective estrogen receptor modulation. Ann NY Acad Sci 949:72–79PubMedCrossRef
Jordan VC (2001b) Selective estrogen receptor modulation: a personal perspective. Cancer Res 61:5683–5687PubMed
Katzenellenbogen BS, Katzenellenbogen JA (2002) Biomedicine. Defining the “S” in SERMs. Science 295:2380–2381PubMedCrossRef
Kos M, Denger S, Reid G, Korach KS, Gannon F (2002) Down but not out? A novel protein isoform of the estrogen receptor alpha is expressed in the estrogen receptor alpha knockout mouse. J Mol Endocrinol 29:281–286PubMedCrossRef
Kousteni S, Bellido T, Plotkin LI, O'Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS et al (2001) Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104:719–730PubMed
Li L, Haynes MP, Bender JR (2003) Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. Proc Natl Acad Sci USA 100:4807–4812PubMedCrossRef
Liaw L, Lindner V, Schwartz SM, Chambers AF, Giachelli CM (1995) Osteopontin and beta 3 integrin are coordinately expressed in regenerating endothelium in vivo and stimulate Arg-Gly-Asp-dependent endothelial migration in vitro. Circ Res 77:665–672PubMedCrossRef
Libby P (2002) Inflammation in atherosclerosis. Nature 420:868–874PubMedCrossRef
Lindner V, Fingerle J, Reidy MA (1993) Mouse model of arterial injury. Circ Res 73:792–796PubMedCrossRef
Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, 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 USA 90:11162–11166PubMedCrossRef
Maret A, Coudert JD, Garidou L, Foucras G, Gourdy P, Krust A, Dupont S, Chambon P, Druet P, Bayard F, Guery JC (2003) Estradiol enhances primary antigen-specific CD4 T cell responses and Th1 development in vivo. Essential role of estrogen receptor alpha expression in hematopoietic cells. Eur J Immunol 33:512–521PubMedCrossRef
Massberg S, Brand K, Gruner S, Page S, Muller E, Muller I, Bergmeier W, Richter T, Lorenz M, Konrad I et al (2002) A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med 196:887–896PubMedCrossRef
Mendelsohn ME (2000) Nongenomic ER-mediated activation of endothelial nitric oxide synthase: how does it work? What does it mean? Circ Res 87:956–960PubMedCrossRef
Pare G, Krust A, Karas RH, Dupont S, Aronovitz M, Chambon P, Mendelsohn ME (2002) Estrogen receptor-alpha mediates the protective effects of estrogen against vascular injury. Circ Res 90:1087–1092PubMedCrossRef
Pendaries C, Darblade B, Rochaix P, Krust A, Chambon P, Korach KS, Bayard F, Arnal JF (2002) The AF-1 activation-function of ERalpha may be dispensable to mediate the effect of estradiol on endothelial NO production in mice. Proc Natl Acad Sci USA 99:2205–2210PubMedCrossRef
Prats H, Kaghad M, Prats AC, Klagsbrun M, Lelias JM, Liauzun P, Chalon P, Tauber JP, Amalric F, Smith JA, Caput D (1989) High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons. Proc Natl Acad Sci USA 86:1836–1840PubMedCrossRef
Ross R (1999) Atherosclerosis-an inflammatory disease. N Engl J Med 340:115–125PubMedCrossRef
Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC et al (2002) Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomized controlled trial. JAMA 288:321–333PubMedCrossRef
Shyy JY, Chien S (2002) Role of integrins in endothelial mechanosensing of shear stress. Circ Res 91:769–775PubMedCrossRef
Smith JD, Trogan E, Ginsberg M, Grigaux C, Tian J, Miyata M (1995) Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E. Proc Natl Acad Sci USA 92:8264–8268PubMedCrossRef
Steinberg D (2002) Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime. Nat Med 8:1211–1217PubMedCrossRef
Strehlow K, Werner N, Berweiler J, Link A, Dirnagl U, Priller J, Laufs K, Ghaeni L, Milosevic M, Bohm M, Nickenig G (2003) Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation. Circulation 107:3059–3065PubMedCrossRef
Tedgui A, Mallat Z (2001) Anti-inflammatory mechanisms in the vascular wall. Circ Res 88:877–887PubMedCrossRef
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E, Chambon P (1989) The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59:477–487PubMedCrossRef
Wagner AH, Schroeter MR, Hecker M (2001) 17beta-Estradiol inhibition of NADPH oxidase expression in human endothelial cells. FASEB J 15:2121–2130PubMedCrossRef
Waters DD, Gordon D, Rossouw JE et al (2004) Women's Ischemic Syndrome Evaluation: current status and future research directions: report of the National Heart, Lung and Blood Institute workshop: October 2–4, 2002: Section 4: lessons from hormone replacement trials. Circulation 17 109(6):e53–e55CrossRef
Yoshitake H, Rittling SR, Denhardt DT, Noda M (1999) Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption. Proc Natl Acad Sci USA 96:8156–8160PubMedCrossRef
Zhang SH, Reddick RL, Piedrahita JA, Maeda N (1992) Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 258:468–471PubMedCrossRef