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【摘要】
Objective— Whereas estradiol prevents fatty streak deposit in immunocompetent apoE –/– or LDLr –/– mice, it is totally ineffective in immunodeficient mice, underlining the key role of immunoinflammation in this effect. In the present work, the role of several major pro- and antiinflammatory cytokines involved in the atheromatous process was evaluated in the effect of estradiol on fatty streak constitution.
Methods and Results— The preventive effect of estradiol was fully maintained in LDLr –/– mice grafted with bone marrow from either IFN- or interleukin (IL)-12–deficient mice, showing that this beneficial effect was not mediated through a specific decrease in the production of these 2 proinflammatory cytokines. Furthermore, IL-10 –/– apoE –/– mice remained protected by estradiol, excluding a significant contribution of this antiinflammatory cytokine. In contrast, the protective effect of estradiol was (1) associated with enhanced aortic expression of TGF-β1 in apoE –/– mice during early steps of atherogenesis; (2) abolished and even reversed in apoE –/– mice administered with a neutralizing anti–TGF-β antibody; (3) abolished in LDLr –/– mice grafted with bone marrow from Smad3-deficient mice.
Conclusions— The status of the TGF-β pathway crucially determines the antiatherogenic effect of estradiol in hypercholesterolemic mice, whereas neither IFN-, IL-12, nor IL-10 are specifically involved in this protection.
The role of several cytokines involved in the atheromatous process was evaluated in the protective effect of estradiol on fatty streak constitution. We demonstrate that the integrity of the TGF-beta pathway is absolutely required for this beneficial property of estradiol, whereas neither IFN-gamma, IL-12 or, IL-10 deficiency altered this effect.
【关键词】 atherosclerosis inflammation cytokine TGFβ estrogen
Introduction
Determining the mechanisms of the vascular effects of estrogens is now identified as a research priority, because epidemiological surveys, experimental observations, and clinical intervention trials have led to conflicting results. 1 Indeed, whereas epidemiological studies suggested that both endogenous and exogenous estrogens protect women against cardiovascular diseases, 2 2 recent controlled prospective and randomized studies did not demonstrate a beneficial effect of hormone replacement therapy (HRT) neither in secondary 3 nor in primary prevention. 4 Although evaluating late steps of atherothrombosis, these clinical trials strikingly contrast with the strong preventive effect of estrogens in all animal models of atherosclerosis, from mice to monkey. 5,6
During the last decade, these hypercholesterolemic mouse models allowed to establish the major contribution of the immune system in the development of early lesions of atherosclerosis, and to highlight the influence of several cytokines in this process. 7 Indeed, proinflammatory cytokines, namely IL-12, IL-18, and IFN-, have been demonstrated to promote fatty streak constitution 7 and probably plaque instability, 8 whereas the prototypic antiinflammatory cytokines TGF-β and IL-10 appear clearly protective. 7,9 TGF-β activity was shown to prevent vascular inflammation and to promote plaque stabilization in mice models of atherosclerosis, including apolipoprotein E–deficient (apoE –/– ) and low-density lipoprotein receptor–deficient (LDLr –/– ) mice. 10–14
We previously reported that, whereas estradiol-17β (E2) strongly prevents fatty streak deposit in immunocompetent hypercholesterolemic mice, it is totally ineffective in both apoE –/– and LDLr –/– mice also deficient in recombination activating gene 2 (RAG-2), demonstrating that lymphocytes are absolutely required for the protective effect of the hormone. 15,16 As lymphocytes are both source and target of cytokines, we hypothesized that the protective effect of E2 could be mediated through a favorable effect on the pro- versus antiinflammatory cytokine balance, because estrogens are known to alter the secretion of several pro- and antiinflammatory cytokines by immune cells in various pathophysiological models. 17–19
In the present work, we separately evaluated the role of major pro- and antiinflammatory cytokines involved in the atheromatous process in the effect of E2 on fatty streak constitution, with an approach consisting in suppressing their expression or activity in hypercholesterolemic mouse models. Whereas neither IFN-, IL-12, nor IL-10 deficiency altered the protective effect of E2, we demonstrate that the integrity of the TGF-β pathway appears to be crucial for this beneficial effect of estrogens.
Materials and Methods
Mice and Experimental Procedures
Female apoE –/– mice, 20 as well as apoE and IL-10 double deficient mice (apoE –/– IL-10 –/–, provided by G.K. Hansson, Karolinska Intitute, Stockholm, Sweden), 21 were ovariectomized at 4 weeks of age, then administered with either 60-day time-release placebo or 0.1 mg estradiol-17β pellets (Innovative Research of America), 20 and maintained on a chow diet. In some experiments, apoE –/– mice were injected intraperitoneally with either an anti–TGF-β1/β2/β3 monoclonal antibody (clone 2G7, provided by D. Fradelizi, INSERM U477, Paris, France) or an isotype matched (IgG2b) control mAb, devoid of neutralizing activity, once a week for 11 weeks, as previously reported. 11
Six-week-old ovariectomized LDLr –/– female mice were lethally irradiated, then were intravenously reconstituted with bone marrow cells from either wild-type C57Bl/6J, IFN- –deficient (IFN- –/– ), or IL-12p40–deficient (IL-12 –/– ) mice, all purchased from Charles River (L?Arbresle, France), or from Smad3-deficient mice (Smad3 –/–, provided by C. Deng, National Institute of Health, Bethesda, Md). 22 Four weeks later, transplanted mice received placebo or E2 pellets and were switched to a high fat atherogenic diet containing 1.25% cholesterol (TD96335, Harlan Teklad). 23
All mice were euthanized after a 12-week period of E2 or placebo treatment, and total and HDL plasma cholesterol concentrations were determined. In some experiments, spleen cells were isolated and stimulated with coated anti-CD3 mAb (1 µg/mL, BD Pharmingen) for 48 hours. Then, IFN-, IL-10, and IL-4 concentrations were determined in supernatants by ELISA as previously described. 24 (For detailed materials and methods, please see supplemental materials available online at http://atvb.ahajournals.org).
Analyses of Fatty Streak Lesion and TGF-β1 Expression
Fatty streak lesion size was estimated at the aortic sinus, as previously described. 20 Collagen content was assessed by Sirius red staining. For immunohistochemical staining, a goat polyclonal anti-CD3 and a rabbit polyclonal anti-TGF-β1/2 (Santa Cruz Biotechnology) were used. TGF-β1mRNA expression was determined by RT-PCR in the aortic arch and the descending aorta from ovariectomized apoE –/– female mice administered with either placebo or E2 pellets for 5 weeks (For detailed materials and methods, please see supplemental materials).
Statistical Analyses
Results were expressed as means±SEM. A 2-way ANOVA was performed to test for the respective effects of E2 treatment, cytokine deficiency, or anti–TGF-β mAb treatment on various parameters. When allowed, paired comparisons were performed with the Fisher PLSD procedure. P <0.05 was considered as statistically significant.
Results
IFN- or IL-12 Deficiency Does Not Alter the Effect of E2 on Fatty Streak Constitution in LDLr –/– Mice
To determine whether the preventive effect of E2 on fatty streak constitution could be mediated by a specific modulation of IFN- or IL-12 production, LDLr –/– female mice received a lethal whole body irradiation and were subsequently reconstituted with bone marrow (BM) from either IFN- –/–, IL-12 –/– or wild-type female mice. In mice grafted with IFN- –/– or IL-12 –/– bone marrow cells, respectively, the abolition of IFN- production by anti-CD3–activated splenocytes and the abolition of IL-12 synthesis by lipopolysaccharide (LPS)-activated peritoneal macrophages were systematically controlled (data not shown). In both cases, cytokine deficiency did not affect total and HDL plasma cholesterol (supplemental Tables I and II). At the level of the aortic sinus, fatty streak lesion area was significantly reduced in placebo-treated IFN- –deficient mice ( Figure 1 A), whereas no significant change ( P =0.18) was observed in IL-12–deficient mice ( Figure 1 B).
Figure 1. Influence of IFN-, IL-12, or IL-10 deficiency on the effect of estradiol on fatty streak constitution in hypercholesterolemic mice. Six-week-old ovariectomized LDLr –/– female mice were lethally irradiated and reconstituted with bone marrow cells from wild-type, IFN- –/– (A), or IL-12 –/– (B) mice, then were administered with placebo or estradiol (E2) and switched to atherogenic diet from the age of 10 to 22 weeks. Six-week-old ovariectomized apoE –/– IL-10 –/– female mice were maintained on a chow diet and treated with placebo or E2 for 12 weeks (C). Mean lesion sizes at the aortic sinus are shown (at least 7 mice per group). Results are expressed as means±SEM. Fisher PLSD test: * P <0.05, *** P <0.001.
E2 treatment significantly decreased body weight and tended to decrease total plasma cholesterol in E2-treated mice irrespective of IFN- or IL-12 deficiency (supplemental Tables I and II), whereas no change in HDL-cholesterol was observed. Finally, E2 treatment decreased fatty streak development in IFN- –/– (BM)/LDL-r –/– mice (-60.6%, P <0.001, Figure 1 A) and in IL-12 –/– (BM)/LDL-r –/– mice (-53.9%, P <0.01, Figure 1 B) as it did in WT(BM)/LDL-r –/– mice, without any interaction between E2 and the respective cytokine deficiency (Two-factor ANOVA). These data clearly indicate that the atheroprotective effect of E2 is independent of the proinflammatory cytokines IFN- and IL-12.
Effect of E2 Treatment on Fatty Streak Development in ApoE –/– /IL-10 –/– Mice
To test the role of the antiinflammatory and antiatherogenic cytokine IL-10 in the preventive effect of E2, apoE –/– /IL-10 –/– female mice were treated with placebo or E2 for 12 weeks. Body weight and total plasma cholesterol were significantly reduced by IL-10 deficiency, as well as by E2 treatment, without interaction between the 2 factors. HDL cholesterol also tended to be decreased, although not significantly, by both IL-10 deficiency and E2 (supplemental Table II I). As previously reported, 21 IL-10 deficiency resulted in a significant increase of atheromatous lesion size at the aortic sinus. However, E2 prevented fatty streak development despite IL-10 deficiency (–43%, P <0.01, Figure 1 C), ruling out a significant participation of this antiinflammatory cytokine in E2 atheroprotective effect.
Table 3. Effect of E2 Treatment in Irradiated LDL-r –/– Female Mice Reconstituted With Bone Marrow From Either Wild-Type (WT) or Smad3 –/– Mice
Effect of TGF-β Activity Neutralization on Fatty Streak Constitution in Placebo- or E2-Treated ApoE –/– Female Mice
To explore the contribution of TGF-β to the protective effect of estrogens, placebo or E2 were administered to ovariectomized apoE –/– female mice simultaneously treated with an anti–TGF-β mAb, according to the procedure reported to neutralize both systemic and local TGF-β activity. 11 Anti–TGF-β mAb treatment did not affect body weight and lipid parameters ( Table 1 ). However, body weight was reduced by E2 in apoE –/– mice treated or not with the anti–TGF-β mAb and, plasma total and HDL cholesterol tended to decrease under E2 treatment in both groups, although not significantly ( Table 1 ). Compared with control mice, administration of either anti–TGF-β mAb or E2 alone resulted in a strong reduction of fatty streak area ( Figure 2 A). In contrast, the combination of E2 and anti–TGF-β mAb treatment was no longer protective, inducing a significant increase in lesion size compared with mice given anti–TGF-β mAb alone (+68%, P <0.05) or E2 alone (+71%, P <0.05) ( Figure 2 A). Indeed, the 2-factor ANOVA showed a strong interaction ( P <0.0001) between E2 and anti–TGF-β mAb ( Table 1 ).
Table 1. Effect of E2 Treatment and Anti–TGF-β mAb Administration in ApoE –/– Female Mice
Figure 2. Effect of estradiol on atherosclerotic lesion size and composition in 18-week-old ovariectomized apoE –/– female mice treated with either anti–TGF-β mAb or control mAb for 12 weeks. A, Oil-red-O staining (original magnification x 40) and quantification of total lesion surface at the aortic sinus. B, Sirius-red staining ( x 125) and quantification of lesion collagen content (sirius-red–stained surface/total lesion surface). C, CD3 staining ( x 200) and quantitative analysis of CD3-positive cells (T lymphocytes, number of positive cells per mm 2 lesion area). Representative photomicrographs from at least 9 mice per group were analyzed. Results are expressed as means±SEM. Fisher?s PLSD test: * P <0.05, ** P <0.01, *** P <0.001.
Influence of TGF-β Activity Neutralization on Lesion Composition and Cytokine Production
As previously reported, neutralizing TGF-β activity influenced lesion composition, especially the balance between collagen and inflammatory cell content. Indeed, anti–TGF-β mAb treatment induced a 2-fold decrease in collagen content and a 2.3-fold increase in lymphocyte infiltration in placebo-treated mice. A significant increase in T lymphocyte density was also observed in the adventitia and adjacent myocardial area (data not shown). A similar effect was observed in E2-treated mice ( Figure 2B and 2 C).
To further assess the inflammatory status of mice from the 4 experimental groups, we analyzed the cytokine production profile by anti–CD3-activated splenocytes. As shown in Table 2, no change was observed in IL-10 production, and IL-4 concentrations were not detectable. In contrast, anti–TGF-β mAb or E2 treatment alone increased IFN- production by splenic T lymphocytes. Furthermore, their combination appeared additive, without interaction between the 2 factors, leading to a further increase in the production of this proinflammatory cytokine.
Table 2. Effect of E2 Treatment and Anti–TGF-β mAb Administration on Cytokine Production by Anti–CD3–Activated Spleen Cells From ApoE –/– Female Mice
Disruption of TGF-β Signaling in Bone Marrow–Derived Cells Abolishes the Protective Effect of E2 on Fatty Streak
To determine whether TGF-β contributes to the atheroprotective effect of E2 through its regulating actions on immune cells, we then grafted irradiated LDLr –/– mice with bone marrow cells from Smad3 –/– or wild-type mice. Indeed, Smad3, one of the intracellular mediators that transduce signals from TGF-β and activin receptors, was previously shown to mediate the antiinflammatory effects of TGF-β1 on immune cells, especially T lymphocytes and monocytes/macrophages. 22,25,26 The deletion of Smad3 gene was systematically controlled by PCR in splenocytes and peritoneal macrophages from Smad3 –/– (BM)/LDL-r –/– mice (data not shown). The 2-factor ANOVA indicates an interaction between E2 and Smad3 genotype on lesion size (P<0.05; Table 3 ). Indeed, whereas the protective effect of E2 was observed as expected in WT(BM)/LDL-r –/– mice (–61.5% in lesion area), it was no longer significant in Smad3 –/– (BM)/LDL-r –/– mice (–16.9%). As observed above under TGF-β activity neutralization, lesion from Smad3 –/– (BM)/LDL-r –/– mice were characterized by an increase in T lymphocyte density and a decrease in collagen content (data not shown).
E2 Treatment Enhances TGF-β1 Expression in the Aorta From apoE –/– Mice
The demonstration that TGF-β activity strongly influences the vascular effect of estrogens led us to finally determine whether E2 could modulate the production of TGF-β in the vascular wall during the atheromatous process. As shown in Figure 3 A, TGF-β 1 mRNA expression was enhanced by a 6-week E2 treatment in both aortic arch (2-fold) and descending aorta (3-fold) from ovariectomized apoE –/– female mice, whereas TGF-β2, TGF-β3, and TGF-βR2 mRNA levels were not affected (data not shown). Furthermore, the expression of TGF-β1/2 was increased in fatty streak lesions from E2-treated ovariectomized apoE –/– mice when compared with lesions from placebo-treated mice, irrespective of fatty streak size ( Figure 3 B).
Figure 3. Estradiol enhances TGF-β1 expression in atherosclerotic lesions and aorta from ovariectomized apoE –/– female mice. A, Quantitative analysis of TGF-β1 mRNA expression in aortic arch and descending aorta from 12-week-old ovariectomized apoE –/– female mice treated with either placebo or E2 for 6 weeks. Data are means±SEM from 6 individual animals in each group and results from Fisher PLSD test are mentioned. B, Representative photomicrographs of TGF-β1/2 immunostaining at the aortic root from 18-week-old ovariectomized apoE –/– female mice treated with either placebo or E2 for 12 weeks (original magnification x 125).
Discussion
The abolition of the protective effect of E2 against fatty streak constitution in immunodeficient mice first underlined the crucial contribution of the immune system to the atheroprotective effect of estrogens. 15,16 According to the key role played by lymphocyte-derived cytokines in atherosclerosis, 7 we logically hypothesized that estrogens prevent atherosclerosis constitution by a direct influence on the intimal production of cytokines, more precisely by decreasing proinflammatory cytokines or increasing the vascular expression of antiinflammatory cytokines. To test this assumption, we decided to assess the functional role of several major cytokines known to influence atheroma development. 7 This experimental strategy allowed us to conclude that the integrity of the TGF-β pathway is absolutely required for the expression of the beneficial effect of E2 on the atheromatous process.
By contrast, our experiments tend to exclude a significant role of IL-10 in this preventive effect of E2, although the expression of this antiinflammatory cytokine is clearly atheroprotective 7 and was reported elsewhere to be influenced by estrogens. 17 Similarly, the atheroprotective effect of E2 in hypercholesterolemic mice does not appear to be mediated by a specific decrease in the expression of one of the proinflammatory cytokines IFN- or IL-12, although their clear-cut proatherogenic effects have been demonstrated. 7 Moreover, we previously showed that the protective effect of E2 was also fully maintained in apoE –/– /IL-6 –/– 27 and in apoE –/– /IL-18 –/– female mice (Elhage et al, unpublished data, 2002). As apoE deletion influences immune responses 28, we cannot exclude that IL-10, IL-6 or IL-18 play some role in the protective effect of E2 in LDLr –/– mice. It is of importance to emphasize that the crucial role of the TGF-β pathway in the protective effect of E2 was demonstrated here in the 2 models of atherosclerosis: apoE –/– mice using a neutralizing anti–TGF-β mAb and LDLr –/– mice using disruption of TGF-β signaling in hematopoietic cells.
Noteworthy, we found here that the administration of the anti–TGF-β mAb in placebo-treated ovariectomized apoE –/– mice led to a substantial decrease in lesion size. This observation differs from the original report by Mallat et al, despite the use of the same anti–TGF-β mAb, 11 but is in perfect agreement with the data reported by Lutgens et al using a recombinant soluble dominant negative form of the TGF-β receptor II. 12 Similarly, Smad3 deficiency in hematopoietic cells did not significantly influence fatty streak area in our graft experiments, contrasting with the strong acceleration of lesion constitution reported by Robertson et al in apoE –/– mice carrying a dominant negative TGF-β receptor 2 in T cells 14. More than the strategy of TGF-β activity inhibition used, the comparison between these series of experimental procedures suggests that the specific conditions of animal care, pathogen-free (in Lutgens? and the present studies) versus conventional (in Mallat?s and Robertson?s studies), could crucially determine the effect of TGF-β blockade on fatty streak deposit, as previously demonstrated for IL-10 deficiency. 29 Overall, our data totally agree with previous observations in mouse models when considering the increased inflammatory components and the decreased collagen content in atheromatous lesions, which reflects the imbalance between inflammation and fibrosis under TGF-β blockade.
The ability of estrogens to increase the expression of TGF-β isoforms has been previously demonstrated in extra-reproductive tissues, especially in the reproductive tract and in bone. 30–32 We report here, for the first time, that chronic E2 administration enhances the expression of TGF-β1 in the aortic wall during the early steps of atherosclerosis in hypercholesterolemic mice, especially in fatty streak lesions. Interestingly, tamoxifen, a selective estrogen receptor modulator, was previously reported to prevent fatty streak constitution in C57Bl/6 mice fed a high-fat diet, with a concomitant increase of both circulating and aortic concentrations of active and latent TGF-β. 33 Noteworthy, almost all the cell types involved in the atheromatous process, namely smooth muscle cells, macrophages, platelets, and lymphocytes including regulatory T cells can be source of TGF-β 7 and target for estrogens. 34 However, neither immunohistochemistry analysis nor mRNA quantification allowed us to determine the specific cell type(s) responsible for the E2-induced increase in TGF-β1 production within the atherosclerotic lesions. Interestingly, natural CD4 + CD25 + regulatory T cells were recently reported to prevent the constitution of fatty streak in apoE –/– mice through the modulation of CD4 + T lymphocyte activity by TGF-β. 35 Because estrogens were shown to expand the CD4 + CD25 + regulatory T cell compartment in mice, 36 it is tempting to speculate that these cells contribute, at least in part, to the enhanced expression of TGF-β observed in E2-treated mice, and thus to the hormonal atheroprotective effect.
The mechanisms responsible for the TGF-β antiatherogenic effects, especially reduction of the intimal inflammatory process and promotion of plaque stabilization, were recently further analyzed. 7,37 Thus, a major part of this protective effect of TGF-β is mediated through its inhibitory action on T cells, as the selective disruption of TGF-β signaling in T cells promotes atherosclerosis. 13,14 Because chronic in vivo E2 administration was recently reported to enhance IFN- production by CD4 + T helper lymphocytes as well as Natural Killer T lymphocytes, it remains possible and even likely that TGF-β contributes to the atheroprotective effect of estrogens by inhibiting the secretion of proinflammatory cytokines in the vessel wall. 18,38 According to this hypothesis, we observed that: (1) the combination of E2 administration and TGF-β blockade in apoE –/– mice resulted in a significant increase in IFN- production by activated splenocytes; (2) the disruption of TGF-β signaling in bone marrow–derived cells abolishes the preventive effect of E2 on fatty streak constitution. Interestingly, E2 was recently reported to prevent bone loss through the silencing of IFN- receptor signaling by TGF-β, 39 suggesting some similarity of interaction between E2, IFN-, and TGF-β in vessel and bone.
Finally, the complex interactions between cytokines, as well as the heterogeneity of the atheromatous tissue, greatly limit the information provided by the assessment of their level. For these reasons, we favored a functional approach consisting in studying the atheroprotective effect of E2 in a context of suppression of cytokine expression or activity. Although most of the cytokines are expressed during the atheromatous process, their precise role is probably not constant at the different steps of atherothrombosis (for instance in asymptomatic fatty streaks and during plaque rupture or erosion leading to clinical events). Whereas several proinflammatory cytokines, which level can be enhanced by E2 in several contexts, does not appear to alter the effect of E2 in asymptomatic fatty streaks (present experimental models), they could contribute to plaque rupture in unstable plaque at the initiation of the hormonal replacement therapy (HERS and WHI trials). This deleterious effect could be largely favored in women with a defect in the TGF-β pathway. 3,4 However, despite numerous studies have suggested that lower levels of TGF-β1 activity predispose to unstable atherosclerotic disease (correlation with serum concentration of active TGF-β1 and association with polymorphisms of the TGF-β1 gene), the precise role of the TGF-β pathway in human atherosclerosis remains to be elucidated. 9
In conclusion, the present work clearly demonstrates the importance of TGF-β status in the vascular effects of estrogens. Thus, it will be of interest to consider the TGF-β pathway in past and future clinical trials concerning menopausal women. More precisely, serum active TGF-β concentrations both at baseline and after initiation of HRT, along with the TGF-β and TGF-β receptors gene polymorphisms, should be assessed in such trials and could represent one of the keys of the individual occurrence of cardiovascular events after initiation of HRT.
Acknowledgments
We thank J.C. Couloumiers and Y. Barreira (IFR31, Animal Facility), J.J. Maoret (IFR31, Molecular Biology Platform), S. Bdioui and M.J. Fouque for technical assistance.
Sources of Funding
This work was supported by INSERM, the Ministère de la Recherche et de la Technologie, Action Concertée Incitative 2001 and 2003, European Vascular Genomics Network No. 503254, Fondation de France and the Conseil Régional Midi-Pyrénées. P. Gourdy has been supported by a grant (Contrat Interface) from INSERM.
Disclosures
None.
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作者单位：Institut National de la Santé et de la Recherche Médicale, INSERM U858 (P.G., A.S., C.F., V.D.E., B.G.S., B.C., F.B., J.F.A.), Institut de Médecine Moléculaire de Rangueil, Toulouse, France; Service de Diabétologie (P.G.), Pôle Cardio-Vasculaire et Mét