[Show abstract][Hide abstract] ABSTRACT: E2 accelerates reendothelialization through estrogen receptor alpha (ER alpha), and we now aimed at defining the precise local and systemic cellular actors of this process.
The respective roles of endothelial and hematopoietic targets of E2 were investigated in a mouse carotid injury model, using confocal microscopy, to follow endothelium repair. Grafting ER alpha(-/-) mice with ER alpha(+/+) bone marrow (BM) was not sufficient to restore the accelerative effect of E2 on reendothelialization, demonstrating the necessary role of extrahematopoietic ER alpha. Using an endothelial-specific inactivation of ER alpha (Cre-Lox system), we showed that endothelial ER alpha plays a pivotal role in this E2 action. Conversely, in ER alpha(+/+) grafted with ER alpha(-/-) BM, the E2 regenerative effect was abolished, demonstrating that ER alpha-expressing hematopoietic cells are also needed. As eNOS expression in BM was required for this action, both endothelial progenitor cells and platelets could be the hematopoietic targets that participate to this beneficial E2 effect.
We demonstrate that endothelial ER alpha plays a pivotal role in E2-mediated reendothelialization. However, endothelial targeting alone is not sufficient because the concomitant stimulation of a subpopulation of BM ER alpha is necessary. This cooperation should be taken into account in strategies aimed at optimizing in-stent reendothelialization.
[Show abstract][Hide abstract] ABSTRACT: Full-length 66-kDa estrogen receptorα (ERα) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ERα isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERαAF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERα A/B domain in the mouse. In these ERαAF-1zero mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-weekold ERαAF-1+/+LDLR-/- (low-density lipoprotein receptor) and ERαAF-1zeroLDLR-/- mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the ic tree of 8-month-old ovariectomized or intact female mice revealed that ERαAF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERαAF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ERα with minimal activation of ERαAF-1 could retain beneficial vascular actions, while minimizing the sexual effects.
[Show abstract][Hide abstract] ABSTRACT: Full-length 66-kDa estrogen receptor alpha (ERalpha) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ERalpha isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERalpha AF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERalpha A/B domain in the mouse. In these ERalphaAF-1(0) mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-week-old ERalphaAF-1(+/+) LDLr(-/-) (low-density lipoprotein receptor) and ERalphaAF-1(0) LDLr (-/-) mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the aortic tree of 8-month-old ovariectomized or intact female mice revealed that ERalpha AF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERalpha AF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ERalpha with minimal activation of ERalpha AF-1 could retain beneficial vascular actions, while minimizing the sexual effects.
Proceedings of the National Academy of Sciences 03/2009; 106(6):2053-8. DOI:10.1073/pnas.0808742106 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Estradiol (E(2)) is known to accelerate reendothelialization and thus prevent intimal thickening and in-stent restenosis after angioplasty. Transplantation experiments with ERalpha(-/-) mice have previously shown that E(2) acts through local and bone marrow cell compartments to enhance endothelial healing. However, the downstream mechanisms induced by E(2) to mediate endothelial repair are still poorly understood.
We show here that after endovascular carotid artery injury, E(2)-enhanced endothelial repair is lost in osteopontin-deficient mice (OPN(-/-)). Transplantation of OPN(-/-) bone marrow into wild-type lethally irradiated mice, and vice versa, suggested that osteopontin plays a crucial role in both the local and the bone marrow actions of E(2). In the vascular compartment, using transgenic mice expressing doxycyclin regulatable-osteopontin, we show that endothelial cell specific osteopontin overexpression mimics E(2)-enhanced endothelial cell migration and proliferation in the regenerating endothelium. In the bone marrow cell compartment, we demonstrate that E(2) enhances bone marrow-derived mononuclear cell adhesion to regenerating endothelium in vivo, and that this effect is dependent on osteopontin.
We demonstrate here that E(2) acceleration of the endothelial repair requires osteopontin, both for bone marrow-derived cell recruitment and for endothelial cell migration and proliferation.
[Show abstract][Hide abstract] ABSTRACT: Although the accelerative effect of 17beta-estradiol (E2) on endothelial regrowth has been clearly demonstrated, the local cellular events accounting for this beneficial vascular action are still uncertain. In the present work, we compared the kinetics of endothelial healing of mouse carotid arteries after endovascular and perivascular injury. Both basal reendothelialization as well as the accelerative effect of E2 were similar in the two models. Three days after endothelial denudation, a regenerative area was observed in both models, characterized by similar changes in gene expression after injury, visualized by en face confocal microscopy (EFCM). A precise definition of the injury limits was only possible with the perivascular model, since it causes a complete and lasting decellularization of the media. Using this model, we demonstrated that the migration of uninjured endothelial cells precedes proliferation (bromodeoxyuridine incorporation) and that these events occur at earlier time points with E2 treatment. We have also identified an uninjured retrograde zone as an intimate component of the endothelial regeneration process. Thus, in the perivascular model, the regenerative area can be subdivided into a retrograde zone and a reendothelialized area. Importantly, both areas are significantly enlarged by E2. In conclusion, the combination of the electric perivascular injury model and EFCM is well adapted to the visualization of the endothelial monolayer and to investigate cellular events involved in reendothelialization. This process is accelerated by E2 as a consequence of the retrograde commitment of an uninjured endothelial zone to migrate and proliferate, contributing to an enlargement of the regenerative area.
[Show abstract][Hide abstract] ABSTRACT: We recently demonstrated that in vitro peroxisome proliferator-activated receptor-gamma (PPARgamma) activation of mouse peritoneal macrophages by IL-13 or PPARgamma ligands promotes uptake and killing of Candida albicans through mannose receptor overexpression. In this study, we demonstrate that i.p. treatment of immunocompetent and immunodeficient (RAG-2(-/-)) mice with natural and synthetic PPARgamma-specific ligands or with IL-13 decreases C. albicans colonization of the gastrointestinal (GI) tract 8 days following oral infection with the yeast. We also showed that Candida GI infection triggers macrophage recruitment in cecum mucosa. These mucosal macrophages, as well as peritoneal macrophages, overexpress the mannose receptor after IL-13 and rosiglitazone treatments. The treatments promote macrophage activation against C. albicans as suggested by the increased ability of peritoneal macrophages to phagocyte C. albicans and to produce reactive oxygen intermediates after yeast challenge. These effects on C. albicans GI infection and on macrophage activation are suppressed by treatment of mice with GW9662, a selective PPARgamma antagonist, and are reduced in PPARgamma(+/-) mice. Overall, these data demonstrate that IL-13 or PPARgamma ligands attenuate C. albicans infection of the GI tract through PPARgamma activation and hence suggest that PPARgamma ligands may be of therapeutic value in esophageal and GI candidiasis in immunocompromised patients.
The Journal of Immunology 05/2008; 180(7):4939-47. DOI:10.4049/jimmunol.180.7.4939 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that estrogen exerts a vasoprotective effect by accelerating reendothelialization after perivascular artery injury through activation of the estrogen receptor alpha. Because 17beta-estradiol (E2) is known to increase the bioavailability of nitric oxide, in this study, we used the same perivascular model to characterize the role of the endothelial nitric oxide synthase (eNOS) pathway in reendothelialization. Surprisingly, we found that the stimulatory effect of E2 on reendothelialization was not altered following pharmacological inhibition of nitric-oxide synthase enzymatic activity by N-nitro-L-arginine methyl ester, whereas it was abolished in eNOS-deficient (eNOS-/-) mice. This discrepancy between eNOS gene inactivation and the pharmacological inhibition of eNOS was confirmed in a classical model of endovascular injury. When assessing the involvement of eNOS in short-term membrane-associated signaling events induced by E2, we found that E2 stimulated phosphorylation of extracellular signal-regulated kinase 1/2 in isolated perfused carotid arteries from wild-type mice in the absence or presence of N-nitro-l-arginine methyl ester, whereas this stimulation was abolished in carotid arteries from eNOS-/- mice. Similar results were obtained in primary cultures of mouse aortic endothelial cells. These data reveal an original and unexpected role of eNOS, in which its presence but not its enzymatic activity appears to be a determinant for estrogen signaling in the endothelium. The consequences of this novel function of eNOS with respect to vascular diseases should be explored.
American Journal Of Pathology 04/2008; 172(3):830-8. DOI:10.2353/ajpath.2008.070439 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The preventive effect of estradiol was fully maintained in LDLr-/- mice grafted with bone marrow from either IFN-gamma 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-beta1 in apoE-/- mice during early steps of atherogenesis; (2) abolished and even reversed in apoE-/- mice administered with a neutralizing anti-TGF-beta antibody; (3) abolished in LDLr-/- mice grafted with bone marrow from Smad3-deficient mice.
The status of the TGF-beta pathway crucially determines the antiatherogenic effect of estradiol in hypercholesterolemic mice, whereas neither IFN-gamma, IL-12, nor IL-10 are specifically involved in this protection.
[Show abstract][Hide abstract] ABSTRACT: 17beta-Estradiol (E2) accelerates reendothelialization and increases the number of circulating endothelial progenitor cells (EPCs), but whether fibroblast growth factor-2 (FGF2) is involved in these processes remains unknown. Here we explored the role of FGF2 in the effect of E2 on reendothelialization and EPC levels in a mouse model. As previously reported, E2 increased both the velocity of reendothelialization and the number of circulating EPCs in ovariectomized wild-type (Fgf2+/+) mice. In contrast, the effect of E2 on both parameters was abolished in FGF2-deficient mice (Fgf2-/-), demonstrating that FGF2 is absolutely required for these effects of E2. To test the implication of medullary and extramedullary FGF2, we developed chimeric mice by grafting Fgf2-/- bone marrow to Fgf2+/+ [Fgf2-/- bone marrow (BM) = > Fgf2+/+] mice and observed that the effect of E2 on both reendothelialization and EPC levels was abolished. In contrast, both effects of E2 in Fgf2+/+BM = >Fgf2-/- mice were similar to those observed in Fgf2+/+ mice, demonstrating that only BM-derived, but not extramedullary, FGF2 is required for both effects. Interestingly, E2 was found to markedly increase both FGF2(lmw) and FGF2(hmw) in bone marrow. In conclusion, FGF2, specifically medullary FGF2, is necessary and sufficient to mediate the accelerative effect of E2 on both reendothelialization and EPC mobilization.
American Journal Of Pathology 11/2006; 169(5):1855-62. DOI:10.2353/ajpath.2006.060260 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Johan Auwerx – Institut de Genetique et Biologie Cellulaire et Moleculaire (IGBMC); Institut Clinique de la Souris, Strasbourgh
Estrogens have puzzling and ill-understood effects on the cardiovascular system in human beings. It is widely believed that estrogens are the prime reason why pre-menopausal women are protected against cardiovascular events, yet substitution of estrogens to post-menopausal women seems not to have similar effects. The group of Arnal and Bayard have been among the first to explore the cardiovascular effects of estrogens in mouse models with various mutations in the estrogen receptor. Their review summarizes these data and underscores the usefulness of these animal models.
Drug Discovery Today Disease Models 12/2004; 1(3):213-221. DOI:10.1016/j.ddmod.2004.11.018