Alteration of plasmalemmal caveolae mimics endothelial dysfunction observed in atheromatous rabbit aorta.

OBJECTIVE: In endothelial cells, nitric oxide (NO) is produced by the endothelial isoform of nitric oxide synthase (eNOS), which is localized in the cholesterol-rich plasmalemmal microdomains involved in signal transduction, known as caveolae. The present study was undertaken to evaluate the effect of hypercholesterolemia and fatty streak formation on the endothelial caveolae and on endothelial function, and attempted to determine to what extent the caveolae were involved in endothelium-derived NO production. METHODS AND RESULTS: We first studied the effect of atheroma on endothelial NO production. Fatty streak infiltrated aorta of cholesterol-fed New Zealand White rabbits demonstrated an impairment of acetylcholine-induced relaxation and nearly normal calcium ionophore A23187-induced maximal relaxation. The abundance of caveolae in the endothelium covering the fatty streak, as well as their 'grape-like' clustering, appeared to be decreased. We therefore investigated the effect, on endothelial NO production, of the cholesterol-binding agents 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CD) and filipin, known to alter caveolae structure and/or function. Treatment with either hp-beta-CD (2%) or filipin (4 microg/ml) did not affect contraction to phenylephrine or relaxant responses to A23187 or to the NO donor sodium nitroprusside. In contrast, both treatments impaired acetylcholine-induced relaxation. Cultured bovine aortic endothelial cells (BAEC) similarly treated with hp-beta-CD demonstrated a 50% decrease of total cellular cholesterol and a decreased abundance of caveolae as well as their 'grape-like' clustering. Cholesterol depletion decreased the bradykinin-induced transient peak of free intracellular calcium and subsequent receptor-stimulated NO production (assessed using reporter cells rich in soluble guanylyl cyclase), whereas that elicited by A23187 remained unaltered. CONCLUSION: Fatty streak deposit is associated with a decrease in caveolae 'transductosomes' abundance which appears to represent a novel mechanism of endothelial dysfunction

Prevention of skin flap necrosis by estradiol involves reperfusion of a protected vascular network.

International audienceAlthough 17beta-estradiol (E2) is protective in experimental models of myocardial and brain ischemia, its effect on skin ischemia remains unknown. Here, we assessed the protective effect of E2 in a mouse model of skin ischemia, mimicking the surgery of skin flaps. Whereas necrosis appeared in the half portion of the skin flap within 1 week after surgery in ovariectomized mice, it was reduced up to 10-fold when mice were pretreated with E2, at least 3 days before the surgery. The beneficial effect of E2 appeared to be attributable to an increase in skin survival, revealed by measuring viability of ex vivo explants and enhancement of the antiapoptotic Bcl-2 protein expression in vivo. This protective effect on the skin contributed to the protection of the vascular network and facilitated reperfusion, which was found to be accelerated in ovariectomized E2-treated mice, whereas hemorrhages were observed in untreated mice. E2 also increased expression of fibroblast growth factor-2 isoforms in the skin and circulating vascular endothelial growth factor in the serum. Finally, this protective effect of E2 was abolished in estrogen receptor-deficient mice (ERalpha(-/-)) but maintained in chimeric mice reconstituted with ERalpha-deficient bone marrow, indicating dispensable action of E2 in bone marrow-derived cells. This protective effect of E2 was mimicked by treatment with tamoxifen, a selective estrogen receptor modulator. In conclusion, we have demonstrated for the first time that E2 exerts a major preventive effect of skin flap necrosis through a prevention of ischemic-induced skin lesions, including those of the vascular network, which contributes to accelerate the reperfusion of the skin flap

Deleting TCRαβ(+) or CD4(+) T Lymphocytes Leads to Opposite Effects on Site-Specific Atherosclerosis in Female Apolipoprotein E-Deficient Mice

Recent studies have demonstrated the importance of lymphocytes, especially CD4(+) T cells, in early lesions of atherosclerosis in hypercholesterolemic mice. However, the role of other T cell subpopulations, like CD8(+) T cells or TCRγδ T lymphocytes, is not yet clear. We have therefore generated apolipoprotein E-deficient mice genetically deficient in specific T lymphocyte subpopulations and measured atherosclerotic lesions in the aortic sinus and en face whole aorta preparation at 18 weeks and at 1 year of age. Whereas TCRγδ(+) T lymphocytes appeared to play a modest role, TCRαβ(+) T lymphocytes played a major role as their deficiency significantly prevented early and late atherosclerosis at all arterial sites. However, neither CD4(+) nor CD8(+) T cells induced any significant decrease of the lesions at the aortic sinus, suggesting that compensatory proatherogenic mechanisms are operating at this site. Interestingly, the absence of CD4(+) T cells led to a dramatic increase in early lesion abundance at the level of the descending thoracic and abdominal aorta, which was still obvious at 1 year. In conclusion, whereas the TCRαβ(+) lymphocyte subset in its whole contribute to aggravate both early and late atherosclerosis, the CD4(+) T subpopulation appears to be critically protective at the level of the lower part of the aorta

The transactivating function 1 of estrogen receptor α is dispensable for the vasculoprotective actions of 17β-estradiol

Full-length 66-kDa estrogen receptor α (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 ERα AF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERα A/B domain in the mouse. In these ERαAF-10 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 ERαAF-1+/+ LDLr−/− (low-density lipoprotein receptor) and ERαAF-10 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 ERα AF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERα 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 ERα with minimal activation of ERα AF-1 could retain beneficial vascular actions, while minimizing the sexual effects

Stromal Estrogen Receptor-α Promotes Tumor Growth by Normalizing an Increased Angiogenesis.

Estrogens directly promote the growth of breast cancers that express the Estrogen Receptor (ERalpha). However, the contribution of stromal expression of ERalpha in the tumor microenvironment to the pro-tumoral effects of estrogen has never been explored. In this study, we evaluated the molecular and cellular mechanisms by which 17beta-estradiol (E2) impacts the microenvironment and modulates tumor development of ERalpha-negative tumors. Using different mouse models of ER-negative cancer cells grafted subcutaneously into syngeneic ovariectomized immunocompetent mice, we found that E2 potentiates tumor growth, increases intratumoral vessel density and modifies tumor vasculature into a more regularly organized structure, thereby improving vessel stabilization to prevent tumor hypoxia and necrosis. These E2-induced effects were completely abrogated in ERalpha-deficient mice, demonstrating a critical role of host ERα. Notably, E2 did not accelerate tumor growth when ERalpha was deficient in Tie2- positive cells, but still expressed by bone marrow derived cells. These results were extended by clinical evidence of ERalpha-positive stromal cell labeling in the microenvironment of human breast cancers. Together, our findings therefore suggest that E2 promotes the growth of ERalpha-negative cancer cells through the activation of stromal ERα (not hematopoiteic but Tie2-dependent expression of ERalpha), which normalizes tumor angiogenesis and allows an adaptation of blood supply to tumor demand preventing hypoxia and necrosis. These findings significantly deepen mechanistic insights into the impact of E2 on tumor development with potential consequences for cancer treatment