Biomedical subjects
Publications and source records attributed to F Bayard.
Estradiol significantly decreases fatty streak formation in the aortic root of chow-fed apolipoprotein E-deficient mice. In contrast, immunodeficient mice with homozygous disruption at the recombinase activating gene 2 loci present fatty streak development that is insensitive to estradiol. Lymphocytes thus appear to be required for development of the atheroprotective effect of estradiol in this mouse model.
Two isoforms of oestrogens receptor (alpha and B) have been identified in the cells of the arterial wall, and an heterogenity of their expression according to the animal species, to the vascular bed and to sex has been reported. Estrogens can thus directly influence the vascular physiology through a genomic mechanism, but extra-genomic mechanisms responsible for a short-term effect have also been suggested. Endothelium appears to be an important target for estradiol, because this hormone potentiates endothelium-dependant relaxation through an increase in NO bioavailability, and accelerates endothelial regrowth. In the model of apolipoprotein E-deficient mice, as the atrhroprotective effect deposit. The immune system appears to play a key role, as the athroprotective effect of estradiol is absent in mice deficient in T and B lymphocytes. Estrogens potentiate the endothelium-dependant relaxation through the increase in nitric oxide bioavailability. Endothelial dysfunction (abnormality of the endothelium-dependent vasodilation) occurs in atheromatous arteries. Estrogens prevent and even correct this endothelial dysfunction. In monkeys, this beneficial effect of estrogens is not altered by coadministration of progesterone, but is abolished.
The expression of the interleukin (IL-6) gene can be regulated by various activating or inhibitory stimuli. This modulation involves several regulatory binding sites on the IL-6 promoter, and appears to be in general cell-specific. We have previously described that the nuclear 24 kDa isoform of fibroblast growth factor-2 (FGF-2) is able to increase IL-6 gene expression in NIH-3T3 cells. The transduction pathway involved was shown to be distinct from the extracellular mode of action of the smallest 18 kDa FGF-2 isoform. In the present study, we show that 24 kDa FGF-2-encoding vectors transfected into HeLa cells inhibit various co-transfected constructs incorporating the promoter element of the IL-6 gene and either the luciferase or the chloramphenicol acetyltransferase units. This down-regulation occurs dose-dependently with the 24 kDa FGF-2, is IL-6-promoter-specific, and does not involve an autocrine loop of the growth factor, since exogenously added FGF-2 fails to modulate the IL-6 promoter activity. Furthermore, 24 kDa FGF-2 inhibits the activity of both the co-transfected deletion mutants IL-6(-224) and IL-6(-158), and the point-mutated IL-6 promoter constructs in which the activating protein-1, nuclear factor (NF)-IL-6 and NF-kappaB elements are disrupted. We identify a responsive region to 24 kDa FGF-2 between positions -158 and -109 on the IL-6 promoter, which notably contains a retinoblastoma control element.
The estrogen receptor protein, in the nuclear receptor superfamily, carries two transactivator domains designated AF1 and AF2. The activity of AF2, localized in the carboxy-terminal region, is ligand-dependent, whereas AF1 (amino-terminal) seems to be activated via the MAPKkinase pathway. Uterine and mammary cells exhibiting large amounts of ERalpha were the first estrogen target organs demonstrated. The response intensity in these tissues is related to the affinity of the receptor and to the number of sites occupied by its ligand. Certain physiological and pharmacological phenomena of estrogen resistance associated with a truncated form of ERalpha (deleted in the AF1 domain) would seem however to challenge this assertion. The 45 kDa truncated form is unable to induce cell proliferation but can still increase the expression of certain genes. In this work we suggest that this 45 kDa ERalpha form may originate from differential regulation of translation of the mRNA encoding the ERalpha. In vitro translation studies and transient expression in COS-7 cells in vivo demonstrated a mechanism of translation regulation that produced from a given mRNA either the wild type ER 67 kDa form or the AF1 deleted ER 45 kDa isoform. Bicistronic vectors were used to demonstrate that the 45 kDa protein originates from translation initiation at AUG 174 induced by an internal ribosome entry.
The antiestrogen binding site (AEBS) is a membranous protein complex that has been shown to be intimately linked with the antiproliferative and antiretroviral effects of certain antiestrogenic compounds such as tamoxifen (Tx). Various specific ligands of AEBS derived from benzylphenoxy ethanamine and a new benzoyl structure were synthesized either by modification of the aminoether side chain or by halogen substitution at the meta-, ortho-, and para position on the benzoyl group. Using the MCF-7 cellular strain and its RTx6 variant (a clone selected for its antigrowth resistance to tamoxifen), it was shown that under high drug concentrations the cytotoxicity of the ligands was directly correlated with their affinity for AEBS. In agreement with previous observations made on triphenylethylenic ligands, modification of the basic ethanamine side chain modulated the ligand affinities. Chloride in meta increased ligand efficacy, whereas chloride substitution in ortho and para decreased it. Effects on AEBS-positive MCF-7 cells were drug concentration- and time-dependent, whereas they were unspecific on the AEBS-negative RTx6 cell line. These cytotoxic effects were confirmed in the absence of estrogen receptor on human AEBS-positive uterine cervix cell carcinoma HeLa cells, but were non-specific on rat fibroblastic AEBS-negative (low concentration) NRK cells. The cytotoxicities of these ligands are related to their affinities for AEBS.
OBJECTIVES: Estradiol is known to exert a protective effect against atherosclerosis, but the mechanism(s) whereby this protection is mediated is/are unclear. However, estradiol-treated castrated animals exhibit increased activity of endothelium-derived relaxing factor (EDRF), which could contribute to vasculoprotection. In the present work, we investigated the molecular mechanism(s) of the enhancement of EDRF activity in the thoracic aorta of oophorectomized female rats given 17 beta-estradiol (E2, 2 or 40 micrograms/kg/day) compared to those given a placebo. METHODS AND RESULTS: The abundance in the thoracic aorta of NO synthase I, II and III mRNA (using RT-PCR) and of NO synthase I, II and III immunoreactive protein (using Western blotting) was unaltered by E2. NO synthase activity (based on arginine/citrulline conversion) in thoracic aorta homogenates did not differ significantly among the three groups, suggesting that NO production was not enhanced by E2. In contrast, lucigenin-enhanced chemiluminescence of aorta from the E2 group was decreased compared to that of the placebo group. Desendothelialization and exogenously added superoxide dismutase suggested that this difference was due to a decrease in extracellular endothelium-derived production of superoxide anion (O2-.). Experiments in cultured bovine aortic endothelial cells confirmed a decreased extracellular production of O2-. in response to ethinylestradiol (1 nM) using both lucigenin-enhanced chemiluminescence and ESR spectroscopy. Luminol-enhanced chemiluminescence revealed that ethinylestradioltreated cultured endothelial cells generated less peroxynitrite (the byproduct of NO-. and O2-. interaction) than control cells. CONCLUSION: Estradiol increases rat aorta EDRF activity in the absence of changes in endothelial NO synthase gene expression. The decreased endothelium-derived generation of O2-. in response to estrogens could account for enhanced EDRF-NO bioactivity and decreased peroxynitrite release. All of these effects could contribute to the vascular protective properties of estrogens.
Vascular smooth muscle cells (SMC) are major constituents of the medial layer of blood vessels and are involved in the development of atherosclerotic plaque. SMC secrete copious IL-6 under basal conditions that can be increased by cytokines such as tumor necrosis factor-alpha and interleukin-1beta (IL-1beta). The goal of our studies was to define the role of estrogen in IL-6 production by SMC. In a first series of experiments, the expression of specific messenger RNAs as well as the production of IL-6 bioactivity by rat SMC in culture could be demonstrated in basal and IL-1-stimulated conditions, but was unaffected by estrogen treatment. Different constructs containing deleted or mutated fragments of the human IL-6 promoter driving luciferase or chloramphenicol acetyltransferase reporter gene were then transiently transfected in these cells. A significant basal activity that was increased 2- to 4-fold after IL-1beta stimulation was observed with the total IL-6 promoter. Deletion analysis indicated that the -158/+11 region containing activator protein-1 and cAMP response element sites was apparently the minimal region of IL-6 promoter to confer both constitutive and IL-1-inducible activities. Site-directed mutagenesis experiments suggest that basal activity is dependent upon the promoter sequence -158 to -112 containing the nuclear factor (NF)-IL6(-153) and Sp1 sites, whereas IL-1beta stimulation would depend on the residual -112 nucleotides containing NF-IL6(-75) and NF-kappaB sites. In contrast to the down-regulation of IL-6 expression by estrogen described in osteoblasts, ethinyl estradiol as well as 17beta-estradiol did not influence stimulated IL-6 activity in our experimental conditions whatever the construct tested, even when either estrogen receptor alpha or beta was overexpressed. Thus, the atheroprotective properties of estrogen are probably not mediated through the regulation of IL-6 production by SMC.
The two isoforms (alpha and beta) of the oestrogen receptor were identified in arterial wall cells. The heterogeneity of their expression is considered according to vascular regions and gender. Oestrogens can have a direct influence on vascular physiology through a "genomic" mechanism of action, although "extragenomic" mechanisms allowing very short-term hormonal action are also possible. Oestrogens potentiate endothelium-dependent relaxation by increasing the bioavailability of nitrogen monoxide (higher production and/or lesser degradation by the superoxide anion as a function of vascular beds). The atheromatous artery is the site of endothelial "dysfunction" (an anomaly of endothelium-dependent vasodilation), which can be prevented and even corrected by administration of oestradiol. In the monkey, this beneficial effect of oestrogens is not altered by addition of progesterone, but abolished by the addition of medroxyprogesterone. Finally, oestrogens prevent formation of the fatty streak. Studies of the mechanism(s) of this effect are now in progress.
We have isolated NIH-3T3 cell lines overexpressing the nuclear 24-kDa isoform of fibroblast growth factor (FGF)-2 and characterized its regulatory effect on the expression of interleukin-6 (IL-6) in these cells. The clone pRF5 expressing the highest level was able to grow in 1% serum medium to a high saturation density and acquired a radioresistance advantage. In pRF5 and another clone pRF1, IL-6 RNA levels were markedly increased. Studies with IL-6 promoter constructs revealed that IL-6 gene up-regulation occurred at the transcriptional level and did not involve the AP-1 binding site. Exogenously added 18-kDa isoform of FGF-2 (100 ng/ml) produced down-regulation of IL-6 involving an AP-1 binding site, thus suggesting a receptor-independent pathway for the intracellular 24-kDa isoform.
A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039-17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.
BACKGROUND: The cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) are secreted by the different cell populations of the vascular wall and have been suggested to promote atherosclerosis. METHODS AND RESULTS: Their respective roles in fatty-streak formation in apolipoprotein E-deficient mice were investigated by use of IL-1 receptor antagonist and TNF binding protein. Estradiol-17beta was used as a positive control. Blocking TNF seemed to be active in female animals but not in males. IL-1 receptor antagonist was as effective as or more effective than estradiol in both sexes. CONCLUSIONS: IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model, and blocking the activity of this cytokine should be considered as a therapeutic possibility.
The atheroprotective properties of estrogens are supported by clinical data from postmenopausal women who use estrogen replacement therapy. However, the mechanisms mediating activity remain unknown, and it has been suggested that estrogens may help to modulate endothelial permeability to atherogenic lipoproteins. In these studies we used bovine vascular endothelial cells as an in vitro model to show that estrogens were able to regulate low-density lipoprotein transport and permeability of the endothelial monolayer. Macromolecular transport was observed to be a second-order polynomial function of estrogen concentration. Moreover, this regulation was correlated with expression of heat shock protein (HSP) 25, which is known to influence fluid phase pinocytosis and cytoskeleton remodeling, thus suggesting a role for HSP 25 in the estrogenic control of transcellular permeability of the endothelium monolayer.
BACKGROUND: Estrogens have atheroprotective properties, the mechanisms of which remain obscure. Estrogens have recently been reported to increase endothelial NO synthase expression in castrated animals and to prevent the degradation of NO by decreasing superoxide anion production in cultured endothelial cells. In both cases, increased NO bioavailability would promote vasodilation, inhibit proliferation of the adjacent vascular smooth muscle, reduce platelet aggregation, and inhibit monocyte adhesion to the endothelium and the inflammatory reaction induced by cytokines, all key contributors in the development of atherosclerosis. METHODS AND RESULTS: In the present work, the respective roles of 17beta-estradiol and NO in the development of the atherosclerotic process were investigated in castrated apolipoprotein E-deficient (apo E KO) mice, which spontaneously develop fatty streak lesions within 3 months. N(omega)-Nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, 50 mg x kg(-1) x d(-1), increased arterial blood pressure and decreased cerebellum cGMP content, demonstrating the blockade of NO production, but did not influence the atherogenic process in castrated apo E KO mice. CONCLUSIONS: 17Beta-estradiol decreased the size of the aortic lesions approximately threefold, and the magnitude of this vasculoprotective effect was not altered by L-NAME. Moreover, L-NAME increased circulating malonyldialdehyde (MDA)-modified LDL, which was not altered by 17beta-estradiol, leading to a complete dissociation between circulating MDA-modified LDL and parietal lesions.
Alternative splicing of vascular endothelial growth factor (VEGF) mRNA results in three distinct molecular forms of 121 or 165 (V165) amino acids that are released in the conditioned medium of cultured cells and one longer isoform of 189 amino acids (V189) that remains cell-associated. V189 has been expressed in wild type CHO-K1 cells and in glycosaminoglycan-deficient pgsA-745 Chinese hamster ovary (CHO) mutant cells. It could be released from CHO-K1 cell membranes by heparin or a synthetic peptide designed on the sequence encoded by exon 6 but was freely released from CHO mutant cells. In both cases, the immunoreactive V189 was mainly released as a 40-kDa cleaved form, provided that the serine protease urokinase, but not plasmin, was active. Recombinant V189 was purified from insect cells infected with a recombinant baculovirus as a nonmitogenic 50-kDa precursor that binds to the receptor Flt-1 but not to Flk-1. It could be matured by urokinase as a 38-kDa fragment able to bind to Flk-1 and to trigger cell proliferation. V165 and V189, however, could be cleaved by plasmin as 34-kDa fragments that exhibit a decreased mitogenic activity. These findings indicate that the carboxyl-terminal domain of V189 masks its binding domain to Flk-1.
The reality of the atheroprotective effect of estrogens is still a matter of debate, and its unknown mechanisms could involve favorable changes in blood lipids and lipoproteins and/or direct action at the level of the arterial wall. We used the recently developed animal model of atherosclerosis constituted by apolipoprotein E-deficient mice in an attempt to clarify these issues. Male and female animals, fed a low-fat chow diet, were treated with increasing doses of 17 beta-estradiol (E2) after castration and compared with testosterone treated and uncastrated (intact) animals. Total serum cholesterol, LDL-cholesterol, and HDL-cholesterol concentrations decreased under E2 treatment in each sex and were weakly correlated with lesion area. However, a highly significant correlation between lesion area and serum E2 levels also suggested a direct action of E2 on cells of the vascular wall. A dose-response curve analysis revealed that these activities were sex-dependent, with females being nearly twice as sensitive to E2 as males. It also revealed that the atheroprotective activity was recruited at higher E2 concentrations than those needed by other E2 target tissues such as uterus or functions such as apoA-1 and LDL production and/or clearance rates.
Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium-derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor-mediated antioxidant effect that enhances the biological activity of endothelium-derived NO. These effects could account at least in part for the vascular protective properties of these hormones.
Four isoforms of the human fibroblast growth factor 2 (FGF-2), with different intracellular localizations and distinct effects on cell phenotype, result from alternative initiations of translation at three CUG and one AUG start codons. We showed here by Western immunoblotting and immunoprecipitation that the CUG-initiated forms of FGF-2 were synthesized in transformed cells, whereas "normal" cells almost exclusively produced the AUG-initiated form. CUG-initiated FGF-2 was induced in primary skin fibroblasts in response to heat shock and oxidative stress. In transformed cells and in stressed fibroblasts, CUG expression was dependent on cis-elements within the 5' region of FGF-2 mRNA and was not correlated to mRNA level, indicating a translational regulation. UV cross-linking experiments revealed that CUG expression was linked to the binding of several cellular proteins to FGF-2 mRNA 5' region. Since translation of FGF-2 mRNA was previously shown to occur by internal ribosome entry, a nonclassical mechanism already described for picornaviruses, the cross-linking patterns of FGF-2 and picornavirus mRNAs were compared. Comigration of several proteins, including a p60, was observed. However, this p60 was shown to be different from the p57/PTB internal entry factor, suggesting a specificity towards FGF-2 mRNA. We report here a process of translational activation of the FGF-2 CUG-initiated forms in direct relation with trans-acting factors specific to transformed and stressed cells. These data favor a critical role of CUG-initiated FGF-2 in cell transformation and in the stress response.
Somatostatin analogs are an alternative medical treatment in patients with TSH-secreting pituitary adenoma. A 31-yr-old infertile woman with a TSH-secreting macroadenoma was treated with continuous sc infusion of 300 micrograms octreotide/day. After 3 months, euthyroid status was restored, and pituitary magnetic resonance imaging showed a reduction of the macroadenoma. Subsequently, the patient was found to be pregnant, and octreotide was stopped after 1 month of gestation. Serum TSH and free thyroid hormone concentrations returned to pretreatment values. At 6 months of pregnancy, a visual field examination was abnormal, and a magnetic resonance imaging scan showed an enlargement of the pituitary adenoma. Reinstitution of octreotide treatment was associated with normalization of TSH and free thyroid hormone concentrations, a rapid improvement of visual fields, and a new reduction in the size of pituitary macroadenoma. Octreotide treatment was continued until an elective cesarean section was performed at 8 months gestation. Despite the presence of immunoreactive octreotide in the umbilical cord, neonatal thyroid parameters were normal, and a physiological rise in TSH with the increase in thyroid hormone concentrations occurred in the neonate. In conclusion, 1) octreotide treatment is effective in controlling TSH-secreting macroadenoma during pregnancy; 2) despite the transplacental passage of immunoreactive octreotide, physiological changes in thyroid parameters occur in the neonate, and 3) exposure of the fetus to octreotide during the first month as well as the last trimester of gestation did not induce any malformation or affect fetal development.