RESUMEN
Typically, inhaled drugs are rapidly absorbed into the bloodstream, which results in systemic side effects and a brief residence time in the lungs. PEGylation was evaluated as a novel strategy for prolonging the retention of small inhaled molecules in the pulmonary tissue. Hydrolysable ester conjugates of PEG1000, PEG2000, 2000, PEG3400 and prednisolone, a model drug cleared from the lungs within a few minutes, were synthesised and thoroughly characterised. The conjugates were stable in buffers with hydrolysis half-lives ranging from 1h to 70 h, depending on the pH and level of substitution. With the exception of PEG3400-prednisolone, conjugates did not induce a significant lactate dehydrogenase (LDH) release from Calu-3 cells after a 20 h exposure. Following nebulisation to isolated perfused rat lungs (IPRL), the PEG2000 and mPEG2000 conjugates reduced the maximum prednisolone concentration in the perfusate (Cmax) by 3.0 and 2.2 fold, respectively. Moreover, while prednisolone was undetectable in the perfusion solution beyond 20 min when the free drug was administered, prednisolone concentrations were still quantifiable after 40 min following delivery of the conjugates. This study is the first to demonstrate hydrolysable PEG drug ester conjugates are a promising approach for optimising the pharmacokinetic profile of small drugs delivered by inhalation.
Asunto(s)
Pulmón/metabolismo , Polietilenglicoles/farmacocinética , Prednisolona/farmacocinética , Administración por Inhalación , Animales , Línea Celular Tumoral , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Ésteres , Humanos , Masculino , Modelos Biológicos , Peso Molecular , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Prednisolona/administración & dosificación , Prednisolona/química , Ratas , Ratas WistarRESUMEN
A new hepatitis B virus (HBV) protein, hepatitis B splice-generated protein (HBSP), has been detected in liver biopsy specimens from patients with chronic active hepatitis. The aim of this study was to characterize the phenotype and functions of peripheral HBSP-specific T cells and to determine whether these T-cell responses may be implicated in liver damage or viral control. Two groups of patients were studied: HBV-infected patients with chronic active hepatitis and HBV-infected patients who were inactive carriers of hepatitis B surface antigen. HBSP-specific T-cell responses were analysed ex vivo and after in vitro stimulation of peripheral blood mononuclear cells. Soluble cytokines and chemokines were analysed in sera and in cell culture supernatants. Few HBSP- or capsid-specific T-cell responses were detected in patients with chronic active hepatitis whereas frequency of HBV-specific T cells was significantly higher in inactive carrier patients. HBSP activated CD8+ and CD4+ T cells that recognized multiple epitopes and secreted inflammatory cytokines. The IL-12 level was significantly lower in sera from asymptomatic carrier patients compared to patients with chronic active hepatitis. IL-12 and IP-10 levels in the sera were significantly and independently correlated with both alanine amino transferase and HBV DNA levels. Our results show that the HBSP protein activates cellular immune responses in HBV-infected patients but has probably no prominent role in liver damage. The pattern of cytokines and chemokines in sera was linked to HBV viral load and was consistent with the level of inflammation during chronic hepatitis.
Asunto(s)
Citocinas/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hígado/patología , Linfocitos T/inmunología , Proteínas Virales/inmunología , Adulto , Anciano , Alanina Transaminasa/sangre , Portador Sano/inmunología , Portador Sano/virología , Células Cultivadas , Citocinas/sangre , Femenino , Hepatitis B Crónica/virología , Humanos , Leucocitos Mononucleares/inmunología , Hígado/virología , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/inmunología , Carga Viral , Adulto JovenRESUMEN
1. Although hormonal therapy (HT) may increase the risk of coronary heart disease (CHD) and stroke in postmenopausal women, epidemiological studies (protection in premenopausal women) suggest and experimental studies (prevention of fatty streak development in animals) demonstrate a major atheroprotective action of estradiol (E2). The understanding of the deleterious and beneficial effects of oestrogens is thus required. 2. The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Although E2 favours an anti-inflammatory effect in vitro (cultured cells), it rather elicits a pro-inflammatory response in vivo involving several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. The functional role of several cytokines was explored in hypercholesterolemic mice. The atheroprotective effect of E2 was fully maintained in mice deficient in interferon-g or interleukin-12, as well as IL-10. In contrast, the protective effect of estradiol was abolished and even reversed in hypercholesterolemic mice given a neutralizing anti-transforming growth factor-b (TGF-b) antibody. Endothelium is another important target for E2, since it not only potentiates endothelial nitric oxide and prostacyclin production, but also controls trafficking of the populations of the immuno-inflammatory system. 3. To conclude, the respective actions of oestrogens on the cell populations involved in the pathophysiology of atherothrombosis may be influenced, among others, by the timing of HT initiation, the status of the vessel wall and, as recently demonstrated the status of the TGF-b pathway.
Asunto(s)
Aterosclerosis/metabolismo , Citocinas/metabolismo , Estradiol/metabolismo , Estradiol/farmacología , Animales , Endotelio/metabolismo , Femenino , Eliminación de Gen , Humanos , Hipercolesterolemia , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Ratones , Factor de Crecimiento Transformador betaRESUMEN
Whereas hormonal replacement/menopause therapy (HRT) in post-menopausal women increases the coronary artery risk, epidemiological studies (protection in pre-menopaused 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 immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the atherosclerotic plaque rupture. Whereas E2 favors an anti-inflammatory effect in vitro (cultured cells), it rather elicits pro-inflammation in vivo, at the level of several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for E2, since it stimulates endothelial NO and prostacyclin production, thus promoting beneficial effects of vasorelaxation and platelet aggregation inhibition. Prostacyclin, but not NO, appears to be involved in the atheroprotective effect of E2. Estradiol accelerates also endothelial regrowth, thus favoring vascular healing. Finally, most of these effects of E2 are mediated by estrogen receptor alpha, and are independent of estrogen receptor beta. In summary, a better understanding of the mechanisms of estrogen action is required not only on the normal and atheromatous arteries, but also on innate and adaptive immune responses. This should help cardiovascular disease prevention optimization after menopause. These mouse models should help to screen existing and future Selective Estrogen Receptor Modulators (SERMs).
Asunto(s)
Estradiol/farmacología , Terapia de Reemplazo de Estrógeno , Animales , Antiinflamatorios/farmacología , Enfermedad de la Arteria Coronaria/inducido químicamente , Enfermedad de la Arteria Coronaria/prevención & control , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Estradiol/efectos adversos , Femenino , Humanos , Mediadores de Inflamación/farmacología , Ratones , Posmenopausia/efectos de los fármacos , Premenopausia/efectos de los fármacos , Factores de Riesgo , Moduladores Selectivos de los Receptores de Estrógeno/farmacologíaRESUMEN
AIM OF THE STUDY: Smooth muscle cells build up the normal media and stabilize atherosclerotic lesions whereas an inflammatory component is determinant for unstable angina. Smooth muscle cells, currently identified by alpha-actin, present a phenotypic heterogeneity and alpha-actin can be reduced in pathology. We tried to characterize vascular cell types, particularly smooth muscle cells, and coronary atherosclerotic tissues, by random genes expression fingerprints. MATERIALS AND METHODS: Expression fingerprints (cDNA electrophoresis) were performed by differential display reverse transcriptase-polymerase chain reaction. Variability of fingerprints was studied for a panel of arterial muscle cell phenotypes and comparisons were made with fingerprints from other cell types (endothelial cells and macrophages). The technique was then applied to human coronary atherectomy samples compared to control human arterial (mammary) smooth muscle. RESULTS: Arterial smooth muscle cells fingerprints were overall similar whatever the cell phenotype (native contractile, dedifferentiated in culture or epithelioid). Moreover, with two primer pairs, the muscular fingerprints markedly differed from the endothelial and the monocytic fingerprints. Application of differential display to coronary atherectomy samples was feasible. Interestingly, the pathological tissues exhibited either smooth muscle-like or smooth muscle-divergent fingerprints. CONCLUSIONS: Smooth muscle cells and inflammatory cells exhibited distinct differential display fingerprint patterns. Thus, a simple expression profile of arbitrary genes provides a molecular bar code tool (pattern signature) useful to characterize vascular cell cultures or tissues. The present work proposes a method to analyze coronary atherectomy samples which estimates their whole quality, muscular versus non muscular (inflammatory), this is of interest for clinical research.
Asunto(s)
Enfermedad de la Arteria Coronaria/patología , Perfilación de la Expresión Génica , Músculo Liso Vascular/química , Enfermedad de la Arteria Coronaria/genética , Dermatoglifia del ADN , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
The mRNA coding for FGF-2 (fibroblast growth factor 2), a major angiogenic factor, is translated by an IRES (internal ribosome entry site)-dependent mechanism. We have studied the role of the IRES in the regulation of FGF-2 expression in vivo, under pathophysiological conditions, by creating transgenic mice lines expressing bioluminescent bicistronic transgenes. Analysis of FGF-2 IRES activity indicates strong tissue specificity in adult brain and testis, suggesting a role of the IRES in the activation of FGF-2 expression in testis maturation and brain function. We have explored translational control of FGF-2 mRNA under diabetic hyperglycaemic conditions, as FGF-2 is implied in diabetes-related vascular complications. FGF-2 IRES is specifically activated in the aorta wall in streptozotocin-induced diabetic mice, in correlation with increased expression of endogenous FGF-2. Thus, under hyperglycaemic conditions, where cap-dependent translation is blocked, IRES activation participates in FGF-2 overexpression, which is one of the keys of diabetes-linked atherosclerosis aggravation. IRES activation under such pathophysiological conditions may involve ITAFs (IRES trans-acting factors), such as p53 or hnRNP AI (heterogeneous nuclear ribonucleoprotein AI), recently identified as inhibitory or activatory ITAFs respectively for FGF-2 IRES.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , Animales , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Codón Iniciador , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ratones , Ratones Transgénicos , Ribosomas/metabolismoRESUMEN
Both 17beta-estradiol (E2) and fibroblast growth factor-2 (FGF2) stimulate angiogenesis and endothelial cell migration and proliferation. The first goal of this study was to explore the potential link between this hormone and this growth factor. E2-stimulated angiogenesis in SC Matrigel plugs in Fgf2+/+ mice, but not in Fgf2-/- mice. Cell cultures from subcutaneous Matrigel plugs demonstrated that E2 increased both migration and proliferation in endothelial cells from Fgf2+/+ mice, but not from in Fgf2-/- mice. Several isoforms of fibroblast growth factor-2 (FGF2) are expressed: the low molecular weight 18-kDa protein (FGF2lmw) is secreted and activates tyrosine kinase receptors (FGFRs), whereas the high molecular weight (21 and 22 kDa) isoforms (FGF2hmw) remains intranuclear, but their role is mainly unknown. The second goal of this study was to explore the respective roles of FGF2 isoforms in the effects of E2. We thus generated mice deficient only in the FGF2lmw (Fgf2lmw-/-). E2 stimulated in vivo angiogenesis and in vitro migration in endothelial cells from Fgf2lmw-/- as it did in Fgf2+/+ mice. E2 increased FGF2hmw protein abundance in endothelial cell cultures from Fgf2+/+ and Fgf2lmw-/- mice. As shown using siRNA transfection, these effects were FGFR independent but involved FGF2-Interacting Factor, an intracellular FGF2hmw partner. This is the first report for a physiological role for the intracellular FGF2hmw found to mediate the effect of E2 on endothelial cell migration via an intracrine action.
Asunto(s)
Endotelio Vascular/fisiología , Estradiol/farmacología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Neovascularización Fisiológica , Animales , División Celular , Movimiento Celular , Células Cultivadas , Endotelio Vascular/citología , Receptor alfa de Estrógeno , Factor 2 de Crecimiento de Fibroblastos/genética , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Receptores de Estrógenos/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
Numerous epidemiological as well as experimental studies have suggested that estradiol (E2) prevents atherosclerosis development. However two controlled prospective and randomized studies in women using hormone replacement therapy (HRT) did not confirm this beneficial effect. We then decided to use mouse models of atherosclerosis to define the possible mechanisms involved and the reasons for the discrepancy. We have shown that, although serum cholesterol decreases, this influence on lipid metabolism is negligible. Surprisingly, E2 induces an inflammatory-immune response towards a T helper cell (Th1) profile with increasing interferon-gamma production that could destabilize atheromatous plaques, and could account for the increase in the frequency of cardiovascular events in women undergoing HRT. At the level of the endothelium, E2 induces an increase in nitric oxide (NO) biodisponibility, but this phenomenon does not concern the development of fatty streaks. Nevertheless, the atheroprotective effect is apparently mediated at the level of the endothelium by a mechanism that has still to be characterized in molecular terms. These new acquisitions constitute a basis for new pharmacological developments allowing the prevention of deleterious effects and preserving the beneficial ones.
Asunto(s)
Arteriosclerosis/fisiopatología , Endotelio Vascular/fisiopatología , Estradiol/metabolismo , Animales , Arteriosclerosis/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/fisiopatología , Ratones , Ratones Noqueados , Células TH1/fisiologíaRESUMEN
The mechanisms that mediate the atheroprotective properties of estrogens remain obscure. In the present study, we evaluated the involvement of the adhesion molecule P-selectin, intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) in the atheroprotective effect of estrogens in murine models evaluating early steps of atherosclerosis. First, we studied the effect of 17 beta-estradiol (E2) administration for 12 weeks on fatty streak constitution at the root aorta of ovariectomized female mice deficient in apolipoprotein E (apoE) alone or deficient in both apoE and either P-selectin or ICAM-1. Compared with respective placebo groups, E2 significantly prevented the development of fatty streak, to a similar extent in all three genotypes (-70.0% in apoE(-/-), -77.4% in apoE(-/-) P-selectin(-/-), and -77.1% in apoE(-/-) ICAM-1(-/-)). Second, the endothelial expression of VCAM-1 at the root aorta was assessed by immunohistochemistry in either placebo or E2-treated ovariectomized C57BL/6 female mice fed an atherogenic diet. Compared with placebo, E2 treatment resulted in a 31.8% decrease of VCAM-1 endothelial expression at this lesion-prone site (P=0.03). These results demonstrate that P-selectin and ICAM-1 are not involved in the atheroprotective effect of estrogens, and suggest that VCAM-1 could play a role in this process.
Asunto(s)
Apolipoproteínas E/deficiencia , Arteriosclerosis/metabolismo , Estradiol/farmacología , Molécula 1 de Adhesión Intercelular/metabolismo , Selectina-P/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Apolipoproteínas E/genética , Arteriosclerosis/patología , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacosAsunto(s)
Aorta/patología , Aorta/trasplante , Rechazo de Injerto/patología , Trasplante Homólogo/patología , Animales , Enfermedad Crónica , Rechazo de Injerto/inmunología , Inmunoglobulina G/sangre , Isoanticuerpos/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Modelos Animales , Trasplante Homólogo/inmunologíaAsunto(s)
Aorta/trasplante , Interleucina-6/inmunología , Linfocitos/inmunología , Bazo/inmunología , Trasplante Homólogo/inmunología , Animales , Aorta/inmunología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/inmunología , Interleucina-6/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones NoqueadosRESUMEN
Although estradiol (E(2)) has been recognized to exert several vasculoprotective effects in several species, its effects in mouse vasomotion are unknown, and consequently, so is the estrogen receptor subtype mediating these effects. We investigated the effect of E(2) (80 microg/kg/day for 15 days) on NO production in the thoracic aorta of ovariectomized C57Bl/6 mice compared with those given placebo. E(2) increased basal NO production. In contrast, the relaxation in response to ATP, to the calcium ionophore A23187, and to sodium nitroprusside was unaltered by E(2), whereas acetylcholine-elicited relaxation was decreased. The abundance of NO synthase I, II, and III immunoreactive proteins (using Western blot) in thoracic aorta homogenates was unchanged by E(2). To determine the estrogen receptor (ER) subtype involved in these effects, transgenic mice in which either the ERalpha or ERbeta has been disrupted were ovariectomized and treated, or not, with E(2). Basal NO production was increased and the sensitivity to acetylcholine decreased in ERbeta knockout mice in response to E(2), whereas this effect was abolished in ERalpha knockout mice. Finally, these effects of E(2) on vasomotion required long-term and/or in vivo exposure, as short-term incubation of aortic rings with 10 nmol/L E(2) in the isolated organ chamber did not elicit any vasoactive effects. In conclusion, this study demonstrates that ERalpha, but not ERbeta, mediates the beneficial effect of E(2) on basal NO production.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Estradiol/administración & dosificación , Óxido Nítrico/metabolismo , Receptores de Estrógenos/metabolismo , Adenosina Trifosfato/farmacología , Animales , Factores Biológicos/metabolismo , Peso Corporal/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Relación Dosis-Respuesta a Droga , Implantes de Medicamentos , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Técnicas In Vitro , Ionóforos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/metabolismo , Nitroprusiato/farmacología , Ovariectomía , Receptores de Estrógenos/deficiencia , Receptores de Estrógenos/genética , Útero/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Two isoforms of estrogen receptor (ER) have been described: ERalpha and ERbeta. The initial gene targeting of ERalpha, consisting in the introduction of a Neo cassette in exon 1 [alphaERKO, hereafter called ERalpha-Neo KO (knockout)], was reported in 1993. More recently, another mouse deficient in ERalpha because of the deletion of exon 2 (ERalphaKO, hereafter called ERalpha-delta2 KO) was generated. In ovariectomized ERalpha-wild-type mice, estradiol (E(2)) increases uterine weight and basal production of endothelial nitric oxide (NO). Both of these effects are abolished in ERalpha-delta2 KO mice. In contrast, we show here that both of these effects of E(2) are partially (uterine weight) or totally (endothelial NO production) preserved in ERalpha-Neo KO. We also confirm the presence of two ERalpha mRNA splice variants in uterus and aorta from ERalpha-Neo KO mice. One of them encodes a chimeric ERalpha protein (ERalpha55), partially deleted in the A/B domain, that was detected in both uterus and aorta by Western blot analysis. The other ERalpha mRNA splice variant codes for an isoform deleted for the A/B domain (ERalpha46), which was detected in uterus of ERalpha-Neo KO, and wild-type mice. This protein isoform was not detected in aorta. The identification of these two N-terminal modified isoforms in uterus, and at least one of them in aorta, probably explains the persistence of the E(2) effects in ERalpha-Neo KO mice. Furthermore, ERalpha-Neo KO mice may help in the elucidation of the specific functions of full-length ERalpha (ERalpha66) and ERalpha46, both shown to be physiologically generated in vivo.
Asunto(s)
Estradiol/farmacología , Óxido Nítrico/biosíntesis , Receptores de Estrógenos/genética , Receptores de Estrógenos/fisiología , Empalme Alternativo , Animales , Aorta/metabolismo , Western Blotting , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Receptor alfa de Estrógeno , Exones , Femenino , Hipertrofia , Inmunohistoquímica , Ratones , Ratones Noqueados , Modelos Genéticos , Mutagénesis Insercional , Tamaño de los Órganos , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Útero/efectos de los fármacos , Útero/metabolismo , Útero/patologíaAsunto(s)
Células Eucariotas/metabolismo , Biosíntesis de Proteínas/fisiología , Animales , Artefactos , Sitios de Unión/fisiología , Genes , Vectores Genéticos , Humanos , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , ARN Viral/metabolismo , Reproducibilidad de los Resultados , Proyectos de Investigación , Ribosomas/metabolismoRESUMEN
The reactions of CpZr(CH(3))(3), 1, and Cp(2)Zr(CH(3))(2), 2, with partially dehydroxylated silica, silica-alumina, and alumina surfaces have been carried out with careful identification of the resulting surface organometallic complexes in order to probe the relationship between catalyst structure and polymerization activity. The characterization of the supported complexes has been achieved in most cases by in situ infrared spectroscopy, surface microanalysis, qualitative and quantitative analysis of evolved gases during surface reactions with labeled surface, solid state (1)H and (13)C NMR using (13)C-enriched compounds, and EXAFS. 1 and 2 react with silica(500) and silica-alumina(500) by simple protonolysis of one Zr-Me bond by surface silanols with formation of a single well-defined neutral compound. In the case of silica-alumina, a fraction of the supported complexes exhibits some interactions with electronically unsaturated surface aluminum sites. 1 and 2 also react with the hydroxyl groups of gamma-alumina(500), leading to several surface structures. Correlation between EXAFS and (13)C NMR data suggests, in short, two main surface structures having different environments for the methyl group: [Al](3)-OZrCp(CH(3))(2) and [Al](2)-OZrCp(CH(3))(mu-CH(3))-[Al] for the monoCp series and [Al](2)-OZrCp(2)(CH(3)) and [Al]-OZrCp(2)(mu-CH(3))-[Al] for the bisCp series. Ethylene polymerization has been carried out with all the supported complexes under various reaction conditions. Silica-supported catalysts in the absence of any cocatalyst exhibited no activity whatsoever for ethylene polymerization. When the oxide contained Lewis acidic sites, the resulting surface species were active. The activity, although improved by the presence of additional cocatalysts, remained very low by comparison with that of the homogeneous metallocene systems. This trend has been interpreted on the basis of various possible parameters, including the (p-pi)-(d-pi) back-donation of surface oxygen atoms to the zirconium center.
RESUMEN
Interleukin-6 (IL-6) gene expressed in bone marrow-derived stromal cells and osteoblasts contributes to the state of mineralization and its control by estradiol may be involved in the development of post-menopausal osteoporosis. Since IL-6 is also expressed in the different cell populations of the arterial wall, the purpose of this study was to gain more insight into its involvement in the atherosclerotic process and the atheroprotective effect of estradiol by studying double deficient mice at the apolipoprotein E and IL-6 loci (IL-6(-/-)/E(-/-)). At 1 year of age, IL-6(-/-)/E(-/-) mice showed similar hypercholesterolemia to IL-6(+/+)/E(-/-) mice but presented significantly larger and more calcified lesions. In younger mice (sixteen weeks of age), no significant difference in fatty streaks could be detected in IL-6(+/+)/E(-/-), IL-6(+/-)/E(-/-) and IL-6(-/-)/E(-/-) mice on a normal chow diet. Estrogen supplementation at this age induced a decrease of fatty streak formation in all three genotypes. The combined data indicate that IL-6 expression is involved at the fibrous plaque stage of the atherosclerotic process but does not constitute a direct target for estradiol to prevent fatty streak formation.
Asunto(s)
Apolipoproteínas E/deficiencia , Arteriosclerosis/patología , Estradiol/metabolismo , Interleucina-6/deficiencia , Interleucina-6/metabolismo , Seno Aórtico/patología , Análisis de Varianza , Animales , Arteriosclerosis/tratamiento farmacológico , Técnicas de Cultivo , Modelos Animales de Enfermedad , Estradiol/uso terapéutico , Femenino , Interleucina-6/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fotomicrografía , Probabilidad , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Enfermedades de la Aorta/fisiopatología , Arteriosclerosis/fisiopatología , Membrana Celular/fisiología , Endotelio Vascular/fisiopatología , beta-Ciclodextrinas , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Aorta Torácica/efectos de los fármacos , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Técnicas de Cultivo de Célula , Membrana Celular/ultraestructura , Ciclodextrinas/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/ultraestructura , Filipina/farmacología , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Masculino , Microscopía Electrónica , Contracción Muscular/efectos de los fármacos , Óxido Nítrico/biosíntesis , ConejosRESUMEN
Angiotensin-converting enzyme (ACE) is mainly responsible for converting angiotensin I (AI) to angiotensin II (AII), and ACE inhibitors prevent atherosclerosis in animal models. Neutral endopeptidase 24.11 (NEP) degrades substance P, kinins and atrial natriuretic peptide (ANP), and aortic wall NEP activity was found to be increased in atherosclerosis. In the present study, we have evaluated the effect of candoxatril, a NEP inhibitor, and of omapatrilat, a dual ACE and NEP inhibitor, on the development of fatty streak in apolipoprotein E (apoE)-deficient mice. Groups of ten male apoE-deficient mice were given either placebo, candoxatril 50 mg/kg per day, or omapatrilat 10, or 100 mg/kg per day for 4 months. None of the treatments influenced body weight, serum total or HDL-cholesterol. Compared with the placebo, candoxatril did not protect the mice from fatty streak deposit. In contrast, omapatrilat dose dependently inhibited the constitution of fatty streak in apoE-deficient mice. The precise advantages of the dual ACE and NEP inhibition versus the inhibition of only ACE should now be considered in the prevention of atherosclerosis as well as in the occurrence of its complications.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Apolipoproteínas E/deficiencia , Arteriosclerosis/prevención & control , Neprilisina/antagonistas & inhibidores , Inhibidores de Proteasas/uso terapéutico , Piridinas/uso terapéutico , Tiazepinas/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/fisiología , Arteriosclerosis/enzimología , Factor Natriurético Atrial/metabolismo , Peso Corporal/efectos de los fármacos , Bradiquinina/metabolismo , Colesterol/sangre , HDL-Colesterol/sangre , Evaluación Preclínica de Medicamentos , Indanos/farmacología , Indanos/uso terapéutico , Masculino , Ratones , Ratones Noqueados , Propionatos/farmacología , Propionatos/uso terapéutico , Inhibidores de Proteasas/farmacología , Piridinas/farmacología , Sustancia P/metabolismo , Tiazepinas/farmacología , Triglicéridos/sangreRESUMEN
BACKGROUND: The atheroprotective effect of 17beta-estradiol (E(2)) has been suggested in women and clearly demonstrated in animals through both an effect on lipid metabolism and a direct effect on the cells of the arterial wall. It has been shown, for example, that E(2) promotes endothelium-dependent relaxation and accelerates reendothelialization in rats. Similar studies have been undertaken in mice to appreciate the molecular mechanism of this process. METHODS AND RESULTS: We report here a model of electric carotid injury adapted from that described by Carmeliet et al (1997) that allows us to precisely evaluate the reendothelialization process. We demonstrate that E(2) accelerates endothelial regeneration in castrated female wild-type mice. In ovariectomized transgenic mice in which either the estrogen receptor (ER)-alpha or ERbeta gene has been disrupted, E(2) accelerated reendothelialization in female ERbeta knockout mice, whereas this effect was abolished in female ERalpha knockout mice. CONCLUSIONS: This study demonstrates that ERalpha but not ERbeta mediates the beneficial effect of E(2) on reendothelialization and potentially the prevention of atherosclerosis.