RESUMEN
Pregnancy represents an immunological challenge for the maternal immune system. Pregnancy augments innate immune responses, and particularly monocytes contribute to maintaining the balance between pro- and anti-inflammatory immune responses required for the successful sequence of distinct immunological phases throughout pregnancy. Nonetheless, studies that focus on the heterogeneity of monocytes and analyze the alteration of monocyte subsets in a longitudinal approach throughout healthy pregnancies have remained scarce. In this study, we characterized the gradual phenotypic changes of monocyte subsets and the secretory potential of bulk monocytes in peripheral blood mononuclear cells of healthy pregnant women from a population-based prospective birth cohort study. Blood samples at predefined time points were analyzed using flow cytometry for in-depth characterization of monocyte subsets, which confirmed a shift from classical towards intermediate monocytes throughout pregnancy. Principal component analysis revealed characteristic phenotypic changes on monocyte subsets, especially on the intermediate monocyte subset, throughout pregnancy. Pregnancy-related hormones were measured in serum and ß-human chorionic gonadotropin levels were significantly associated with expression of CD11b, CD116 and CCR2 on monocyte subsets. TLR4 and TLR7/8 stimulation of monocytes furthermore showed reduced polycytokine production towards the end of pregnancy. These data provide a comprehensive overview of phenotypic changes and secretory potential of monocytes in healthy pregnant women and establish a selective contribution of different monocyte subsets to healthy pregnancy. The results from this study therefore build a basis for future comparisons and evaluation of women with adverse pregnancy outcomes.
Asunto(s)
Inmunidad Innata , Monocitos/inmunología , Embarazo/sangre , Adulto , Antígeno CD11b/metabolismo , Gonadotropina Coriónica Humana de Subunidad beta/sangre , Femenino , Humanos , Monocitos/metabolismo , Embarazo/inmunología , Trimestres del Embarazo/sangre , Trimestres del Embarazo/inmunología , Estudios Prospectivos , Receptores CCR2/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Adulto JovenRESUMEN
BACKGROUND: This pilot study investigated the effect of sufentanil titration by 3 different analgesia monitoring devices or clinical signs during general anesthesia. METHODS: Forty-eight patients undergoing radical retropubic prostatectomy with sevoflurane/sufentanil anesthesia were randomly assigned into 4 groups and received sufentanil guided either by 1 of 3 analgesia monitoring devices (Surgical Pleth Index [SPI], Pupillary Pain Index [PPI], Nociception Level [NoL]) or by clinical judgment (control). The primary end point was intraoperative sufentanil consumption. Adrenocorticotropic hormone (ACTH) and cortisol were measured at 4 time points during the day of surgery. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests and by mixed model and area under the curve (AUC) analyses for group comparisons and time effects of stress hormones. RESULTS: The total amount of sufentanil administration (µg·kg·minute·10) differed between the groups (median [quartiles]: control = 5.6 [4.4-6.4], SPI = 7.2 [4.8-8.4], PPI = 2.0 [1.8-2.9], NoL = 3.8 [3.3-5.1]; PPI versus SPI, -5.1 [-6.6 to -1.3], P < .001; NoL versus SPI, -3.0 [-5.2 to 0.2], P = .024; control versus SPI, -1.6 [-3.7 to 1.7], P = .128; NoL versus PPI, 1.7 [0.6-3.4], P < .001; control versus PPI, 3.4 [2.0-4.6], P < .001; control versus NoL, 1.6 [-0.2 to 3.3], P = .017) (Hodges-Lehmann estimator [99% confidence interval {CI}], P values). The AUC analysis indicated differences among groups in cumulative ACTH levels (ng·liter·minute, natural logarithm (ln)-transformed data) of NoL versus PPI (-1.079 [-1.950 to -0.208], P = .001) and PPI versus SPI (1.192 [0.317-2.068], P= .001), as well as differences in cortisol levels (µg·liter·minute) for PPI versus SPI (46,710 [21,145-72,274], P < .001), NoL versus SPI (27,645 [3163-52,126], P = .003), and control versus SPI (31,824 [6974-56,675], P = .001) (differences in means [99% CI], P value). Secondary end points (postoperative recovery, pain level, and analgesia medication) showed no differences. CONCLUSIONS: The type of analgesia nociception monitoring affected the total amount of sufentanil administered. Lower sufentanil doses in the PPI group were associated with an increased endocrine stress response. Titration by SPI caused no opioid reduction compared to the control but was associated with a reduced endocrine stress response.
Asunto(s)
Hormona Adrenocorticotrópica/sangre , Analgésicos Opioides/administración & dosificación , Anestesia General/métodos , Monitoreo Intraoperatorio/métodos , Dimensión del Dolor/métodos , Sufentanilo/administración & dosificación , Anciano , Hormonas/sangre , Humanos , Masculino , Persona de Mediana Edad , Dolor Postoperatorio , Proyectos Piloto , Estudios Prospectivos , Prostatectomía/efectos adversos , Prostatectomía/tendencias , Método Simple Ciego , Estrés Psicológico/sangre , Estrés Psicológico/diagnósticoRESUMEN
Mouse models are important and versatile tools to study mechanisms and novel therapies of human disease in vivo. Both, the number and the complexity of murine models are constantly increasing and modification of genes of interest as well as any exogenous challenge may lead to unanticipated biological effects. Laboratory diagnostics of blood samples provide a comprehensive and rapid screening for multiple organ function and are fundamental to detect human disease. Here, we adapt an array of laboratory medicine-based tests commonly used in humans to establish a platform for standardized, multi-parametric, and quality-controlled diagnostics of murine blood samples. We determined sex-dependent reference intervals of 51 commonly used laboratory medicine tests for samples obtained from the C57BL/6J mouse strain. As a proof of principle, we applied these diagnostic tests in a mouse cytomegalovirus (MCMV) infection model to screen for organ damage. Consistent with histopathological findings, plasma concentrations of liver-specific enzymes were elevated, supporting the diagnosis of a virus-induced hepatitis. Plasma activities of aminotransferases correlated with viral loads in livers at various days after MCMV infection and discriminated infected from non-infected animals. This study provides murine blood reference intervals of common laboratory medicine parameters and illustrates the use of these tests for diagnosis of infectious disease in experimental animals.
Asunto(s)
Análisis Químico de la Sangre/métodos , ADN Viral/sangre , Pruebas Diagnósticas de Rutina/métodos , Hepatitis Viral Animal/diagnóstico , Infecciones por Herpesviridae/veterinaria , Muromegalovirus/aislamiento & purificación , Enfermedades de los Roedores/diagnóstico , Animales , Hepatitis Viral Animal/virología , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/virología , Pruebas de Función Hepática , Ratones Endogámicos C57BL , Enfermedades de los Roedores/virología , Transaminasas/sangreRESUMEN
During pregnancy the maternal immune system has to develop tolerance towards the developing fetus. These changes in maternal immunity can result in increased severity of certain infections, but also in amelioration of autoimmune diseases. Pregnancy-related hormones have been suggested to play a central role in the adaptation of the maternal immune system, but their specific effects on innate immune function is not well understood. In a longitudinal study of pregnant women, we investigated innate immune cell function in response to toll-like receptors (TLR) 4 and 7 stimulation, two TLR pathways playing a critical role in early innate immune recognition of bacteria and viruses. IFNα production by TLR7-stimulated pDCs was decreased in early pregnancy, and increased towards the end of pregnancy. In contrast, pro-inflammatory TLR4-induced TNFα production by monocytes was increased during early pregnancy, but declined after the first trimester. Changes in cytokine production were associated with changes in pregnancy-related hormones and monocyte subpopulations over the course of pregnancy. These data demonstrating a significant association between pregnancy-related hormones and modulation of innate immune responses mediated by TLRs provide novel insights into the immunological adaptations occurring during pregnancy.
Asunto(s)
Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Monocitos/inmunología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 7/metabolismo , Gonadotropina Coriónica/sangre , Femenino , Humanos , Inmunidad Innata/inmunología , Estudios Longitudinales , Masculino , Embarazo , Estudios Prospectivos , Factor de Necrosis Tumoral alfa/sangreRESUMEN
OBJECTIVES: Nitric oxide synthases (NOSs) mediate the first window of anesthetic-induced preconditioning (APC). The authors tested the hypothesis that endothelial NOS (eNOS) mediates the first window and inducible NOS (iNOS) mediates the second window of APC. DESIGN: Randomized, prospective, blinded laboratory investigation. SETTING: Experimental laboratory. PARTICIPANTS: Mice. INTERVENTIONS: Mice were subjected to a 45-minute coronary artery occlusion (CAO) and a 180-minute reperfusion. C57BL/6 mice received desflurane, 1.0 minimum alveolar concentration, for 30 minutes or 12, 24, 48, or 96 hours before CAO. In eNOS(-/-) and iNOS(-/-) mice, desflurane was given 30 minutes and 48 hours before CAO. In the control groups, no desflurane was administered. Myocardial infarct size (IS) was determined after staining with Evans blue and triphenyltetrazolium chloride. MEASUREMENTS AND MAIN RESULTS: The second window of APC was detectable at 48 hours but not at 12, 24, and 96 hours after preconditioning. In the control groups, IS was not different among the wild-type (50 ± 10%), eNOS(-/-) (52 ± 14%), and iNOS(-/-) (46 ± 10%) mice. The IS decreased significantly (p < 0.05) when desflurane was administered 30 minutes (10 ± 6%) or 48 hours (16 ± 7%) before CAO in wild-type mice, 48 hours (21 ± 13%) before CAO in eNOS(-/-) mice, and 30 minutes (13 ± 6%) before CAO in iNOS(-/-) mice. Desflurane given 30 minutes before CAO in eNOS(-/-) mice (60 ± 10%) and 48 hours before CAO in iNOS(-/-) mice (48 ± 21%) did not decrease the IS significantly compared with controls. CONCLUSIONS: Endothelial NOS and iNOS work independently to mediate the first and second windows of APC, respectively. Endothelial NOS is not necessary to trigger the second window of APC.
Asunto(s)
Anestésicos por Inhalación/uso terapéutico , Precondicionamiento Isquémico Miocárdico/métodos , Isoflurano/análogos & derivados , Óxido Nítrico Sintasa de Tipo III/fisiología , Óxido Nítrico Sintasa de Tipo I/fisiología , Animales , Presión Arterial/fisiología , Peso Corporal/fisiología , Vasos Coronarios/fisiología , Desflurano , Frecuencia Cardíaca/fisiología , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Isoflurano/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo III/genéticaRESUMEN
Fabry disease is an X-chromosomal recessive deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal). This results in an accumulation of globotriaosylceramide (GL-3) in a variety of cells often with subsequent functional impairment. Here, the impact of Fabry disease on the biology of circulating angiogenic cells (CACs) and the endothelial response to transient ischemia was investigated. Untreated patients with Fabry disease (n = 26), patients after initiation of alpha-Gal enzyme replacement therapy (ERT) (n = 16) and healthy controls (n = 26) were investigated. Endothelial function was assessed by the EndoPAT2000 device. CAC numbers were assessed by flow-cytometry, CAC function by a modified Boyden chamber assay. Fabry patients showed a pathologic endothelial response, which normalized after ERT. CACs were increased in number, but functionally impaired. Immunofluorescence and electron microscopy identified an accumulation of GL-3 in Fabry CACs. ERT attenuated CAC dysfunction and improved markers of oxidative stress response in Fabry patients via a reduction in GL-3 accumulation in vitro and in vivo. Silencing of alpha-Gal in healthy CACs impaired their migratory capacity underlining a key role of this enzyme for CAC function. CAC supernatant as well as CACs from Fabry patients impaired angiogenesis and migratory capacity of HUVECs providing a mechanistic link between CAC and endothelial dysfunction. CAC adhesion to TNF-α pre-stimulated HUVECs and tube formation was impaired by alpha-Gal knockdown. Fabry patients show a dysfunction of CAC and a pathologic endothelial response. ERT improves CAC and endothelial function and thus may attenuate development of cardiovascular disease in the long term in this patient population.
Asunto(s)
Endotelio Vascular/fisiología , Enfermedad de Fabry/fisiopatología , Células Madre Hematopoyéticas/fisiología , Neovascularización Fisiológica , Adolescente , Adulto , Anciano , Células Cultivadas , Terapia de Reemplazo Enzimático , Enfermedad de Fabry/terapia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/genética , Trihexosilceramidas/metabolismo , alfa-Galactosidasa/fisiologíaRESUMEN
MicroRNAs (miRs) are small non- coding RNA molecules controlling a plethora of biological processes such as development, cellular survival and senescence. We here determined miRs differentially regulated during cardiac postnatal development and aging. Cardiac function, morphology and miR expression profiles were determined in neonatal, 4 weeks, 6 months and 19 months old normotensive male healthy C57/Bl6N mice. MiR-22 was most prominently upregulated during cardiac aging. Cardiac expression of its bioinformatically predicted target mimecan (osteoglycin, OGN) was gradually decreased with advanced age. Luciferase reporter assays validated mimecan as a bona fide miR-22 target. Both, miR-22 and its target mimecan were co- expressed in cardiac fibroblasts and smooth muscle cells. Functionally, miR-22 overexpression induced cellular senescence and promoted migratory activity of cardiac fibroblasts. Small interference RNA-mediated silencing of mimecan in cardiac fibroblasts mimicked the miR-22-mediated effects. Rescue experiments revealed that the effects of miR-22 on cardiac fibroblasts were only partially mediated by mimecan. In conclusion, miR-22 upregulation in the aging heart contributed at least partly to accelerated cardiac fibroblast senescence and increased migratory activity. Our results suggest an involvement of miR-22 in age-associated cardiac changes, such as cardiac fibrosis.
Asunto(s)
Envejecimiento/genética , Fibroblastos/patología , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , ARN/genética , Regulación hacia Arriba/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Animales Recién Nacidos , Fibroblastos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/biosíntesis , Reacción en Cadena de la PolimerasaRESUMEN
The volatile anesthetic desflurane (DES) effectively reduces cardiac infarct size following experimental ischemia/reperfusion injury in the mouse heart. We hypothesized that endogenous estrogens play a role as mediators of desflurane-induced preconditioning against myocardial infarction. In this study, we tested the hypothesis that desflurane effects local estrogen synthesis by modulating enzyme aromatase expression and activity in the mouse heart. Aromatase metabolizes testosterone to 17ß- estradiol (E2) and thereby significantly contributes to local estrogen synthesis. We tested aromatase effects in acute myocardial infarction model in male mice. The animals were randomized and subjected to four groups which were pre-treated with the selective aromatase inhibitor anastrozole (A group) and DES alone (DES group) or in combination (A+DES group) for 15 minutes prior to surgical intervention whereas the control group received 0.9% NaCl (CON group). All animals were subjected to 45 minutes ischemia following 180 minutes reperfusion. Anastrozole blocked DES induced preconditioning and increased infarct size compared to DES alone (37.94 ± 15.5% vs. 17.1 ± 3.62%) without affecting area at risk and systemic hemodynamic parameters following ischemia/reperfusion. Protein localization studies revealed that aromatase was abundant in the murine cardiovascular system with the highest expression levels in endothelial and smooth muscle cells. Desflurane application at pharmacological concentrations efficiently upregulated aromatase expression in vivo and in vitro. We conclude that desflurane efficiently regulates aromatase expression and activity which might lead to increased local estrogen synthesis and thus preserve cellular integrity and reduce cardiac damage in an acute myocardial infarction model.
Asunto(s)
Inhibidores de la Aromatasa/efectos adversos , Aromatasa/metabolismo , Precondicionamiento Isquémico Miocárdico , Infarto del Miocardio/metabolismo , Anestésicos por Inhalación/farmacología , Animales , Aromatasa/genética , Inhibidores de la Aromatasa/farmacología , Desflurano , Regulación de la Expresión Génica/efectos de los fármacos , Hemodinámica , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Isoflurano/análogos & derivados , Isoflurano/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Transporte de Proteínas , RatasRESUMEN
OBJECTIVE: MicroRNAs are a class of small ribonucleotides regulating gene/protein targets by transcript degradation or translational inhibition. Transforming growth factor-ß (TGF-ß) is involved in cardiac fibrosis partly by stimulation of endothelial-to-mesenchymal transition (EndMT). Here, we investigated whether microRNA (miR)-21, a microRNA enriched in fibroblasts and involved in general fibrosis, has a role in cardiac EndMT. METHODS AND RESULTS: TGF-ß treatment of endothelial cells significantly increased miR-21 expression and induced EndMT characterized by suppression of endothelial and increase of fibroblast markers. Overexpression of miR-21 alone also stimulated EndMT. Importantly, miR-21 blockade by transfection of specific microRNA inhibitors partly prevented TGF-ß-induced EndMT. Mechanistically, miR-21 silenced phosphatase and tensin homolog in endothelial cells, resulting in activation of the Akt-pathway. Akt inhibition partly restored TGF-ß-mediated loss of endothelial markers during EndMT. In vivo, pressure overload of the left ventricle led to increased expression of miR-21 in sorted cardiac endothelial cells, which displayed molecular and phenotypic signs of EndMT. This was attenuated by treatment of mice subjected to left ventricular pressure overload with an antagomir against miR-21. CONCLUSIONS: TGF-ß-mediated EndMT is regulated at least in part by miR-21 via the phosphatase and tensin homolog/Akt pathway. In vivo, antifibrotic effects of miR-21 antagonism are partly mediated by blocking EndMT under stress conditions.
Asunto(s)
Transdiferenciación Celular/efectos de los fármacos , Endotelio Vascular/citología , Mesodermo/citología , MicroARNs/fisiología , Factor de Crecimiento Transformador beta/farmacología , Transdiferenciación Celular/fisiología , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Humanos , Mesodermo/efectos de los fármacos , Mesodermo/fisiología , Proteínas de Microfilamentos/fisiología , Fosfohidrolasa PTEN/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tensinas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. METHODS AND RESULTS: Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. CONCLUSIONS: Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.
Asunto(s)
Células Endoteliales/metabolismo , MicroARNs/fisiología , Infarto del Miocardio/fisiopatología , Animales , Apoptosis/efectos de los fármacos , Arteriolas/patología , Capilares/patología , Hipoxia de la Célula , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Colágeno , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Células Endoteliales/patología , Factor de Transcripción GATA2/biosíntesis , Factor de Transcripción GATA2/genética , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/etiología , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Laminina , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Infarto del Miocardio/complicaciones , Infarto del Miocardio/genética , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Oligorribonucleótidos/farmacología , Proteoglicanos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Esferoides Celulares , Remodelación Ventricular , Pez Cebra/embriología , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genética , Quinasas p21 Activadas/biosíntesis , Quinasas p21 Activadas/genéticaRESUMEN
AIMS: Hyperaldosteronism is associated with vascular injury and increased cardiovascular events. Bone marrow-derived endothelial progenitor cells (EPCs) play an important role in endothelial repair and vascular homeostasis. We hypothesized that hyperaldosteronism impairs EPC function and vascularization capacity in mice and humans. METHODS AND RESULTS: We characterized the effects of aldosterone and mineralocorticoid receptor (MR) blockade on EPC number and function as well as vascularization capacity and endothelial function. Treatment of human EPC with aldosterone induced translocation of the MR and impaired multiple cellular functions of EPC, such as differentiation, migration, and proliferation in vitro. Impaired EPC function was rescued by pharmacological blockade or genetic ablation of the MR. Aldosterone protein kinase A (PKA) dependently increased reactive oxygen species formation in EPC. Aldosterone infusion in mice impaired EPC function, EPC homing to vascular structures and vascularization capacity in a MR-dependent but blood pressure-independent manner. Endothelial progenitor cells from patients with primary hyperaldosteronism compared with controls of similar age displayed reduced migratory potential. Impaired EPC function was associated with endothelial dysfunction. MR blockade in patients with hyperaldosteronism improved EPC function and arterial stiffness. CONCLUSION: Endothelial progenitor cells express a MR that mediates functional impairment by PKA-dependent increase of reactive oxygen species. Normalization of EPC function may represent a novel mechanism contributing to the beneficial effects of MR blockade in cardiovascular disease prevention and treatment.
Asunto(s)
Aldosterona/fisiología , Células Endoteliales/fisiología , Hiperaldosteronismo/patología , Células Madre/fisiología , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endotelio Vascular/citología , Eplerenona , Femenino , Humanos , Hiperaldosteronismo/fisiopatología , Masculino , Ratones , Persona de Mediana Edad , Antagonistas de Receptores de Mineralocorticoides/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Mineralocorticoides/metabolismo , Espironolactona/análogos & derivados , Espironolactona/farmacología , VasodilataciónRESUMEN
RATIONALE: The endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) is increased in patients with coronary artery disease and may regulate function of circulating angiogenic progenitor cells (APCs) by small regulatory RNAs. OBJECTIVES: To study the role of microRNAs in ADMA-mediated impairment of APCs. METHODS AND RESULTS: By using microarray analyses, we established microRNA expression profiles of human APCs. We used ADMA to induce APC dysfunction and found 16 deregulated microRNAs. We focused on miR-21, which was 3-fold upregulated by ADMA treatment. Overexpression of miR-21 in human APCs impaired migratory capacity. To identify regulated miR-21 targets, we used proteome analysis, using difference in-gel electrophoresis followed by mass spectrometric analysis of regulated proteins. We found that transfection of miR-21 precursors significantly repressed superoxide dismutase 2 in APCs, which resulted in increased intracellular reactive oxygen species concentration and impaired nitric oxide bioavailability. MiR-21 further repressed sprouty-2, leading to Erk Map kinase-dependent reactive oxygen species formation and APC migratory defects. Small interference RNA-mediated superoxide dismutase 2 or sprouty-2 reduction also increased reactive oxygen species formation and impaired APC migratory capacity. ADMA-mediated reactive oxygen species formation and APC dysfunction was rescued by miR-21 blockade. APCs from patients with coronary artery disease and high ADMA plasma levels displayed >4-fold elevated miR-21 levels, low superoxide dismutase 2 expression, and impaired migratory capacity, which could be normalized by miR-21 antagonism. CONCLUSIONS: We identified a novel miR-21-dependent mechanism of ADMA-mediated APC dysfunction. MiR-21 antagonism therefore emerges as an interesting strategy to improve dysfunctional APCs in patients with coronary artery disease.
Asunto(s)
Inhibidores de la Angiogénesis/fisiología , Arginina/análogos & derivados , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , MicroARNs/fisiología , Neovascularización Patológica/patología , Neovascularización Patológica/prevención & control , Células Madre/metabolismo , Arginina/fisiología , Enfermedad de la Arteria Coronaria/genética , Regulación de la Expresión Génica/fisiología , Humanos , MicroARNs/biosíntesis , MicroARNs/genética , Neovascularización Patológica/genética , Células Madre/citología , Células Madre/patologíaRESUMEN
MicroRNA-21 (miR-21) expression is activated in multiple types of cancers, such as breast, liver, brain, prostate, myometrial cancers but also in cardiovascular diseases. MiR-21 regulates a plethora of target proteins which are involved in cellular survival, apoptosis and cell invasiveness. MiR-21 regulation is complex due to an own promoter that is target for various transcription factors and hormones. The consistent miR-21 overexpression under pathophysiological conditions points to miR-21 as a valuable tool for new therapeutic strategies. In this review, we present and analyze current data about miR-21 expression in various pathologies ranging from cancer to cardiovascular disease. Further, miR-21 regulatory mechanisms and miR-21 downstream targets are discussed. Finally, we highlight the particular role of miR-21 as a therapeutic target in various diseases.
Asunto(s)
Enfermedades Cardiovasculares/fisiopatología , MicroARNs/metabolismo , Neoplasias/fisiopatología , Animales , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/terapia , Supervivencia Celular , Sistemas de Liberación de Medicamentos , Regulación de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/terapiaRESUMEN
Estrogen receptor alpha (ERalpha) is present in the nucleus, the cytosol and in mitochondria. The rat ERalpha ligand binding domain was employed as bait in a bacterial two-hybrid screening of a human heart cDNA library to detect novel protein-protein interaction partners of ERalpha in the heart. 17beta-Hydroxysteroid dehydrogenase type 10 (17beta-HSD10), which converts potent (17beta-estradiol) to less potent estrogens (estrone), co-localized with 17beta-HSD10 in the mitochondria of rat cardiac myocytes. GST pull-down experiments confirmed the interaction of ERalpha and 17beta-HSD10. These findings suggest that the ERalpha estrogen receptor might be involved in regulating intracellular estrogen levels by modulating 17beta-HSD10 activity.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Receptor alfa de Estrógeno/metabolismo , Mitocondrias Cardíacas/enzimología , Miocitos Cardíacos/enzimología , 3-Hidroxiacil-CoA Deshidrogenasas/genética , Animales , Núcleo Celular/enzimología , Receptor alfa de Estrógeno/genética , Biblioteca de Genes , Humanos , Inmunoprecipitación , Miocitos Cardíacos/ultraestructura , Ratas , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Anesthetic preconditioning is mediated by beta- adrenergic signaling. This study tested the hypotheses that desflurane-induced preconditioning is dose-dependently blocked by metoprolol and mediated by calcium/calmodulin-dependent protein kinase II (CaMK II). METHODS: Pentobarbital-anesthetized New Zealand White rabbits were instrumented for measurement of systemic hemodynamics and subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. Rabbits were assigned to receive vehicle (control), 0.2, 1.0, 1.75, or 2.5 mg/kg metoprolol for 30 min, or the CaMK II inhibitor KN-93 in the absence or presence of 1.0 minimum alveolar concentration desflurane. Protein expression of CaMK II, phospholamban, and phospho-phospholamban was measured by Western blotting. Myocardial infarct size and area at risk were measured with triphenyltetrazolium staining and patent blue, respectively. RESULTS: Baseline hemodynamics were not different among groups. Infarct size was 60 +/- 3% in control and significantly (* P < 0.05) decreased to 33 +/- 2%* by desflurane. The CaMK II inhibitor KN-93 did not affect infarct size (55 +/- 4%) but blocked desflurane-induced preconditioning (57 +/- 3%). Metoprolol at 0.2 and 1.0 mg/kg had no effect on infarct size (55 +/- 3% and 53 +/-3%), whereas metoprolol at 1.75 and 2.5 mg/kg reduced infarct size to 48 +/- 4%* and 39 +/- 5%*, respectively. Desflurane-induced preconditioning was attenuated by metoprolol at 0.2 mg/kg, leading to an infarct size of 46 +/- 5%*, and was completely abolished by metoprolol at 1.0, 1.75, and 2.5 mg/kg, resulting in infarct sizes of 51 +/- 3%, 52 +/- 3%, and 55 +/- 3%, respectively. CONCLUSIONS: Desflurane-induced preconditioning is dose-dependently blocked by metoprolol and mediated by CaMK II.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Cardiotónicos/farmacología , Precondicionamiento Isquémico Miocárdico/métodos , Isoflurano/análogos & derivados , Metoprolol/farmacología , Infarto del Miocardio/prevención & control , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Cardiotónicos/uso terapéutico , Desflurano , Isoflurano/antagonistas & inhibidores , Isoflurano/farmacología , Isoflurano/uso terapéutico , Masculino , Metoprolol/uso terapéutico , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Fosforilación/efectos de los fármacos , ConejosRESUMEN
BACKGROUND: The biological functions of estrogens extend beyond the female and male reproductive tract, affecting the cardiovascular and renal systems. Traditional views on the role of postmenopausal hormone therapy (HT) in protecting against heart disease, which were challenged by clinical end point studies that found adverse effects of combined HT, are now being replaced by more differentiated concepts suggesting a beneficial role of early and unopposed HT that does not include a progestin. OBJECTIVE: We reviewed recent insights, concepts, and research results on the biology of both estrogen receptor (ER) subtypes, ERalpha and ERbeta, in cardiac and vascular tissues. Knowledge of these ER subtypes is crucial to understanding gender and estrogen effects and to developing novel, exciting strategies that may have a profound clinical impact. METHODS: This review focuses on in vivo studies and includes data presented at the August 2007 meeting of the American Physiological Society as well as data from a search of the MEDLINE and Ovid databases from January 1986 to November 2007. Search results were restricted to English-language publications, using the following search terms: estrogen, estrogen receptor alpha, estrogen receptor beta, estrogen receptor alpha agonist, estrogen receptor alpha antagonist, estrogen receptor beta agonist, estrogen receptor beta antagonist, PPT, DPN, heart, vasculature, ERKO mice, BERKO mice, transgenic mice, and knockout mice. RESULTS: Genetic mouse models and pharmacologic studies that employed selective as well as nonselective ER agonists support the concept that both ER subtypes confer protective effects in experimental models of human heart disease, including hypertension, cardiac hypertrophy, and chronic heart failure. CONCLUSIONS: Genetic models and novel ligands hold the promise of further improving our understanding of estrogen action in multiple tissues and organs. These efforts will ultimately enhance the safety and efficacy of HT and may also result in new applications for synthetic female sex hormone analogues.
Asunto(s)
Fenómenos Fisiológicos Cardiovasculares , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/fisiología , Estrógenos/fisiología , Animales , Insuficiencia Cardíaca/fisiopatología , Hipertensión/fisiopatología , Ratones , Ratones Noqueados , Ratones Transgénicos , Infarto del Miocardio/fisiopatología , Receptores de Estrógenos/agonistasRESUMEN
BACKGROUND: ATP-regulated K+ channels are involved in anesthetic-induced preconditioning (APC). The role of other K+ channels in APC is unclear. We tested the hypothesis that APC is mediated by large-conductance calcium-activated K+ channels (K(Ca)). METHODS: Pentobarbital-anesthetized male C57BL/6 mice were subjected to 45 min of coronary artery occlusion and 3 h reperfusion. Thirty minutes before coronary artery occlusion, 1.0 MAC desflurane was administered for 15 min alone or in combination with the large-conductance K(Ca) channel activator NS1619 (1 microg/g i.p.), its respective vehicle dimethylsulfoxide (10 microL/g i.p.), the large-conductance K(Ca) channel blocker iberiotoxin (0.05 microg/g i.p.), or the protein kinase A (PKA) inhibitor H-89 (0.5 microg/g intraventricular). Infarct size was determined with triphenyltetrazolium chloride and area at risk with Evans blue. Mitochondrial and sarcolemmal localization of large-conductance K(Ca) channels in cardiac myocytes was investigated with immunocytochemical staining of isolated cardiac myocytes. RESULTS: Desflurane significantly reduced infarct size compared with control animals (7.4% +/- 0.8% vs 51.3% +/- 6.1%; P < 0.05). Activation of large-conductance K(Ca) channels by NS1619 (7.5% +/- 1.8%; P < 0.05) mimicked and blockade of large-conductance K(Ca) channels by iberiotoxin (49.1% +/- 7.5%) abrogated desflurane-induced preconditioning. PKA blockade by H-89 abolished desflurane-induced (45.1% +/- 4.0%) but not NS1619-induced (9.0% +/- 2.4%, P < 0.05) preconditioning. Immunocytochemical staining revealed that large-conductance K(Ca) channels were localized in the mitochondria but not in the sarcolemma of cardiac myocytes. CONCLUSION: These data suggest that desflurane-induced APC is mediated in part by activation of mitochondrial large-conductance K(Ca) channels, and that activation of these channels by desflurane is mediated by PKA.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Precondicionamiento Isquémico Miocárdico , Isoflurano/análogos & derivados , Mitocondrias/metabolismo , Canales de Potasio/metabolismo , Animales , Desflurano , Precondicionamiento Isquémico Miocárdico/métodos , Isoflurano/farmacología , Isoflurano/uso terapéutico , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/metabolismoRESUMEN
AIMS: The biological effects of oestrogens are mediated by two different oestrogen receptor (ER) subtypes, ERalpha and ERbeta, which might play different, redundant, or opposing roles in cardiovascular disease. Previously, we have shown that the selective ERalpha agonist 16alpha-LE2 improves vascular relaxation, attenuates cardiac hypertrophy, and increases cardiac output without lowering elevated blood pressure in spontaneously hypertensive rats (SHR). Because ERbeta-deficient mice exhibit elevated blood pressure and since the ERbeta agonist 8beta-VE2 attenuated hypertension in aldosterone-salt-treated rats, we have now tested the hypothesis that the isotype-selective ERbeta agonist 8beta-VE2 might be capable of lowering elevated blood pressure in ovariectomized SHR. METHODS AND RESULTS: Treatment of ovariectomized SHR with 8beta-VE2 for 12 weeks conferred no uterotrophic effects but lowered elevated systolic blood pressure (-38 +/- 5 mmHg, n = 31, P < 0.001 vs. placebo) as well as peripheral vascular resistance (-31.3 +/- 4.6%, P < 0.001 vs. placebo). 8beta-VE2 enhanced aortic ERbeta expression (+75.7 +/- 7.1%, P < 0.01 vs. placebo), improved NO-dependent vasorelaxation, augmented phosphorylation of the vasodilator-stimulated phosphoprotein in isolated aortic rings (P < 0.05 vs. placebo), increased cardiac output (+20.4 +/- 2.5%, P < 0.01 vs. placebo), and attenuated cardiac hypertrophy (-22.2 +/- 3.2%, p < 0.01 vs. placebo). 8beta-VE2, in contrast to oestradiol, did not enhance cardiac alpha-myosin heavy chain expression. CONCLUSION: Ligand-dependent activation of ERbeta confers blood pressure lowering effects in SHR that are superior to those of 17beta-estradiol or the ERalpha agonist 16alpha-LE2 and attenuates cardiac hypertrophy primarily by a reduction of cardiac afterload without promoting uterine growth.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Cardiomegalia/prevención & control , Estradiol/farmacología , Receptor beta de Estrógeno/agonistas , Hipertensión/tratamiento farmacológico , Miocardio/metabolismo , Ovariectomía , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Modelos Animales de Enfermedad , Estradiol/análogos & derivados , Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Hemodinámica/efectos de los fármacos , Hipertensión/complicaciones , Hipertensión/metabolismo , Hipertensión/fisiopatología , Ligandos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Miocardio/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratas , Ratas Endogámicas SHR , Útero/efectos de los fármacos , Útero/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
The murine in vivo model of acute myocardial infarction is increasingly used to study signal transduction pathways. However, methodological details of this model are rarely published, and durations of ischemia and reperfusion (REP) time vary considerably among different laboratories. In this study, we tested the hypothesis that infarct size (IS) is dependent on both duration of ischemia and REP time. Pentobarbital-anesthetized male C57BL/6 mice were intubated, mechanically ventilated, and instrumented for continuous monitoring of mean arterial blood pressure and heart rate. After left fourth thoracotomy, the left anterior descending coronary artery was ligated. Mice were randomly assigned to receive 30, 45, or 60 mins of coronary artery occlusion (CAO) and 120, 180, or 240 mins of REP, respectively. IS was determined with triphenyltetrazolium chloride and area at risk (AAR) with Evans blue, respectively. Arterial blood gas analysis and hemodynamics were not different among groups. Prolongation of CAO from 30 to 60 mins significantly (*P<0.05) increased IS from 18% +/- 5% to 69% +/- 3%*, from 20% +/- 2% to 69% +/- 6%* and from 42% +/- 10% to 75% +/- 2%* after 120, 180, and 240 mins REP, respectively. Moreover, IS was increased from 18% +/- 5% to 42% +/- 10%* (30 mins CAO) and from 40% +/- 3% to 72% +/- 6%* (45 mins CAO) when REP time was prolonged from 120 to 240 mins. IS was not increased when REP was prolonged from 120 to 240 mins at 60 mins CAO (69% +/- 3% vs. 75% +/- 2%). In the present study, we describe important methodological aspects of the murine in vivo model of acute myocardial infarction and provide evidence that, in this model, IS depends both on duration of ischemia and on REP time.
Asunto(s)
Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Animales , Análisis de los Gases de la Sangre , Electrocardiografía , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
Controversial results obtained from human and animal studies on the prevention of heart disease by estrogens and progestins warrant a better understanding of nuclear hormone receptor function and interaction. To address this issue and taking into account that effects of synthetic progestins are not only referable to action through the progesterone receptor but may also be mediated by other steroid receptors, we characterized cardiovascular function and inflammatory gene expression in aldosterone salt-treated rats on long-term administration of 17beta-estradiol, medroxyprogesterone acetate, and drospirenone, a new progestogen exhibiting antimineralocorticoid activity. The complex pattern of cardiovascular injury in ovariectomized Wistar rats induced by chronic aldosterone infusion plus a high-salt diet was significantly attenuated in sham-ovariectomized rats and by coadministration of 17beta-estradiol in ovariectomized animals after 8 weeks of continuous treatment. The beneficial role of 17beta-estradiol on blood pressure, cardiac hypertrophy, vascular osteopontin expression, perivascular fibrosis, and impaired NO-dependent relaxation of isolated aortic rings was completely abrogated by coadministration of medroxyprogesterone acetate. In contrast, drospirenone was either neutral or additive to 17beta-estradiol in protecting against aldosterone salt-induced cardiovascular injury and inflammation. The current results support the hypothesis of complex interactions among estrogen, progesterone, glucocorticoid, androgen, and mineralocorticoid receptor signaling in cardiovascular injury and inflammation. Novel progestins, such as drospirenone, confer superior effects compared with medroxyprogesterone acetate in a model of aldosterone-induced heart disease because of its antimineralocorticoid properties.