RESUMEN
BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). We demonstrated that MBG induces fibrosis via mechanism involving inhibition of Fli1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. We hypothesized that PE blockade of increased MBG with antibody would lessen the fibrosis of umbilical arteries and lower the blood pressure in rats with PE. METHODS: We tested 36 pregnant Sprague-Dawley rats in which 12 were made hypertensive by 1.8% Na supplementation (days 6-19 of gestation), 12 pregnant rats served controls. At day 19, PE rats received one intraperitoneal injection of polyclonal anti-MBG-4 antibody (0.5 ug/ml) for 4 hours. RESULTS: PE was associated with higher blood pressure (117 ± 2 vs. 107 ± 2 mm Hg; P < 0.01), plasma MBG levels (1.54 ± 0.34 vs. 0.49 ± 0.11 nmol/L; P < 0.01), protein excretion (26 vs. 12 mg/24 hours), sFlt-1 (3-fold), decrease in Fli1 (7-fold) and increase in collagen-1 in aorta (4-fold) vs. control rats (all P < 0.01). In 12 rats treated with polyclonal anti-MBG-4 antibody blood pressure dropped (93 ± 3 mm Hg) and Fli1 was decreased much less (2-fold; P < 0.01 vs. nontreated rats). CONCLUSIONS: These results demonstrate that in experimental PE elevated MBG level is implicated in umbilical fibrosis via suppression of Fli1.
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Anticuerpos/farmacología , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Bufanólidos/antagonistas & inhibidores , Preeclampsia/prevención & control , Proteína Proto-Oncogénica c-fli-1/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Arterias Umbilicales/efectos de los fármacos , Animales , Bufanólidos/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrosis , Preeclampsia/enzimología , Preeclampsia/patología , Preeclampsia/fisiopatología , Embarazo , Ratas Sprague-Dawley , Cloruro de Sodio Dietético , Arterias Umbilicales/enzimología , Arterias Umbilicales/patología , Arterias Umbilicales/fisiopatología , Regulación hacia ArribaRESUMEN
Ultraviolet (UV) filters are chemicals widely used in personal care products (PCPs). Due to their effect as endocrine disruptor compounds (EDCs), the toxicity of UV filters is a current concern for human health. EDC exposure may be correlated to cardiovascular diseases (CVD), but to our knowledge, no studies assessed the UV filters effects as human EDCs at the vascular level. Octylmethoxycinnamate (OMC) is the world's most widely used UV-B filter, present in more than 90% of PCPs. Due to its demonstrated multiple hormonal activities in animal models, this substance is also suspected to be a human EDC. The purpose of this study was to assess the rapid/short-term effects of OMC on arterial tonus and analyse its mode of action (MOA). Using human umbilical arteries, the endocrine effects of OMC were evaluated in in vitro (cellular and organ) experiments by planar cell surface area (PCSA) and organ bath, respectively. Our data show that OMC induces a rapid/short-term smooth muscle relaxation acting through an endothelium-independent MOA, which seems to be shared with oestrogens, involving an activation of soluble guanylyl cyclase (sGC) that increases the cyclic guanosine monophosphate (cGMP) intracellular levels and an inhibition of L-type voltage-operated Ca2+ channels (L-Type VOCC).
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Bloqueadores de los Canales de Calcio/farmacología , Cinamatos/farmacología , Guanilil Ciclasa Soluble/metabolismo , Rayos Ultravioleta , Arterias Umbilicales/enzimología , Arterias Umbilicales/fisiología , Vasodilatación/efectos de los fármacos , GMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Modelos Biológicos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Vasoconstricción/efectos de los fármacosRESUMEN
OBJECTIVES: Preeclampsia is the most common disorder associated with pregnancy. Our earlier findings revealed a substantial increase in the amount of matrix metalloproteinase-26 (matrilysin 2; MMP-26) in preeclamptic umbilical cord blood. The role of MMP-26 in preeclamptic umbilical cord tissue has not been fully elucidated. Some reports have indicated that the expression of matrilysin 2 and tissue inhibitor of matrix metalloproteinase 4 (TIMP-4) is coordinately regulated during progression of various diseases. STUDY DESIGN: Therefore, we decided to assess the expression and activity of MMP-26 and TIMP-4 in normal and preeclamptic umbilical cord tissues - umbilical cord arteries (UCA), vein (UCV) and Wharton's jelly (WJ). Tissues obtained from 10 normal (control material) and 10 preeclamptic umbilical cords were assessed using immunoenzymatic assay, Western immunoblotting, reverse transcriptase - polymerase chain reaction and fluorometric determination of the enzyme activity. RESULTS: All umbilical cord tissues, both control and preeclamptic, expressed MMP-26 and TIMP-4 in macromolecular complexes. Preeclampsia induced a significant increase in the content and actual activity of MMP-26 in UCV and WJ, as compared to control. The content of TIMP-4 in preeclamptic UCV and WJ was reduced. The content of MMP-26 mRNA was lower in UCA and UCV, whereas higher in WJ in preeclampsia. CONCLUSIONS: Divergent changes in MMP-26 mRNA and protein expression suggest a difference in the factors controlling the matrilysin synthesis in healthy and preeclamptic subjects. The decrease in TIMP-4 content in preeclamptic UCV might be the main reason for significantly higher actual activity of MMP-26 in that tissue. Only in preeclamptic Wharton's jelly the changes were compatible in terms of the content and activity of MMP-26 and TIMP-4. It cannot be excluded that similar alterations can be observed for the whole vascular system of newborns delivered by mothers with preeclampsia.
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Metaloproteinasas de la Matriz Secretadas/análisis , Preeclampsia/enzimología , Inhibidores Tisulares de Metaloproteinasas/análisis , Cordón Umbilical/enzimología , Adulto , Femenino , Edad Gestacional , Humanos , Metaloproteinasas de la Matriz Secretadas/genética , Embarazo , ARN Mensajero/análisis , Arterias Umbilicales/enzimología , Venas Umbilicales/enzimología , Gelatina de Wharton/enzimología , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
The role of primary cilia in mechanosensation is essential in endothelial cell (EC) shear responsiveness. Here, we find that venous, capillary, and progenitor ECs respond to shear stress in vitro in a cilia-dependent manner. We then demonstrate that primary cilia assembly in human induced pluripotent stem cell (hiPSC)-derived ECs varies between different cell lines with marginal influence of differentiation protocol. hiPSC-derived ECs lacking cilia do not align to shear stress, lack stress fiber assembly, have uncoordinated migration during wound closure in vitro, and have aberrant calcium influx upon shear exposure. Transcriptional analysis reveals variation in regulatory genes involved in ciliogenesis among different hiPSC-derived ECs. Moreover, inhibition of histone deacetylase 6 (HDAC6) activity in hiPSC-ECs lacking cilia rescues cilia formation and restores mechanical sensing. Taken together, these results show the importance of primary cilia in hiPSC-EC mechano-responsiveness and its modulation through HDAC6 activity varies among hiPSC-ECs.
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Cilios/enzimología , Células Endoteliales/enzimología , Histona Desacetilasa 6/metabolismo , Células Madre Pluripotentes/enzimología , Calcio/metabolismo , Movimiento Celular/fisiología , Citoesqueleto/enzimología , Células Endoteliales/citología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Mecanotransducción Celular , Técnicas Analíticas Microfluídicas , Células Madre Pluripotentes/citología , Arterias Umbilicales/citología , Arterias Umbilicales/enzimologíaRESUMEN
INTRODUCTION: Serum gamma glutamyl transferase (GGT), produced and released mostly from the liver and bile duct, is an enzyme involved in response to oxidative stress, and has been used as a maker for prediction of cardiovascular events. Umbilical artery blood flow resistance index, e.g., the systolic/diastolic ratio (S/D ratio) as determined by ultrasound, has been used to assess the fetal intrauterine conditions. While changes of GGT and S/D ratio in preeclampsia are found to be associated with the risk for adverse perinatal outcome, the potential value of combined use of the two measurements for the prediction of adverse perinatal outcome has not been determined. MATERIALS AND METHODS: This study included severe preeclampsia patients in late pregnancy and determined their serum GGT levels and ultrasonic flow resistance index of umbilical artery within a week before delivery. Demographic data and perinatal outcomes including perinatal death, five-minute Apgar score, admission to neonatal intensive care unit, respiratory distress syndrome, and intrauterine growth restriction, are documented and analyzed. RESULTS: It was found that serum GGT combined with umbilical artery S/D ratio predicted perinatal adverse outcomes in severe preeclampsia patients with a sensitivity of 94.30% and a specificity of 80.00%. Moreover, absent or reversed UA diastolic blood flow was found to be an independent risk factor for intrauterine growth restriction. CONCLUSION: GGT in combination with umbilical artery S/D ratio is a potentially useful marker for the prediction of adverse outcome in severe preeclampsia patients. Future studies in a larger cohort of patients should be performed to verify the efficacy of the strategy. Early and accurate prediction of adverse perinatal events can facilitate the efforts to improve the perinatal outcomes of neonates associated with preeclamptic pregnancies.
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Retardo del Crecimiento Fetal/diagnóstico , Unidades de Cuidado Intensivo Neonatal/estadística & datos numéricos , Preeclampsia/diagnóstico por imagen , Síndrome de Dificultad Respiratoria del Recién Nacido/diagnóstico , Arterias Umbilicales/diagnóstico por imagen , gamma-Glutamiltransferasa/sangre , Adulto , Puntaje de Apgar , Biomarcadores/sangre , Velocidad del Flujo Sanguíneo , Diástole , Femenino , Retardo del Crecimiento Fetal/fisiopatología , Feto , Edad Gestacional , Humanos , Recién Nacido , Parto/fisiología , Muerte Perinatal/etiología , Preeclampsia/sangre , Embarazo , Síndrome de Dificultad Respiratoria del Recién Nacido/fisiopatología , Índice de Severidad de la Enfermedad , Sístole , Ultrasonografía , Arterias Umbilicales/enzimologíaRESUMEN
RATIONALE: Arterial endothelial cells are morphologically, functionally, and molecularly distinct from those found in veins and lymphatic vessels. How arterial fate is acquired during development and maintained in adult vessels is incompletely understood. OBJECTIVE: We set out to identify factors that promote arterial endothelial cell fate in vivo. METHODS AND RESULTS: We developed a functional assay, allowing us to monitor and manipulate arterial fate in vivo, using arteries isolated from quails that are grafted into the coelom of chick embryos. Endothelial cells migrate out from the grafted artery, and their colonization of host arteries and veins is quantified. Here we show that sympathetic innervation promotes arterial endothelial cell fate in vivo. Removal of sympathetic nerves decreases arterial fate and leads to colonization of veins, whereas exposure to sympathetic nerves or norepinephrine imposes arterial fate. Mechanistically, sympathetic nerves increase endothelial ERK (extracellular signal-regulated kinase) activity via adrenergic α1 and α2 receptors. CONCLUSIONS: These findings show that sympathetic innervation promotes arterial endothelial fate and may lead to novel approaches to improve arterialization in human disease.
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Fibras Adrenérgicas/enzimología , Arterias/enzimología , Arterias/inervación , Endotelio Vascular/enzimología , Endotelio Vascular/inervación , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Animales , Arterias/crecimiento & desarrollo , Movimiento Celular/fisiología , Embrión de Pollo , Membrana Corioalantoides/enzimología , Membrana Corioalantoides/crecimiento & desarrollo , Membrana Corioalantoides/inervación , Coturnix , Endotelio Vascular/crecimiento & desarrollo , Activación Enzimática/fisiología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Técnicas de Cultivo de Órganos , Sistema Nervioso Periférico/enzimología , Sistema Nervioso Periférico/crecimiento & desarrollo , Trasplante de Tejidos/métodos , Arterias Umbilicales/enzimología , Arterias Umbilicales/crecimiento & desarrolloRESUMEN
BACKGROUND: Modified expression of nitric oxide synthases (NOSs) and an imbalance between the pro-oxidative and the antioxidative system accompany endothelial dysfunction, the first stage of atherosclerosis. Humans born small (SGA) or large (LGA) for gestational age are at higher risk of developing atherosclerosis later in life than humans born appropriate for gestational age (AGA). We hypothesized that indicators of endothelial dysfunction could be detectable at birth. The purpose of this study was to find out whether the expression patterns of NO synthases (endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS)), pro-oxidative enzymes (components of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, NADPH oxidase 1 (NOX1), NOX2, NOX4, p22phox, and p47phox), and antioxidative enzymes (superoxide dismutase 1-3 (SOD1-3), glutathione peroxidase 1 (GPX1), and catalase) in umbilical arteries differ among SGA, LGA, and AGA newborns. METHODS: Thirty-six umbilical cords were obtained from healthy, normal, full-term SGA, AGA, and LGA newborns. The umbilical arteries were dissected and homogenized. mRNA expression was analyzed with quantitative real-time PCR. Western blotting was performed to determine protein expression. RESULTS: mRNA and protein expression of NO synthases, pro-oxidative enzymes, and antioxidative enzymes did not differ in the umbilical arteries from newborns of the three groups. CONCLUSION: Indicators of endothelial dysfunction in terms of differences in enzyme expression in SGA or LGA newborns vs. AGA newborns were not present at birth.
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Peso al Nacer , Enzimas/análisis , Recién Nacido de Bajo Peso , Recién Nacido Pequeño para la Edad Gestacional , Arterias Umbilicales/enzimología , Análisis de Varianza , Western Blotting , Catalasa/análisis , Enzimas/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Edad Gestacional , Glutatión Peroxidasa/análisis , Humanos , Recién Nacido , Isoenzimas , Masculino , NADPH Oxidasas/análisis , Óxido Nítrico Sintasa/análisis , Oxidación-Reducción , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Superóxido Dismutasa/análisis , Glutatión Peroxidasa GPX1RESUMEN
The main vasodilator in the placenta is nitric oxide (NO), which is synthesized by endothelial NO synthase (eNOS). Arginase-2 competes with eNOS for l-arginine, and its activity has been related with vascular dysfunction. Recently, we showed that hypoxia induces arginase-2, and decreases eNOS activity in human umbilical vein endothelial cells (HUVEC). However there is evidence that vascular responses to hypoxia are not similar throughout the placental vascular tree. We studied whether arginase-2 plays a role controlling vascular tone in human umbilical vessels, and the changes in the expression of arginase-2 and eNOS proteins by hypoxia in endothelial cells from umbilical arteries (HUAEC) and veins (HUVEC). In isolated umbilical vessels the presence of eNOS and arginase-2 was determined in the endothelium, and the NO-dependent vasoactive responses in the presence and absence of S-(2-boronoethyl)-L-cysteine (BEC, arginase inhibitor) were studied. Additionally, HUAEC and HUVEC were exposed (0-24 h) to hypoxia (2% O2) or normoxia (5% O2), and protein levels of eNOS (total and phosphorylated at serine-1177) and arginase-2 were determined. In umbilical arteries and veins arginase-2 and eNOS were detected mainly at the endothelium. BEC induced a higher concentration-dependent relaxation in umbilical arteries than veins, and these responses were NOS-dependent. In HUAEC exposed to hypoxia there were no changes in eNOS and arginase-2 levels, however there was a significant increase of p-eNOS. In contrast, HUVEC showed an increase in arginase-2 and a reduction of p-eNOS in response to hypoxia. These results show that arginases have a vascular role in placental vessels counteracting the NOS-dependent relaxation, which is differentially regulated in placental artery and vein endothelial cells.
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Arginasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Embarazo/metabolismo , Arterias Umbilicales/enzimología , Venas Umbilicales/enzimología , Vasodilatación , Células Endoteliales/enzimología , Endotelio Vascular/enzimología , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hipoxia/enzimología , MiografíaRESUMEN
BACKGROUND: The importance of rosuvastatin at therapeutic dosage in regulating the release, activity, protein level, and expression of matrix metalloproteinases (MMP)-2 and MMP-9 was investigated. METHODS: Human umbilical artery smooth muscle cells were stimulated, in vitro, in a serum-free medium with rosuvastatin at various concentrations (2, 4, 7, and 10 ng/mL, which correspond to the maximal plasma concentration observed in healthy men after a daily oral intake of 5, 10, 20, and 40 mg, respectively). The release of MMP-2 and MMP-9 in the conditioned medium was assessed by enzyme-linked immunosorbent assay and confirmed by Western blot, the activity and expression were determined by zymography and polymerase chain reaction, respectively. RESULTS: Human umbilical artery smooth muscle cells stimulated with rosuvastatin at 7 and 10 ng/mL had a significant lower release, activity, protein level, and expression of MMP-2 and MMP-9, when compared with those stimulated at 2 and 4 ng/mL (MMP-2 =p < 0.0001 and p < 0.0001, respectively; MMP-9 =p < 0.0001 and p < 0.0001, respectively). CONCLUSION: The effects of rosuvastatin in reducing MMP-2 and MMP-9, which might stabilize the atherosclerotic plaques, are dose-dependent.
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Fluorobencenos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Pirimidinas/farmacología , Sulfonamidas/farmacología , Western Blotting , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosuvastatina Cálcica , Arterias Umbilicales/efectos de los fármacos , Arterias Umbilicales/enzimologíaRESUMEN
Our objective was to investigate the endothelial nitric oxide synthase (eNOS) immuno-reactivity and the ultrastructure of endothelial cells of a human umbilical artery in both normal and preeclamptic pregnancies. The umbilical cords from normal and preeclamptic pregnancies were collected immediately after vaginal and abdominal deliveries. Umbilical arteries were isolated and fixed in 10% neutral formaline solution, embedded in paraffin, and then stained with hematoxylin and eosin (H&E) for the histologic investigation, and eNOS activation were examined in samples by streptavidine-biotine immunohistochemical methods. The arterial sections were also fixed in phosphate-buffered 2.5% glutaraldehyde solution (pH 7.2) for 3 h and post-fixed with 1% osmium tetroxide at 4°C for 2 h for the investigation of the ultrastructural examination. In the umbilical artery of preeclamptic pregnancies, endothelial cells were oval, triangular, or polygonal, and were disorganized. Some endothelial cells were separated by enlarged intercellular spaces. A dilated endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. The nuclei of some endothelial cells displayed deep invaginations and irregular outlines. Most endothelial cells had a high number of cytoplasmic vacuoles. In preeclampsia, eNOS immunoreactivity increased considerably in endothelial cells when compared to normal pregnancies. We believe that preeclampsia plays an important role in the pathogenesis of endothelial cell dysfunction and activation in the umbilical artery. However, the disturbance mechanism of endothelial cells is not known, and further studies are necessary to clarify the exact mechanism.
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Óxido Nítrico Sintasa de Tipo III/análisis , Preeclampsia/enzimología , Preeclampsia/patología , Embarazo , Células Endoteliales/enzimología , Células Endoteliales/ultraestructura , Endotelio Vascular/enzimología , Endotelio Vascular/ultraestructura , Femenino , Humanos , Inmunohistoquímica , Óxido Nítrico/metabolismo , Orgánulos/ultraestructura , Preeclampsia/fisiopatología , Transducción de Señal , Arterias Umbilicales/enzimología , Arterias Umbilicales/ultraestructura , Venas Umbilicales/enzimología , Venas Umbilicales/ultraestructuraRESUMEN
The cyclic nucleotides involvement in the vasorelaxation induced by testosterone in human umbilical artery was investigated. The effect of this hormone on denuded human umbilical arteries contracted by serotonin (5-HT), histamine or KCl was analysed. Testosterone effect on potassium current (IK) was also studied in human umbilical artery vascular smooth muscle cells (HUASMC). In general, the relaxant effects of testosterone, sodium nitroprusside (SNP) and atrial natriuretic peptide (ANP) are similar. The testosterone relaxant effect is not different to the induced by the conjoint application of ANP and testosterone. However, the effects of SNP and testosterone seem additive. The inhibition of protein kinase A (PKA) did not modify the testosterone relaxant effect, but protein kinase G (PKG) inhibition significantly reduced the testosterone effect independently of the contractile stimuli. In HUASMC, the IK is mainly constituted by potassium exit through voltage sensitive (KV) and large-conductance Ca2+ activated (BKCa) potassium channels. Testosterone significantly activates the basal IK. SNP does not induce a significant modification in basal or testosterone stimulated IK. In contrast, ANP stimulates the basal IK, but does not increase the testosterone stimulation on IK. The IK increases induced by testosterone or by ANP are not significantly affected by the PKA inhibition, but are completely inhibited by the PKG inhibition. Our results show that testosterone and ANP stimulate the activity of BKCa and KV channels due to PKG activation and suggest that this hormone relaxes by activating particulate guanylate cyclase which increases the cGMP intracellular level.
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Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Testosterona/farmacología , Arterias Umbilicales/irrigación sanguínea , Arterias Umbilicales/enzimología , Vasodilatación/efectos de los fármacos , GMP Cíclico/metabolismo , Conductividad Eléctrica , Femenino , Guanilato Ciclasa/metabolismo , Humanos , Técnicas In Vitro , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Embarazo , Arterias Umbilicales/efectos de los fármacos , Arterias Umbilicales/metabolismo , Vasoconstricción/efectos de los fármacosRESUMEN
Semicarbazide-sensitive amine oxidase (SSAO; E.C.1.4.3.6.) is widely distributed in different tissues, particularly in vascular smooth muscle and adipose tissue. Its physiological function remains unclear. Up to now, the common method to determine SSAO is based on enzymatic activity measurement. However, enzymatic activity could be easily influenced by the temperature, pH, and circumstance. In the present study, we have developed the single-reaction monitoring (SRM) approach for measuring the absolute amount of SSAO expression in human umbilical artery based on LC-ESI-MS/MS. The measurement of protein was converted to the measurement of a unique peptide of SSAO from Homo sapiens. The peptide (YQLAVTQR) was confirmed to be unique to the SSAO in human using the ExPasy blast tools, and thus the synthetic peptide was used as the standard which can produce abundant parent ion (m/z = 490.0) and daughter ion (m/z = 687.4) in the mass spectrometry. Trap drive and fragmentation energy of MS/MS of the unique peptide was 60 V and 0.6 V, respectively. The calibration curve was linear over the range of 1.99-127.8 fmol/microL, with 1.99 fmol/microl of the lower limit of quantification. The inter- and intra-day precisions and recoveries for all samples were satisfactory. The results demonstrated SSAO protein concentration was 7.75 fmol/g wet weight. It proved that the novel assay was sensitive and selective to measure the amount of SSAO protein originated from H. sapiens.
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Amina Oxidasa (conteniendo Cobre)/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Arterias Umbilicales/enzimología , Tejido Adiposo/enzimología , Amina Oxidasa (conteniendo Cobre)/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Péptidos/análisis , Ratas , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: NO and NO synthases (NOS) play an important role in the physiology of the fetomaternal blood circulation, although their expression in pathological conditions is unclear. Intrauterine growth retardation (IUGR) is a disorder most probably caused by abnormality of the fetomaternal bloodflow. MATERIALS AND METHODS: The expression of endothelial NOS (ecNOS) from arteria umbilicalis and the nitrite and peroxynitrite level of umbilical blood were determined. Major consequences of peroxynitrite toxicity are lipid peroxidation and glutathione depletion; these parameters were also measured. Finally, superoxide dismutase (SOD) activity was assayed to evaluate the level of superoxide anions. RESULTS: Elevated expression of ecNOS was found to be coupled with significantly lower SOD activity and glutathione level, and increased lipid peroxidation in IUGR neonates. CONCLUSION: The increased NO indices could represent a compensatory effort to improve placental bloodflow, but in IUGR neonates it is coupled with inadequate antioxidant defence, resulting in significant oxidative stress.
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Endotelio Vascular/enzimología , Retardo del Crecimiento Fetal/enzimología , Óxido Nítrico Sintasa de Tipo III/genética , Arterias Umbilicales/enzimología , Adulto , Deformación Eritrocítica , Femenino , Sangre Fetal/química , Retardo del Crecimiento Fetal/sangre , Expresión Génica , Glutatión/análisis , Humanos , Recién Nacido , Peroxidación de Lípido , Masculino , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/sangre , Estrés Oxidativo , Ácido Peroxinitroso/sangre , Embarazo , ARN Mensajero/metabolismo , Superóxido Dismutasa , Regulación hacia ArribaRESUMEN
The expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was examined in the umbilical vessels of the patients with pre-eclampsia (PE) to explore its possible role in the pathogenesis of PE. The NOSTRIN mRNA in umbilical tissues was determined by RT-PCR. The eNOS activity in umbilical vessels was spectrophotometrically detected. NO2-/NO3-, the stable metabolic end products of NO, was measured by using nitrate reductase. RT-PCR showed that the expression level of NOSTRIN was significantly higher in women with PE than in the normal group (P<0.01). The activity of eNOS was significantly decreased in PE group [(12.83+/-3.61) U/mg] than in normal group [(21.72+/-3.83) U/mg] (P<0.01). The level of NO2-/NO3- in PE patients (27.53+/-7.48) micromol/mg was significantly lower than that of normal group (54.27+/-9.53) micromol/mg (P<0.01). The significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in umbilical vessels of women with PE (r=-0.58, P<0.01). It was concluded that the level of NOSTRIN expression was increased in umbilical vessel of women with PE, indicating that it may be involved in the pathogenesis of PE.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Preeclampsia/enzimología , Arterias Umbilicales/enzimología , Venas Umbilicales/enzimología , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Unión al ADN , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Preeclampsia/etiología , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Arterias Umbilicales/citología , Venas Umbilicales/citologíaRESUMEN
The expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was examined in the umbilical vessels of the patients with pre-eclampsia (PE) to explore its possible role in the pathogenesis of PE. The NOSTRIN mRNA in umbilical tissues was determined by RT-PCR. The eNOS activity in umbilical vessels was spectrophotometrically detected. NO2-/NO3-, the stable metabolic end products of NO, was measured by using nitrate reductase. RT-PCR showed that the expression level of NOSTRIN was significantly higher in women with PE than in the normal group (P<0.01). The activity of eNOS was significantly decreased in PE group [(12.83+/-3.61) U/mg] than in normal group [(21.72+/-3.83) U/mg] (P<0.01). The level of NO2-/NO3- in PE patients (27.53+/-7.48) micromol/mg was significantly lower than that of normal group (54.27+/-9.53) micromol/mg (P<0.01). The significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in umbilical vessels of women with PE (r=-0.58, P<0.01). It was concluded that the level of NOSTRIN expression was increased in umbilical vessel of women with PE, indicating that it may be involved in the pathogenesis of PE.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Preeclampsia/enzimología , Preeclampsia/etiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Arterias Umbilicales/citología , Arterias Umbilicales/enzimología , Venas Umbilicales/citología , Venas Umbilicales/enzimologíaRESUMEN
Fibroblast growth factor (FGF2), but not vascular endothelial growth factor (VEGF), upregulates endothelial nitric oxide synthase (eNOS) protein expression, at least partially, via activation of extracellular signal-regulated kinase 2/1 (ERK2/1) in ovine fetoplacental artery endothelial (oFPAE) cells. Herein we further investigated the temporal effects of FGF2 and VEGF on other signalling pathways including members (Jun N-terminal kinase JNK1/2 and p38MAPK) of mitogen-activated protein kinases (MAPK), phosphatidylinositol-3 kinase/v-akt murine thymoma viral oncogene homologue 1 (PI3K/AKT1), and the tyrosine kinase c-SRC, and examined if either one or more of these pathways play a role in the differential regulation of eNOS by FGF2 and VEGF. We first confirmed that in oFPAE cells, FGF2, but not VEGF, increased eNOS protein. FGF2 stimulated eNOS protein in a time- and concentration-dependent manner, which also depended on cell density. FGF2 provoked sustained (5min to 12h) whereas VEGF only stimulated transient (5min) ERK2/1 phosphorylation. FGF2 was 1.7-fold more potent in stimulating ERK2/1 phosphorylation than VEGF. FGF2 and VEGF only transiently activated JNK1/2 and AKT1 within 5min; however, FGF2 was a stronger stimulus than VEGF. FGF2 and VEGF did not significantly activate p38MAPK at 5min; however, VEGF stimulated p38MAPK phosphorylation at 60min. VEGF but not FGF2 significantly stimulated c-SRC phosphorylation. Inhibitors of MEK-ERK2/1 (PD98059), JNK1/2 (SP600125) and PI3K (wortmannin), but not p38MAPK (SB203580) and SRC (PP2), decreased the FGF2-increased eNOS protein expression. Thus, the FGF2-induced eNOS protein expression requires activation of multiple signalling pathways including ERK2/1, JNK1/2 and PI3K/AKT1. Differences in intensity and temporal patterns of activation of these pathways by FGF2 and VEGF may account for their differential effects on eNOS expression in OFPAE cells.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Placenta/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Células Cultivadas , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Femenino , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Placenta/irrigación sanguínea , Placenta/metabolismo , Circulación Placentaria/efectos de los fármacos , Circulación Placentaria/fisiología , Embarazo , Proteínas Quinasas/metabolismo , Ovinos , Transducción de Señal/efectos de los fármacos , Arterias Umbilicales/efectos de los fármacos , Arterias Umbilicales/enzimología , Arterias Umbilicales/metabolismoRESUMEN
OBJECTIVE: To investigate the expression and significance of Rho-associated protein kinase II (Rock II) in preeclamptic placenta and umbilical artery. METHODS: Semiquantitative RT-PCR and Western blot were used to investigate the expression of Rock II mRNA and Rock II protein in placenta and umbilical artery from 35 women with moderate preeclampsia (MPE group), 38 women with severe preeclampsia (SPE group) and 45 normal third trimester pregnant women (control group), the S/D value of umbilical artery was examined by ultrasound. RESULTS: (1) The expression of Rock II mRNA of placenta in MPE group (0.82+/-0.14) and SPE group (0.93+/-0.13) were significantly higher than that in control group (0.70+/-0.12, P<0.01). The expression of Rock II protein of placenta in MPE group (0.79+/-0.15) and SPE group (0.92+/-0.12) were significantly higher compared with control group (0.68+/-0.11, P<0.01). The expression of Rock II mRNA and protein of placenta in SPE group were higher compared with MPE group (P<0.01). (2) The expression of Rock II mRNA of umbilical artery in MPE group (0.69+/-0.13) and SPE group (0.55+/-0.12) were significantly lower than that in control group (0.76+/-0.10, P<0.01). The expression of Rock II protein of umbilical artery in MPE group (0.68+/-0.10) and SPE group (0.51+/-0.12) were lower compared with control group (0.75+/-0.13, P<0.01). The expression of Rock II mRNA and protein of umbilical artery in SPE group were significantly lower compared with MPE group (P<0.01). (3) There were no correlations between the expression of Rock II mRNA and protein in placenta and umbilical artery and the S/D value and birth weight (P>0.05). CONCLUSION: The upregulated expression of Rock II in placentas and downregulated expression in umbilical artery may be a compensation in preeclampsia.
Asunto(s)
Placenta/enzimología , Preeclampsia/enzimología , Arterias Umbilicales/enzimología , Quinasas Asociadas a rho/biosíntesis , Adulto , Western Blotting , Femenino , Edad Gestacional , Humanos , Inmunohistoquímica , Placenta/metabolismo , Preeclampsia/patología , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/enzimología , Trofoblastos/metabolismo , Trofoblastos/patología , Arterias Umbilicales/metabolismo , Quinasas Asociadas a rho/genéticaRESUMEN
Cyclic nucleotides (cAMP and cGMP) are the main second messengers linked to vasodilatation. They are synthesized by cyclases and degraded by different types of phosphodiesterases (PDE). The effect of PDE inhibition and cyclases stimulation on 5-hydroxytryptamine (5-HT; 1 microM) and histamine (10 microM) contracted arteries was analysed. Stimulation of guanylate cyclase or adenylate cyclase relaxed the histamine- and 5-HT-induced contractions indicating that intracellular increase of cyclic nucleotides leads to vasodilatation of the human umbilical artery. We investigated the role of different PDE families in the regulation of this effect. The presence of the different PDE types in human umbilical artery smooth muscle was analysed by RT-PCR and the expression of PDE1B, PDE3A, PDE3B, PDE4C, PDE4D and PDE5A was detected. The unspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 50 microM) relaxed histamine-contracted human umbilical artery on 47.4+/-7.2%. This effect seems to be due to PDE4 and PDE5 inhibition because among the selective PDE inhibitors used only the PDE4 inhibitor (rolipram; 1 microM) and the PDE5 inhibitors (dipyridamole and T0156; 3 microM and 1 microM respectively) induced significant relaxation (39.0+/-8.7, 30.4+/-6.0 and 36.3+/-2.8 respectively). IBMX, dipyridamole and T0156 produced similar relaxation on 5-HT-induced contraction. After forskolin, the addition of IBMX or rolipram increased the effect of the adenylate cyclase stimulator and almost completely relaxed the human umbilical artery contracted by histamine (92.5+/-4.9 and 90.9+/-4.7 respectively), suggesting a main role of PDE4. The data obtained with 5-HT contracted arteries confirmed this, because only rolipram and IBMX significantly increased the forskolin vasodilator effect. The administration of dipyridamole and T0156 after sodium nitroprusside (SNP) induced a significant increase of the SNP relaxant effect on histamine-contracted arteries, but PDE1 and PDE3 inhibition did not increase the effect of the guanylate cyclase stimulator. Similar effects were obtained in 5-HT contracted arteries, the SNP induced relaxation was increased by the PDE5 inhibition, but not by PDE1 or PDE3 inhibition. In summary, our results demonstrate that: 1) the increase of cAMP and/or cGMP levels induces relaxation of the human umbilical vascular smooth muscle; 2) four families of PDE are expressed in this smooth muscle: PDE1, PDE3, PDE4 and PDE5; 3) between these families, PDE4 and PDE5 are the key enzymes involved in the regulation of the relaxation associated to cAMP and cGMP, respectively.
Asunto(s)
AMP Cíclico/fisiología , GMP Cíclico/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/fisiología , Músculo Liso Vascular/fisiología , Arterias Umbilicales/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Adenilil Ciclasas/metabolismo , Colforsina/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/biosíntesis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/biosíntesis , Dipiridamol/farmacología , Femenino , Guanilato Ciclasa/metabolismo , Histamina/farmacología , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/fisiología , Contracción Muscular , Relajación Muscular , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Naftiridinas/farmacología , Nitroprusiato/farmacología , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa 5 , Pirimidinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rolipram/farmacología , Serotonina/farmacología , Arterias Umbilicales/efectos de los fármacos , Arterias Umbilicales/enzimología , Vasodilatadores/farmacologíaRESUMEN
Catecholamines have been shown to be involved in vascular remodeling through the stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% O(2)) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce a trophic effect in human endothelial cells through stimulation of the alpha(1D)-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective alpha(1D)-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that alpha(1D)-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by N(G)-nitro-L-arginine methyl ester. Conversely, the alpha(1A)-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the alpha(1B)-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of alpha(1D)-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of alpha(1D)-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Tamaño de la Célula/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Fenilefrina/farmacología , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Arterias Umbilicales/efectos de los fármacos , Antagonistas Adrenérgicos/farmacología , Hipoxia de la Célula , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Técnicas de Cultivo de Órganos , Piperazinas/farmacología , Receptores Adrenérgicos alfa 1/metabolismo , Arterias Umbilicales/enzimología , Arterias Umbilicales/metabolismoRESUMEN
OBJECTIVE: The objective of the study was to evaluate endothelial nitric oxide synthase concentration in the placenta, uterine, and umbilical vessels near term in an ovine model of intrauterine growth restriction induced by hyperthermia beginning in early gestation. STUDY DESIGN: Four pregnant ewes were exposed to hyperthermia conditions for 80 days beginning at 35 days gestation to induce intrauterine growth restriction. Four ewes were kept in ambient conditions as controls. Umbilical artery Doppler systolic to diastolic ratios were calculated. At 128 days gestation, fetal catheters were placed for aortic blood pressure measurements and blood gas determination. At 132 days gestation, fetal mean systemic blood pressure and gases were determined. Sheep placentomes, umbilical artery and vein, and uterine artery were assessed for endothelial nitric oxide synthase concentration and immunolocalization. RESULTS: Compared with control pregnancies, the intrauterine growth restriction pregnancies showed: (1) reduced fetal and placental weights (P < or = .01); (2) elevated systemic blood pressure (41 +/- 1.53 mm Hg versus 44.3 +/- 1.71 mm Hg; P < or = .05) and systolic to diastolic ratios (3.0 +/- 0.34 versus 3.8 +/- 0.18; P < or = .01); (3) reduced fetal O2 saturation (52.2 +/- 7.03% versus 33.05 +/- 10.98%; P < or = .008); and (4) decreased endothelial nitric oxide synthase protein concentration in the umbilical artery (2.7-fold; P < or = .01) and a trend for a decrease in the uterine artery (1.4-fold; P < or = .1). CONCLUSION: We conclude that placental endothelial nitric oxide synthase protein concentration is increased near term in our ovine model of intrauterine growth restriction, and that this increase may be secondary to hypoxia.