Aislamiento y función de células progenitoras endoteliales de sangre periférica durante la evolución clínica de pacientes con infarto agudo de miocardio. Efecto de las micropartículas Shh+ en la función celular

Las células endoteliales (CE) recubren el interior de los vasos sanguíneos constituyendo el endotelio, actualmente considerado un órgano muy activo e implicado en la regulación del tono vascular, el tráfico de células sanguíneas, el equilibrio hemostático, la permeabilidad, la proliferación, la supervivencia, y la respuesta inmunitaria tanto adaptativa como innata. Los factores de riesgo cardiovascular como la hipercolesterolemia, la diabetes y el tabaquismo pueden causar lesiones endoteliales, uno de los principales desencadenantes de la patogénesis en la aterosclerosis, predisponiendo al desarrollo y progresión de enfermedades cardiovasculares. Hoy en día, las enfermedades cardiovasculares son la principal causa de muerte en todo el mundo, y la manifestación más temida es el síndrome coronario agudo. Éste tiene lugar cuando el flujo sanguíneo coronario es interrumpido, secundario a la ruptura de una placa aterosclerótica. En 1997, el descubrimiento de las células progenitoras endoteliales (CPE) supuso un cambio en el paradigma de la reparación vascular. Por primera vez, se describió un mecanismo alternativo a la angiogénesis para el mantenimiento y reparación del endotelio, contrarrestar las lesiones endoteliales, reemplazar el endotelio no funcional y potenciar la reparación tisular tras una lesión vascular isquémica. El proceso por el cual las CPE contribuyen a la formación de nuevos vasos sanguíneos es conocida como vasculogenesis postnatal y tiene lugar a través de 4 etapas diferentes: movilización, emplazamiento, invasión y diferenciación. La mayoría de estudios han centrado su atención en la definición molecular y en la determinación cuantitativa de las CPE por citometría de flujo, atendiendo a la expresión de un marcador progenitor, el CD34, un marcador endotelial, el KDR y la ausencia de expresión del marcador panleucocitario CD45. Sin embargo, el análisis de muestras de sangre por citometría de flujo únicamente aporta información cuantitativa sobre las CPE y la expresión de un número limitado de marcadores en su superficie. En muestras de sangre periférica de humanos las CPE solamente representan un 0.0001% del total de células mononucleadas (CMN). Para obtener información cualitativa, las CPE necesitan ser aisladas y expandidas en cultivo. Todos los protocolos existentes para el aislamiento de las CPE difieren en el tiempo y en las condiciones de cultivo. Los protocolos con tiempos de cultivo corto, 14 días, dan lugar a células con un fenotipo más endotelial, una capacidad proliferativa mayor y capaces de diferenciarse a CE maduras. Las CPE obtenidas bajo diferentes condiciones pueden mostrar pequeñas o grandes diferencias en su fenotipo y función biológica. Por este motivo, es necesaria una comparación sistemática de las condiciones de cultivo más utilizadas con el fin de establecer un protocolo consenso. La importancia biológica de las CPE ha sido demostrada en numerosos estudios que describen su participación en procesos de vasculogenesis postnatal, tanto en condiciones fisiológicas como en condiciones patológicas como la isquemia miocárdica y periférica y la aterosclerosis. El número de CPE está reducido en muestras de sangre de sujetos que presentan algún factor de riesgo cardiovascular y/o aterosclerosis. La reducción de los niveles de CPE, acompañada de un incremento de los niveles de células endoteliales circulantes (CEC), marcador de la existencia y severidad de una lesión vascular, son actualmente considerados marcadores de daño vascular. Además, la reducción del número de CPE ha demostrado ser un predictor de futuros eventos cardiovasculares. De la misma manera, los factores de riesgo cardiovascular y diferentes patologías como las cardiovasculares pueden afectar las capacidades funcionales de las CPE. Las micropartículas (MP) son pequeñas vesículas liberadas por la membrana plasmática de células activadas o apoptóticas. Presentan un diámetro comprendido entre 0.1 y 1 µm y son heterogéneas en cuanto a su composición. Las MP representan un nuevo mecanismo de comunicación intercelular y actúan como vectores transportando diferentes mensajes biológicos que participan en la patofisiología de, principalmente, enfermedades cardiovasculares. Sin embargo, debido a la capacidad de las MP de transportar diferentes moléculas como citoquinas, quimioquinas, factores de crecimiento y ácidos nucleicos exógenos, éstas pueden ser herramientas terapéuticas potencialmente útiles para el tratamiento de diferentes condiciones patológicas. Las MP generadas por linfocitos T bajo condiciones mitógenas contienen el morfogen Sonic Hedgehog (Shh) (MPShh+). Se ha demostrado que las MPShh+ tienen un efecto beneficioso en el modelo de isquemia animal y en CE de origen humano en cultivo, restableciendo la lesión endotelial y favoreciendo la angiogénesis. Las MPShh+ son también capaces de inducir, en CE, la producción de óxido nítrico (NO) y reducir las especies reactivas del oxígeno, reforzando el efecto beneficioso sobre el sistema cardiovascular. Uno de los objetivos de esta tesis es el de establecer las condiciones óptimas para el aislamiento y cultivo de CPE a partir de muestras de sangre periférica humana y determinar las características morfológicas, fenotípicas y funcionales de las CPE obtenidas utilizando dichas condiciones. Otro objetivo de esta tesis es el de analizar la movilización de las CEC y CPE y estudiar las capacidades funcionales de las CPE en sujetos sanos y pacientes con IAM durante 6 meses de evolución clínica. El último objetivo es el de determinar el efecto de las MPShh+ sobre la capacidad funcional de las CPE aisladas de sujetos sanos. Para la determinación de las condiciones óptimas de aislamiento y cultivo de CPE se utilizaron muestras de sangre periférica de 50 sujetos sanos obtenidas en el Servicio de Cardiología del Hospital Clínico Universitario de la Universidad de Valencia. En primer lugar se determinaron las condiciones óptimas para la recogida de las muestras de sangre atendiendo a los criterios de volumen de muestra, anticoagulante y tiempo de procesado. Después, las condiciones de cultivo (matriz extracelular y medio de cultivo). La capacidad funcional de las CPE, aisladas bajo las condiciones óptimas, se evaluó a través de los parámetros de adhesión, crecimiento, proliferación y vasculogenesis celular y se comparó con las células endoteliales de vena de cordón umbilical (HUVEC), la referencia estándar para cultivos de CE. Nuestros resultados pusieron de manifiesto que para la obtención de cultivos de CPE con un comportamiento funcional óptimo es necesario controlar una serie de parámetros durante la recogida de las muestras de sangre y en las condiciones de cultivo utilizadas. Las condiciones testadas en esta tesis indican que: (i) las muestras de sangre deben ser recogidas en tubos de heparina; (ii) en un volumen mínimo de 30 mL; (iii) y con un tiempo de procesado inferior a 2 horas tras su obtención; (iv) la fibronectina es la mejor matriz extracelular y (v) el medio de cultivo tiene que estar enriquecido con suero bovino fetal (SBF) al 20%. Además, las CPE aisladas bajo las condiciones óptimas mostraron unas características morfológicas, fenotípicas y funcionales similares a CE de otros orígenes, como lo son las HUVEC. La movilización de las CEC y las CPE se estudió en muestras de sangre periférica en 50 sujetos sanos y 50 pacientes con infarto agudo de miocardio (IAM) durante 6 meses de evolución clínica, todos ellos reclutados por el Hospital Clínico Universitario de Valencia. El criterio para la inclusión de los pacientes con IAM fue: edad ≤ 75 años, primer episodio de IAM y la presencia de uno o mas de los factores tradicionales de riesgo cardiovascular. Las CEC y las CPE se definieron por citometría de flujo como células que expresaban los marcadores CD45-CD31+CD146+ y CD45-CD34+KDR+ respectivamente. Los resultados mostraron que, comparado con sujetos sanos, los niveles de CEC aumentaban el día del infarto, y que estos niveles elevados se mantenían sin cambios durante 6 meses de evolución clínica, revelando la existencia de daño vascular. Por otra parte en pacientes con IAM, comparado con niveles encontrados en sujetos sanos, la CPE aumentaron 3 veces el día del infarto, 6 veces a los 30 días del infarto que se redujeron con 6 meses de evolución clínica. Este hecho indica la activación de un mecanismo de reparación endotelial en etapas tempranas del IAM que es atenuado después de 6 meses de evolución clínica. La capacidad funcional de las CPE se determinó en 10 sujetos sanos y 10 pacientes con IAM, un subconjunto de la muestra de estudio anteriormente mencionada. Los parámetros funcionales se analizaron en cultivos de CPE, aislados utilizando las condiciones óptimas determinadas en la primera parte de esta tesis, en términos de adhesión, crecimiento, proliferación y vasculogenesis celular. Los resultados obtenidos revelaron una modificación funcional de las CPE de pacientes con IAM durante 6 meses de evolución clínica. La capacidad de adhesión de las CPE de los pacientes con IAM aumentó el día del infarto, alcanzó valores máximos a los 30 días del infarto y se reestableció a niveles encontrados en sujetos sanos tras 6 meses de evolución clínica. A nivel proteico, los niveles de expresión de las integrinas α5 and αv siguieron un patrón similar al que seguía la capacidad de adhesión de las CPE en la evolución del IAM. Sin embargo, las capacidades de crecimiento, proliferación y vasculogenesis de las CPE de pacientes con infarto estaban reducidas en las fases agudas del IAM y se restablecieron a los valores encontrados en sujetos sanos a los 6 meses de evolución clínica. El efecto de las MPShh+ en la función celular de las CPE se determinó en 5 cultivos de CPE aislados de muestras de sangre periférica de sujetos sanos. En primer lugar, las CPE se trataron con MPShh+ (10 µg/mL ) durante 24h y, a continuación, se estudió la expresión génica, la capacidad vasculogénica in vitro e in vivo, la producción de NO y la reorganización del esqueleto de actina, tanto en presencia como en ausencia de inhibidores específicos de la ruta de señalización de Sonic Hedgehog. Las MPShh+ incrementaron la expresión de los genes PTCH1, SMO, GLI1, VEGFA, KDR, NOS3 y KLF2. El tratamiento con MPShh+ incrementó la expresión proteica y la activación de la eNOS, aumentando consequentemente la producción de NO, y la capacidad vasculogénica in vitro y in vivo de los cultivos de CPE a través de mecanismos dependientes de SMO y PI3K. Por último, las MPShh+ indujeron la reorganización del citosqueleto de actina de CPE en cultivo a través de un mecanismo dependiente de la Rho quinasa (ROCK) pero independiente de SMO. Conjuntamente, proponemos un protocolo consenso para el aislamiento y cultivo de CPE, a partir de muestras de sangre periférica humana, con una morfología, fenotipo y comportamiento funcional endotelial. Nuestros resultados sugieren la existencia de una lesión endotelial tras el infarto, acompañada de la activación de los mecanismos de reparación endotelial, principalmente mediado por las CPE, en las fases iniciales después de un evento isquémico. Además, las CPE sufren un cambio en su comportamiento funcional durante la evolución clínica del IAM, con un predominio de la capacidad de adhesión en las fases iniciales que es atenuada en fases más tardías a favor de las capacidades de crecimiento, proliferación y vasculogénicas. Las MPShh+ pueden representar una nueva herramienta terapéutica para el tratamiento de enfermedades isquémicas ya que mejoran la producción de NO y la capacidad vasculogénica de las CPE.Endothelial cells (EC) line up the inner surface of blood vessels and conform the endothelium, now considered a very active organ in regulating the vasomotor tone, blood cell trafficking, hemostatic balance, permeability, proliferation, survival, and innate and adaptive immunity. Traditional cardiovascular risk factors such as hypercholesterolemia, hypertension, diabetes, and smoking may cause endothelial injury, one of the major initiating steps in the pathogenesis of atherosclerosis, predisposing to the development and progression of cardiovascular diseases. Nowadays, cardiovascular disease is the leading cause of death worldwide, and its most threatening manifestation is the acute coronary syndrome that occurs when blood flow is prevented to part of the heart secondary to the rupture of an atherosclerotic plaque. In 1997, with the discovery of endothelial progenitor cells (EPC), an alternative mechanism to angiogenesis for the maintenance and repair of the endothelium to counteract ongoing endothelial cell injury, replace dysfunctional endothelium, and enhance tissue repair after ischemic vascular injury became apparent. The process by which EPCs contribute to formation of new vessels is known as postnatal vasculogenesis and takes place through four steps, namely mobilization, homing, invasion and differentiation. Most studies have centered their attention on the molecular definition and quantitative determination of EPC by flow cytometry attending to the expression of a progenitor marker, CD34, an endothelial marker, the Kinase-insert Domain Receptor (KDR) and the lack of expression the panleucocyte marker CD45. However, analysis of blood samples by flow cytometry only provides quantitave information about EPC and the expression of a limited number of markers in their surface. In adult peripheral blood samples EPC represent only 0.0001% of total mononuclear cells (MNC). To obtain qualitative data, EPC need to be isolated and expanded in culture. All the existing protocols for isolating EPC differ in culture time and conditions. The short-term protocols, 14 days, yield cells with a more EC phenotype, higher proliferative capacity and able to differentiate to mature EC. EPC obtained under these different culture conditions exhibit slight or profound differences on their phenotype and biological function. A systematic comparison of the most used conditions for EPC culture is still required in order to obtain a consensus protocol. The biological significance of EPC has been demonstrated by increasing evidence of their role in postnatal vasculogenesis in physiological conditions and pathological conditions such myocardial and limb ischemia and atherosclerosis. EPC are reduced in peripheral blood samples of subjects with cardiovascular risk factors and established atherosclerosis, and this reduction, together with increased circulating endothelial cell (CEC) levels, a marker of the presence and severity of vascular injury, are considered a marker of the ongoing vascular damage. Additionally, EPC reduction has been found to be an independent predictor of future cardiovascular events. Similarly, EPC functional capacities are also impaired with the presence of cardiovascular risk factors and in different pathological settings such in cardiovascular diseases. Microparticles (MP) are small vesicles shed from plasma membrane of activated or apoptotic cells. They are 0.1 to 1 µm in diameter and heterogeneous in composition. They represent a novel way for intercellular communication as vectors by transporting and delivering biological messages that actively participate in the pathophysiology, notably in cardiovascular diseases. However, because MP’s capability to deliver secretory molecules such as cytokines, chemokines, growth factors as well as exogenous nucleic acids they can be potential useful therapeutic tools for treating different pathological conditions. MP generated from T lymphocytes under mitogenic conditions carry the morphogen Sonic Hedgehog (Shh) (MPShh+). MPShh+ have previously been shown to have beneficial effect on ischemic murine model as well as on cultured EC of human origin by correcting endothelial injury and favor angiogenesis. MPShh+ are able to induce NO production and to reduce reactive oxygen species on EC further supporting the beneficial effect on cardiovascular system. This thesis aims to set the optimal conditions for EPC isolation and culture from human peripheral blood samples and to determine the morphology, phenotype and functional capacity of EPC obtained under these optimal conditions. Another aim, is to analyze the mobilization of CEC and EPC and to study EPC functional capacities in healthy subjects and AMI patients during 6 months of clinical evolution. Our last aim is to determine the effect of MPShh+ on functional capacities of EPC isolated from healthy subjects. To determine the optimal conditions for EPC isolation and culture, peripheral blood samples from 50 healthy subjects were obtained at the Cardiology Service in the Hospital Clínico of Valencia. First, we selected the optimal blood sample withdrawal conditions in terms of volumen, anticoagulant and blood sample procesing time. Then, culture conditions (extracellular matrix, culture media composition) were analysed. Functional capacity of EPC isolated under optimal conditions was measured in terms of cell adhesion, growth, proliferation and vasculogenesis and compared to HUVEC, the standard reference for EC culture. Our results showed the need to control a number of parameters related to sample collection and to culture conditions to obtain an optimal functional performance of EPC. The conditions tested here indicated that: (i) peripheral blood should be recovered in heparin tubes; (ii) in a minimum volume of 30 ml; (iii) blood samples should be processed within 2 hours after collection; (iv) fibronectin was the best extracellular matrix and (v) culture media should be supplemented with 20% FBS. EPC isolated under optimal conditions showed morphological, phenotypic and functional characteristics comparable to endothelial cells from other sources, such as HUVEC. The mobilization of CEC and EPC was studied in peripheral blood samples of 50 healthy subjects and 50 acute myocardial infarction patients (AMI) during 6 months of clinical evolution, all recruited at the Cardiology Service in the Hospital Clínico of Valencia. AMI patient inclusion criteria were: age≤ 75 years, first AMI and the presence of more than one traditional vascular risk factor. CEC and EPC were defined by flow cytometry as cells expressing CD45-CD31+CD146+ and CD45-CD34+KDR+ respectively. We found that, when compared to healthy subjects, CEC levels were increased at the day of infarction, and this elevated CEC levels were maintained without changes during 6 months of clinical evolution revealing an ongoing vascular damage. On the other hand, EPC levels in patients suffering AMI triplicated control levels at the day of infarction, represented 6 times EPC control levels at 30 days of infarction, and showed a reduction after 6 months of clinical evolution. This indicates the activation of endothelial reparatory mechanism at very early stages of the AMI that is attenuated after 6 month of clinical evolution. EPC functional capacities were determined in a subset of 10 healthy subjects and 10 AMI patients during 6 months of clinical evolution of the above mention study sample. Functional parameters were analyzed in cultures of EPC, isolated by using the optimal set of conditions determined in the first part of this thesis, in terms of cell adhesion, growth, proliferation and vasculogenesis. Our results revealed functional modifications on EPC isolated from AMI patients during 6 months of clinical evolution. When compared with healthy subjects, adhesive capacity of EPC from AMI patients was increased at the day of infarction, peaked at 30 days after infarction, and was restored to control values after 6 months of clinical evolution. Protein levels of integrin α5 and αv followed a pattern similar to EPC adhesive capacity in the AMI. Howerever, proliferative, growth and vasculogenic capacities were found to be reduced in the acute stages of AMI and were reestablished to values found in healthy subjects after 6 months of clinical evolution. The effect of MPShh+ on EPC cell function was determined on EPC isolated from peripheral blood samples of 5 healthy subjects. EPC were treated with MPShh+ (10 µg/mL ) for 24 hours and, then, gene expression, in vitro and in vivo vasculogenesis, nitric oxide production and actin cytoskeleton remodeling were studied, either with the presence or in the absence of specific inhibitors for the Sonic Hedgehog signaling pathway. We found that MPShh+ increased PTCH1, SMO, GLI1, VEGFA, KDR, NOS3 y KLF2 gene expression. MPShh+ treatment increased eNOS protein expression and activation, increasing consequently NO production, and in vitro and in vivo vasculogenic capacity of EPC cultures in a SMO and PI3K dependent mechanisms. Finally, MPShh+ induced the remodeling of the actin cytoskeleton in a Rho kinase (ROCK)-dependent but SMO-independent way. Together, we proposed a consensus procedure for isolating and culturing EPC from human perpipheral blood samples with an endothelial-like morphology, phenotype and functional behavior. Our results suggest the existence of an endothelial injury as a consequence of the infarction that is accompanied with the activation of the endothelial reparatory mechanisms, mainly mediated by EPC, at very early stages after the ischemic event. Additionally, EPC suffer a functional behavioral change throughout AMI clinical evolution, with a predominant adhesive capacity at early stages of AMI th

An affordable method to obtain cultured endothelial cells from peripheral blood

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell-like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above-mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols

Extracellular histones trigger oxidative stress-dependent induction of the NF-kB/CAM pathway via TLR4 in endothelial cells.

Extracellular histones have been reported to aggravate different pathophysiological processes by increasing vascular permeability, coagulopathy, and inflammation. In the present study, we elucidate how extracellular histones (10-100 mu g/mL) concentration dependently increase cytosolic reactive oxygen species (ROS) production using human umbilical vein endothelial cells (HUVECs). Furthermore, we identify cyclooxygenase (COX) and NADPH oxidase (NOX) activity as sources of ROS production in extracellular histone-treated HUVEC. This COX/NOX-mediated ROS production is also involved in enhanced NF-kB activity and cell adhesion molecules (VCAM1 and ICAM1) expression in histone-treated HUVEC. Finally, by using different toll-like receptor (TLR) antagonists, we demonstrate the role of TLR4 in CAMs overexpression triggered by extracellular histones in endothelial cells. In conclusion, our data suggest that through TLR4 signaling, extracellular histones increase endothelial cell activation, a mechanism involving increased COX- and NOX-mediated ROS. These findings increase our understanding on how extracellular histones enhance systemic inflammatory responses in diseases in which histone release occurs as part of the pathological processes

Extracellular histones disarrange vasoactive mediators reléase through COX-NOS interaction in human endothelial cells

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies

Microparticles Harboring Sonic Hedgehog Morphogen Improve the Vasculogenesis Capacity of Endothelial Progenitor Cells Derived from Myocardial Infarction Patients.

Endothelial progenitor cells (EPC) play a role in endothelium integrity maintenance and regeneration. Decreased numbers of EPC or their impaired function correlates with an increase in cardiovascular events. Thus, EPC are important predictors of cardiovascular mortality and morbidity. Microparticles carrying Sonic hedgehog (Shh) morphogen (MPShhþ) trigger pro-angiogenic responses, both in endothelial cells and in ischaemic rodent models. Here, we propose that MPShhþ regulates EPC function, thus enhancing vasculogenesis, and correcting the defects in dysfunctional EPC obtained from acute myocardial infarction (AMI) patients

Estradiol, acting through estrogen receptor alpha, restores dimethylarginine dimethylaminohydrolase activity and nitric oxide production in oxLDL-treated human arterial endothelial cells

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase. ADMA accumulation, mainly due to a decreased dimethylarginine dimethylaminohydrolase (DDAH) activity, has been related to the development of cardiovascular diseases. We investigate whether estradiol prevents the alterations induced by oxidized low density lipoprotein (oxLDL) on the DDAH/ADMA/NO pathway in human umbilical artery endothelial cells (HUAEC). HUAEC were exposed to estradiol, native LDL (nLDL), oxLDL and their combinations for 24h. In some experiments, cells were also exposed to the unspecific estrogen receptor (ER) antagonist ICI 182780, or the specific ERα antagonist MPP. ADMA concentration was measured by HPLC and concentration of NO by amperometry. Protein expression and DDAH activity were measured by immunoblotting and an enzymatic method, respectively. oxLDL, but not to nLDL, increased ADMA concentration with a concomitant decrease of DDAH activity. oxLDL reduced eNOS protein and NO production. Estradiol alone had no effects on DDAH/ADMA/NO pathway, but increased the attenuated endothelial NO production induced by oxLDL by restoring the DDAH activity. ICI 182780 and MPP completely abolished these effects of estradiol on oxLDL-exposed cells. In conclusion, estradiol restores DDAH activity ADMA levels and NO production impaired by oxLDL in HUAEC acting through ERα

Extracellular histones activate autophagy and apoptosis via mTOR signaling in human endothelial cells

Circulating histones have been proposed as targets for therapy in sepsis and hyperinflammatory symptoms. However, the proposed strategies have failed in clinical trials. Although different mechanisms for histone-related cytotoxicity are being explored, those mediated by circulating histones are not fully understood. Extracellular histones induce endothelial cell death, thereby contributing to the pathogenesis of complex diseases such as sepsis and septic shock. Therefore, the comprehension of cellular responses triggered by histones is capital to design effective therapeutic strategies. Here we report how extracellular histones induce autophagy and apoptosis in a dose-dependent manner in cultured human endothelial cells. In addition, we describe how histones regulate these pathways via Sestrin2/AMPK/ULK1-mTOR and AKT/mTOR. Furthermore, we evaluate the effect of Toll-like receptors in mediating autophagy and apoptosis demonstrating how TLR inhibitors do not prevent apoptosis and/or autophagy induced by histones. Our results confirm that histones and autophagic pathways can be considered as novel targets to design therapeutic strategies in endothelial damage

Microparticles harbouring Sonic hedgehog morphogen improve the vasculogenesis capacity of endothelial progenitor cells derived from myocardial infarction patients

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Estradiol, acting through ERα, induces endothelial non-classic renin-angiotensin system increasing angiotensin 1-7 production.

Intracellular renin-angiotensin system (RAS) can operate independently of the circulating RAS. Estrogens provide protective effects by modulating the RAS. Our aim was to investigate the effect of estradiol (E2) on angiotensin converting enzymes (ACE) 1 and ACE2 expression and activities in human endothelial cells (HUVEC), and the role of estrogen receptors (ER). The results confirmed the presence of active intracellular RAS in HUVEC. Physiological concentrations of E2 induced a concentration-dependent increase of ACE1 and ACE2 mRNA expression and ACE1, but not ACE2, protein levels. ACE1 and ACE2 enzymatic activities were also induced with E2. These effects were mediated through ERα activation, since ER antagonists ICI 182780 and MPP completely abolished the effect of E2. Moreover, the ERα agonist PPT mirrored the E2 effects on ACE1 and ACE2 protein expression and activity. Exposure of endothelial cells to E2 significantly increased Ang-(1–7) production. In conclusion, E2 increases Ang-(1–7) production, through ERα, involving increased ACE1 and ACE2 mRNA expression and activity and ACE1 protein levels.Fil: Mompeón, Ana. Universidad de Valencia; EspañaFil: Lázaro Franco, Macarena. Universidad de Valencia; EspañaFil: Bueno Betí, Carlos. Universidad de Valencia; EspañaFil: Pérez Cremades, Daniel. Universidad de Valencia; EspañaFil: Vidal Gómez, Xavier. Universidad de Valencia; EspañaFil: Monsalve, Elena. Universidad de Valencia; EspañaFil: Gironacci, Mariela Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Hermenegildo, Carlos. Universidad de Valencia; EspañaFil: Novella, Susana. Universidad de Valencia; Españ