Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro

BACKGROUND Platelet-rich concentrates are used as a source of growth factors to improve the healing process. The diverse preparation protocols and the gaps in knowledge of their biological properties complicate the interpretation of clinical results. QUESTIONS/PURPOSES In this study we aimed to (1) analyze the concentration and kinetics of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF), leukocyte- and platelet-rich plasma (L-PRP), and natural blood clot during in vitro culture; (2) investigate the migration of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as a functional response to the factors released; and (3) uncover correlations between individual growth factors with the initial platelet/leukocyte counts or the induced cell migration. METHODS L-PRF, L-PRP, and natural blood clot prepared from 11 donors were cultured in vitro for 28 days and media supernatants collected after 8 hours and 1, 3, 7, 14, and 28 days. Released transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin growth factor (IGF-1), platelet-derived growth factor AB (PDGF-AB), and interleukin-1β (IL-1β) were measured in the supernatants with enzyme-linked immunosorbent assay. Migration of MSC and HUVEC induced by the supernatants was evaluated in Boyden chambers. RESULTS More TGF-ß1 was released (mean ± SD in pg/mL of blood) from L-PRF (37,796 ± 5492) compared with L-PRP (23,738 ± 6848; p < 0.001) and blood clot (3739 ± 4690; p < 0.001), whereas more VEGF and IL-1ß were released from blood clot (1933 ± 704 and 2053 ± 908, respectively) compared with both L-PRP (642 ± 208; p < 0.001 and 273 ± 386; p < 0.001, respectively) and L-PRF (852 ± 376; p < 0.001 and 65 ± 56, p < 0.001, respectively). No differences were observed in IGF-1 and PDGF-AB released from any of the concentrates. TGF-β1 release peaked at Day 7 in L-PRF and at 8 hours and Day 7 in L-PRP and 8 hours and Day 14 in blood clot. In all concentrates, main release of VEGF occurred between 3 and 7 days and of IL-1β between Days 1 and 7. IGF-1 and PDGF-AB were released until Day 1 in L-PRP and blood clot, in contrast to sustained release over the first 3 days in L-PRF. The strongest migration of MSC occurred in response to L-PRF, and more HUVEC migration was seen in L-PRF and blood clot compared with L-PRP. TGF-β1 correlated with initial platelet counts in L-PRF (Pearson r = 0.66, p = 0.0273) and initial leukocyte counts in L-PRP (Pearson r = 0.83, p = 0.0016). A positive correlation of IL-1β on migration of MSC and HUVEC was revealed (Pearson r = 0.16, p = 0.0208; Pearson r = 0.31, p < 0.001). CONCLUSIONS In comparison to L-PRP, L-PRF had higher amounts of released TGF-β1, a long-term release of growth factors, and stronger induction of cell migration. Future preclinical studies should confirm these data in a defined injury model. CLINICAL RELEVANCE By characterizing the biologic properties of different platelet concentrates in vitro, we may gain a better understanding of their clinical effects and develop guidelines for specific future applications.

Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue-Like Environments in PEG Hydrogels.

In vitro engineered tissues which recapitulate functional and morphological properties of bone marrow and bone tissue will be desirable to study bone regeneration under fully controlled conditions. Among the key players in the initial phase of bone regeneration are mesenchymal stem cells (MSCs) and endothelial cells (ECs) that are in close contact in many tissues. Additionally, the generation of tissue constructs for in vivo transplantations has included the use of ECs since insufficient vascularization is one of the bottlenecks in (bone) tissue engineering. Here, 3D cocultures of human bone marrow derived MSCs (hBM-MSCs) and human umbilical vein endothelial cells (HUVECs) in synthetic biomimetic poly(ethylene glycol) (PEG)-based matrices are directed toward vascularized bone mimicking tissue constructs. In this environment, bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) promotes the formation of vascular networks. However, while osteogenic differentiation is achieved with BMP-2, the treatment with FGF-2 suppressed osteogenic differentiation. Thus, this study shows that cocultures of hBM-MSCs and HUVECs in biological inert PEG matrices can be directed toward bone and bone marrow-like 3D tissue constructs.

Normal values for pancreatic stone protein in different age groups.

BACKGROUND Pancreatic stone protein (PSP) has been identified as a promising sepsis marker in adults, children and neonates. However, data on population-based reference values are lacking. This study aimed to establish age-specific reference values for PSP. METHODS PSP was determined using a specific ELISA. PSP serum concentrations were determined in 372 healthy subjects including 217 neonates, 94 infants and children up to 16 years, and 61 adults. The adjacent categories method was used to determine which age categories had significantly different PSP concentrations. RESULTS PSP circulating levels were not gender-dependent and ranged from 1.0 to 99.4 ng/ml with a median of 9.2 ng/ml. PSP increased significantly between the age categories, from a median of 2.6 ng/ml in very preterm newborns, to 6.3 ng/ml in term newborns, to 16.1 ng/ml in older children (p < 0.001). PSP levels were higher on postnatal day three compared to levels measured immediately post delivery (p < 0.001). Paired umbilical artery and umbilical vein samples were strongly correlated (p < 0.001). Simultaneously obtained capillary heel-prick versus venous samples showed a good level of agreement for PSP (Rho 0.89, bias 19 %). CONCLUSIONS This study provides age-specific normal values that may be used to define cut-offs for future trials on PSP. We demonstrate an age-dependent increase of PSP from birth to childhood.

The Gas6-Axl Interaction Mediates Endothelial Uptake of Platelet Microparticles.

Upon activation, platelets release plasma-membrane derived microparticles (PMPs) exposing phosphatidylserine (PS) on their surface. The function and clearance mechanism of these MPs are incompletely understood. As they are pro-coagulant and potentially pro-inflammatory, rapid clearance from the circulation is essential for prevention of thrombotic diseases. The tyrosine kinase receptors Tyro3, Axl and Mer (TAMs) and their ligands protein S and Gas6 are involved in the uptake of PS-exposing apoptotic cells in macrophages and dendritic cells. Both TAMs and their ligands are expressed in the vasculature, the functional significance of which is poorly understood. In this study we investigated how vascular TAMs and their ligands may mediate endothelial uptake of PMPs. PMPs, generated from purified human platelets, were isolated by ultracentrifugation and labeled with biotin or PKH67. The uptake of labeled MPs in the presence of protein S and Gas6 in human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) was monitored by flow cytometry, western blotting and confocal/electron microscopy. We found that both endothelial cell types can phagocytose PMPs, and using TAM-blocking antibodies or siRNA knock-down of individual TAMs we show that the uptake is mediated by endothelial Axl and Gas6. As circulating PMPs-levels were not altered in Gas6-/- mice compared to Gas6+/+ mice, we hypothesize that the Gas6-mediated uptake is not a means to clear the bulk of circulating PMPs but may serve to phagocytose PMPs locally generated at sites of platelet activation and as a way to affect endothelial responses.

Targeted cancer diagnosis with radiolabeled endostatin

Nuclear imaging is used for non-invasive detection, staging and therapeutic monitoring of tumors through the use of radiolabeled probes. Generally, these probes are used for applications in which they provide passive, non-specific information about the target. Therefore, there is a significant need for actively-targeted radioactive probes to provide functional information about the site of interest. This study examined endostatin, an endogenous inhibitor of tumor angiogenesis, which has affinity for tumor vasculature. The major objective of this study was to develop radiolabeled analogues of endostatin through novel chemical and radiochemical syntheses, and to determine their usefulness for tumor imaging using in vitro and in vivo models of vascular, mammary and prostate tumor cells. I hypothesize that this binding will allow for a non-invasive approach to detection of tumor angiogenesis, and such detection can be used for therapeutic monitoring to determine the efficacy of anti-angiogenic therapy. ^ The data showed that endostatin could be successfully conjugated to the bifunctional chelator ethylenedicysteine (EC), and radiolabeled with technetium-99m and gallium-68, providing a unique opportunity to use a single precursor for both nuclear imaging modalities: 99mTc for single photon emission computed tomography and 68Ga for positron emission tomography, respectively. Both radiolabeled analogues showed increased binding as a function of time in human umbilical vein endothelial cells and mammary and prostate tumor cells. Binding could be blocked in a dose-dependent manner by unlabeled endostatin implying the presence of endostatin receptors on both vascular and tumor cells. Animal biodistribution studies demonstrated that both analogues were stable in vivo, showed typical reticuloendothelial and renal excretion and produced favorable absorbed organ doses for application in humans. The imaging data provide evidence that the compounds quantitate tumor volumes with clinically-useful tumor-to-nontumor ratios, and can be used for treatment follow-up to depict changes occurring at the vascular and cellular levels. ^ Two novel endostatin analogues were developed and demonstrated interaction with vascular and tumor cells. Both can be incorporated into existing nuclear imaging platforms allowing for potential wide-spread clinical benefit as well as serving as a diagnostic tool for elucidation of the mechanism of action of endostatin. ^

Studies of biological rhythms in responses to influenza infection and vaccination

Three studies examined seasonal or circadian variations in selected responses to influenza infection or vaccination. The first, a seroepidemiologic study, evaluated temporal patterns of antibody titers to influenza A/Texas. Human umbilical cord bloods were sampled over a two-year period when the virus was not present in the community. No endogenous seasonal pattern was detected. The second study included three experiments on circadian rhythms in mice. Neither susceptibility nor protection from inactivated or attenuated vaccine varied significantly according to time of administration. A slight effect, however, was suggested with inactivated vaccine. Three human vaccine trials comprised the third study. Outcome variables included rise in antibody titer, final antibody titer, incidence of adverse reactions, and protection from community infection. Patterns in antibody response and protection variables were inconsistent, and generally not clinically significant. Local reactions to inactivated vaccine were more frequent if injections were received in the afternoon as compared to morning. This was true to adults that had been previously vaccinated. ^

Células madres, un desafío para todos

Fil: Glorio, Roberto. Universidad de Buenos Aires

A sedimentological, faunal, and isotopic record of the middle-to-late Pliocene transition at DSDP Hole 80-548

Hydraulic piston coring at DSDP Site 548, on the upper continental slope southwest of Ireland, recovered a nearly complete Pliocene section spanning 103 m of sediment. The sediments are greenish gray carbonate-rich hemipelagites containing abundant nannofossils and foraminifers. Grain-size analysis demonstrates that the texture of the section is fairly constant, with most of the variation occurring in 63- to 32-µm and < 2-µm fractions. Previous research has shown that the middle-to-late Pliocene transition in the North Atlantic was marked by the appearance of the planktonic foraminiferal species Globorotalia inflata and by the first occurrence of significant quantities of ice-rafted sediment grains in deep-sea sediments. The latter is taken to represent the first important development of Northern Hemisphere glaciation. The first appearance of G. inflata is carefully documented for Site 548 and is demonstrated to be an evolutionary datum at this site, rather than an ecologically controlled first appearance. Surface ocean conditions represented in the sediment section spanning the appearance of G. inflata were strongly cyclic, resulting in large periodic changes in the abundances of Globorotalia puncticulata and N. acostaensis. The benthic foraminiferal population was studied in detail over the middle-to-upper Pliocene transition to establish the nature and behavior of the intermediate-depth water mass in the northeastern Atlantic at the time of ice-sheet growth in the Northern Hemisphere. This water mass is presently warm and saline, having its source in the Mediterranean Sea. The benthic data show that the intermediate-depth water mass was undergoing a series of progressive changes over the interval including the first appearance of G. inflata. These changes are particularly reflected in the relative abundances of Globocassidulina subglobosa (Brady), Uvigerina, and Ehrenbergina. Also, the mean size of individuals in the G. subglobosa populations shows systematic variation, indicating changing intermediate-depth water properties. Oxygen-isotope analyses show that the intermediate-depth water mass was cold during the middle-to-late Pliocene transition. This interpretation is supported by the relative abundances of benthic foraminiferal species. Hence, the intermediate-depth northeastern Atlantic water mass of the middle to late Pliocene was considerably different from the intermediate-depth water mass of the present.

Desarrollo de tecnologías de telemanipulación con alto grado de escalado orientadas a la interacción hombre-robot en entornos nucleares

Esta tesis se centra en desarrollo de tecnologías para la interacción hombre-robot en entornos nucleares de fusión. La problemática principal del sector de fusión nuclear radica en las condiciones ambientales tan extremas que hay en el interior del reactor, y la necesidad de que los equipos cumplan requisitos muy restrictivos para poder aguantar esos niveles de radiación, magnetismo, ultravacío, temperatura... Como no es viable la ejecución de tareas directamente por parte de humanos, habrá que utilizar dispositivos de manipulación remota para llevar a cabo los procesos de operación y mantenimiento. En las instalaciones de ITER es obligatorio tener un entorno controlado de extrema seguridad, que necesita de estándares validados. La definición y uso de protocolos es indispensable para regir su buen funcionamiento. Si nos centramos en la telemanipulación con algo grado de escalado, surge la necesidad de definir protocolos para sistemas abiertos que permitan la interacción entre equipos y dispositivos de diversa índole. En este contexto se plantea la definición del Protocolo de Teleoperación que permita la interconexión entre dispositivos maestros y esclavos de distinta tipología, pudiéndose comunicar bilateralmente entre sí y utilizar distintos algoritmos de control según la tarea a desempeñar. Este protocolo y su interconectividad se han puesto a prueba en la Plataforma Abierta de Teleoperación (P.A.T.) que se ha desarrollado e integrado en la ETSII UPM como una herramienta que permita probar, validar y realizar experimentos de telerrobótica. Actualmente, este Protocolo de Teleoperación se ha propuesto a través de AENOR al grupo ISO de Telerobotics como una solución válida al problema existente y se encuentra bajo revisión. Con el diseño de dicho protocolo se ha conseguido enlazar maestro y esclavo, sin embargo con los niveles de radiación tan altos que hay en ITER la electrónica del controlador no puede entrar dentro del tokamak. Por ello se propone que a través de una mínima electrónica convenientemente protegida se puedan multiplexar las señales de control que van a través del cableado umbilical desde el controlador hasta la base del robot. En este ejercicio teórico se demuestra la utilidad y viabilidad de utilizar este tipo de solución para reducir el volumen y peso del cableado umbilical en cifras aproximadas de un 90%, para ello hay que desarrollar una electrónica específica y con certificación RadHard para soportar los enormes niveles de radiación de ITER. Para este manipulador de tipo genérico y con ayuda de la Plataforma Abierta de Teleoperación, se ha desarrollado un algoritmo que mediante un sensor de fuerza/par y una IMU colocados en la muñeca del robot, y convenientemente protegidos ante la radiación, permiten calcular las fuerzas e inercias que produce la carga, esto es necesario para poder transmitirle al operador unas fuerzas escaladas, y que pueda sentir la carga que manipula, y no otras fuerzas que puedan influir en el esclavo remoto, como ocurre con otras técnicas de estimación de fuerzas. Como el blindaje de los sensores no debe ser grande ni pesado, habrá que destinar este tipo de tecnología a las tareas de mantenimiento de las paradas programadas de ITER, que es cuando los niveles de radiación están en sus valores mínimos. Por otro lado para que el operador sienta lo más fielmente posible la fuerza de carga se ha desarrollado una electrónica que mediante el control en corriente de los motores permita realizar un control en fuerza a partir de la caracterización de los motores del maestro. Además para aumentar la percepción del operador se han realizado unos experimentos que demuestran que al aplicar estímulos multimodales (visuales, auditivos y hápticos) aumenta su inmersión y el rendimiento en la consecución de la tarea puesto que influyen directamente en su capacidad de respuesta. Finalmente, y en referencia a la realimentación visual del operador, en ITER se trabaja con cámaras situadas en localizaciones estratégicas, si bien el humano cuando manipula objetos hace uso de su visión binocular cambiando constantemente el punto de vista adecuándose a las necesidades visuales de cada momento durante el desarrollo de la tarea. Por ello, se ha realizado una reconstrucción tridimensional del espacio de la tarea a partir de una cámara-sensor RGB-D, lo cual nos permite obtener un punto de vista binocular virtual móvil a partir de una cámara situada en un punto fijo que se puede proyectar en un dispositivo de visualización 3D para que el operador pueda variar el punto de vista estereoscópico según sus preferencias. La correcta integración de estas tecnologías para la interacción hombre-robot en la P.A.T. ha permitido validar mediante pruebas y experimentos para verificar su utilidad en la aplicación práctica de la telemanipulación con alto grado de escalado en entornos nucleares de fusión. Abstract This thesis focuses on developing technologies for human-robot interaction in nuclear fusion environments. The main problem of nuclear fusion sector resides in such extreme environmental conditions existing in the hot-cell, leading to very restrictive requirements for equipment in order to deal with these high levels of radiation, magnetism, ultravacuum, temperature... Since it is not feasible to carry out tasks directly by humans, we must use remote handling devices for accomplishing operation and maintenance processes. In ITER facilities it is mandatory to have a controlled environment of extreme safety and security with validated standards. The definition and use of protocols is essential to govern its operation. Focusing on Remote Handling with some degree of escalation, protocols must be defined for open systems to allow interaction among different kind of equipment and several multifunctional devices. In this context, a Teleoperation Protocol definition enables interconnection between master and slave devices from different typologies, being able to communicate bilaterally one each other and using different control algorithms depending on the task to perform. This protocol and its interconnectivity have been tested in the Teleoperation Open Platform (T.O.P.) that has been developed and integrated in the ETSII UPM as a tool to test, validate and conduct experiments in Telerobotics. Currently, this protocol has been proposed for Teleoperation through AENOR to the ISO Telerobotics group as a valid solution to the existing problem, and it is under review. Master and slave connection has been achieved with this protocol design, however with such high radiation levels in ITER, the controller electronics cannot enter inside the tokamak. Therefore it is proposed a multiplexed electronic board, that through suitable and RadHard protection processes, to transmit control signals through an umbilical cable from the controller to the robot base. In this theoretical exercise the utility and feasibility of using this type of solution reduce the volume and weight of the umbilical wiring approximate 90% less, although it is necessary to develop specific electronic hardware and validate in RadHard qualifications in order to handle huge levels of ITER radiation. Using generic manipulators does not allow to implement regular sensors for force feedback in ITER conditions. In this line of research, an algorithm to calculate the forces and inertia produced by the load has been developed using a force/torque sensor and IMU, both conveniently protected against radiation and placed on the robot wrist. Scaled forces should be transmitted to the operator, feeling load forces but not other undesirable forces in slave system as those resulting from other force estimation techniques. Since shielding of the sensors should not be large and heavy, it will be necessary to allocate this type of technology for programmed maintenance periods of ITER, when radiation levels are at their lowest levels. Moreover, the operator perception needs to feel load forces as accurate as possible, so some current control electronics were developed to perform a force control of master joint motors going through a correct motor characterization. In addition to increase the perception of the operator, some experiments were conducted to demonstrate applying multimodal stimuli (visual, auditory and haptic) increases immersion and performance in achieving the task since it is directly correlated with response time. Finally, referring to the visual feedback to the operator in ITER, it is usual to work with 2D cameras in strategic locations, while humans use binocular vision in direct object manipulation, constantly changing the point of view adapting it to the visual needs for performing manipulation during task procedures. In this line a three-dimensional reconstruction of non-structured scenarios has been developed using RGB-D sensor instead of cameras in the remote environment. Thus a mobile virtual binocular point of view could be generated from a camera at a fixed point, projecting stereoscopic images in 3D display device according to operator preferences. The successful integration of these technologies for human-robot interaction in the T.O.P., and validating them through tests and experiments, verify its usefulness in practical application of high scaling remote handling at nuclear fusion environments.

O ESTUDANTE DA EAD (EDUCAÇÃO A DISTÂNCIA): UM ESTUDO DE PERFIL E INTERAÇÃO GERACIONAL

A aprendizagem formal e tradicional tem dado lugar a um cenário desafiador no qual educador e educando não comungam do mesmo espaço físico. A Educação a Distância (EAD), ainda é vista como uma solução que agrega cada vez mais alunos de diferentes idades que desejam uma graduação de ensino superior ou a continuidade dela. A pesquisa com o título: “O estudante da EAD (educação a distância): um estudo de perfil e interação geracional” propõe conhecer as características do perfil atual do estudante da EAD, abordando o diálogo entre as gerações no ambiente social escolar. O enfoque da pesquisa é qualitativa, exploratória e descritiva com dados que foram coletados através de entrevista com 08 alunos das gerações X e Y para assim entender se este perfil tem sido renovado com alunos mais jovens, do que a faixa etária de 25 a 45 anos. O resultado demonstra que alunos na faixa de 17 a 24 anos a cada ano aumentam 1% das matrículas. Já a faixa de 25 a 45 anos prevalece com 70% das matrículas. Portanto, este resultado revela que o perfil do aluno EAD ainda é o do jovem adulto, para adulto mais experiente, que busca a graduação com o propósito de progressão no ambiente profissional. As duas gerações citadas geração X e geração Y, mesmo em contextos históricos diferenciados de valores, crenças e comportamentos participam atualmente de uma transformação social que contempla os meios de produção do trabalho, a formação educacional e as relações sociais. O diálogo intergeracional direciona a um aprendizado compartilhado, participativo na troca de experiências mutuas. Para a geração X o jovem atual não é mais nomeado como o que precisa escutar e aprender, mas tem muito a partilhar, principalmente diante da facilidade com os meios tecnológicos. E para a geração Y, na partilha não há barreiras de idade, mas a segurança de interagir e se comunicar diante da troca de experiências

Alloantigen-induced unresponsiveness in cord blood T lymphocytes is associated with defective activation of Ras

Human umbilical cord blood T lymphocytes (CBTL) respond to primary allostimulation but they do not proliferate upon rechallenge with alloantigen. Using PKH-26-labeled cells created a proliferative block that was observed only in CBTL that have divided during primary stimulation (PKH-26dim) but not in unstimulated (PKH-26bright) CBTL. CBTL’s secondary unresponsiveness resembles anergy and can be overcome by treatment with phorbol myristate acetate (PMA) and ionomycin or by high doses (50–100 units/ml) of interleukin 2. Addition of interleukin 2 to the primary cultures does not prevent the induction of secondary unresponsiveness. Defective Ras activation is detected in PKH-26dim CBTL during secondary response to alloantigen or after antibody-mediated T cell receptor stimulation whereas Ras is activated and proliferation is induced in CBTL during primary alloantigenic stimulation. Upon stimulation with PMA plus ionomycin, PMA plus alloantigen, but not alloantigen plus ionomycin, Ras is activated in PKH-26dim CBTL, and the block in proliferation is overcome. Correction of PKH-26dim CBTL’s proliferative defect correlates with PMA-induced Ras activation, suggesting a defect in the signaling pathway leading to Ras. Ras-independent signals, necessary but not sufficient to induce PKH-26dim CBTL proliferation, are provided by alloantigen exposure, as evident by the ability of PMA plus alloantigen but not PMA alone to overcome the proliferative block. Functional signal transduction through CD28 in PKH-26dim CBTL is supported by detectable CD28-mediated PI-3 kinase activation after PKH-26dim CBTL’s exposure to alloantigen or CD28 cross-linking. These results suggest that defective activation of Ras plays a key role in PKH-26dim CBTL’s secondary unresponsiveness and point to a defect along the T cell receptor rather than the CD28 signaling pathway.

Immunotargeting of liposomes to activated vascular endothelial cells: A strategy for site-selective delivery in the cardiovascular system

Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1β (IL-1β). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1β-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2–4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.

Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na+ and K+ and urinary Na+ and K+ excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO2−/NO3− (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-d,l-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60–70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to ≈70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.

Tumor necrosis factor-α induces adhesion molecule expression through the sphingosine kinase pathway

The signaling pathways that couple tumor necrosis factor-α (TNFα) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFα induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFα resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFα on endothelial cells leading to extracellular signal-regulated kinases and NF-κB activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFα-induced extracellular signal-regulated kinases and NF-κB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFα-induced endothelial cell activation.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.