En este trabajo de tesis se ha investigado la relación de las enzimas esfingosina quinasa 1 (SphK1) y hemoxigenasa 1 (HO-1) con el cáncer. En el caso de la primera de estas enzimas se ha podido demostrar que se encuentra sobre expresada en carcinomas escamosos de cabeza y cuello y que su presencia se correlaciona con una menor sobrevida de los pacientes. Se ha aportado evidencia indicando que dicha correlación puede ser la manifestación de una relación de causa y efecto en la que esta quinasa estimula la proliferación celular disminuyendo los niveles de p21Cip1, e inhibe la apoptosis al disminuir los niveles de caspasa 3 clivada e incrementar los de p-Bad. En cuanto a HO-1 se ha explorado su relación con el cáncer en el carcinoma escamoso de cabeza y cuello y en cáncer de pul-món y de mama, tanto en tumores humanos como en modelos animales. En carcinomas escamosos de cabeza y cuello se ha demostrado que HO-1 se encuentra sobre expresada y que se correlaciona con el grado tumoral. Se ha comprobado que la enzima presenta localización nuclear además de su típica localización citoplasmática, que dicha localización es mayor en las células tumorales que en los tejidos epiteliales adyacentes y que se correlaciona con tumores más agresivos y de peor pronóstico y con la progresión tumoral. En cáncer de pulmón HO-1 se detectó en las células epiteliales del tumor mos-trando una localización principalmente citoplasmática en comparación con tejidos pulmonares de aspecto normal donde los niveles de expresión fueron menores y con localización nuclear más frecuente. También se observó que se correla-cionaba con estadios más avanzados de la enfermedad, con el tamaño tumoral y con la presencia de metástasis ganglionares. En cáncer de mama se han realizado por primera vez estudios de expresión de HO-1 en tumores humanos comprobándose que se correlaciona con una mayor sobrevida de los pacien-tes. Se ha identificado un modelo animal adecuado para estu-diar la relación de la enzima con este tipo de cáncer. Utilizan-do este modelo se ha comprobado que la activación de la misma conduce a un menor desarrollo tumoral. Se ha aportado evidencia indicando que los mecanismos que conducen a esta disminución del volumen tumoral, y tal vez también a la mayor sobrevida global de los pacientes, son la disminución en la supervivencia celular, debida a un efecto pro apoptótico, y la inhibición de la migración celular producidas por esta enzima. También se ha comprobado en este carcinoma la localización nuclear de la enzima y se ha aportado evidencia indicando que una forma truncada en el extremo carboxilo terminal se localiza en el núcleo. Esta localización nuclear no se correla-ciona con la sobrevida global de los pacientes. Finalmente, los resultados obtenidos en esta tesis sugieren que ambas enzi-mas pueden ser de potencial interés como factores pronós-ticos y/o blancos terapéuticos y señalan la necesidad de continuar investigando los mecanismos celulares y moleculares que la desregulación de estas enzimas podría estar afectan-do contribuyendo así al desarrollo del cáncer. / In this thesis the relation between the enzymes sphingosine kinase 1 and heme oxygenase 1 with cancer has been inves-tigated. In the case of the former one it has been demons-trated that it is over expressed in head neck squamous cell carcinoma and that its presence correlates with a shorter patient survival. Evidence has been provided showing that such correlation could be due to a cause-effect relationship in which this kinase stimulates cellular proliferation by down regulation of p21Cip1 levels and inhibits apoptosis through a decrease in the levels of cleaved caspase 3 and an increase in p-Bad. Regarding heme oxigenase 1, its relation with cancer has been investigated in head neck squamous cell carcinoma, lung and breast cancer both in human tumors and animal models. In head and neck squamous cell carcinoma the enzyme was shown to be over expressed and to correlate with tumor grade. Furthermore, nuclear localization of the enzyme has also been demonstrated. It has also been detec-ted that such nuclear localization is more frequent in tumor cells that in adjacent non malignant tissues. Furthermore this nuclear localization correlates with lower differentiation gra-des and with tumor progression. In lung cancer the heme oxygenase 1 has been detected mainly in the cytoplasm of epithelial cells whereas it was more frequently localized in the nucleus in normal lung tissues. Additionally, enzyme levels have been demonstrated to be higher in tumor tissues than in non malignant adjacent ones and to correlate with advanced stages of the disease, with tumor size and with lymph node metastasis. This is the first study showing heme oxygenase 1 protein expression in human breast tumor tissues and its correlation with longer patient survival. This research has also identified an adequate animal model to study the relation between the enzyme and this type of cancer. In this animal model the enzyme activation leads to a decrease in tumor burden. Evidence has also been provided showing that the mechanisms responsible for this decrease in the tumor volume are the reduction in cellular survival due to a pro apoptotic effect and the inhibition of cellular migration. A nuclear locali-zation of the enzyme in this tumor type has also been de-monstrated. Evidence has also been provided that this nuclear heme oxygenase 1 is a C-terminal truncated protein. This nuclear localization does not correlate with patient sur-vival. Finally, the results obtained in this thesis suggest that both enzymes could be potentially interesting as prognostic factors and/or therapeutic targets and they encourage future investigations regarding the cellular and molecular mecha-nisms modulated by these enzymes and whose deregulation could contribute to cancer development.

Orientador: Carlos Roque Duarte Correia / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-11T11:57:03Z (GMT). No. of bitstreams: 1 Azevedo_LuizFabriciodaSilva_M.pdf: 1610950 bytes, checksum: 7d31330e73f1ffeb923f119448edcc2f (MD5) Previous issue date: 2008 / Resumo: Esfingolipídios são compostos naturais que apresentam uma miríade de atividades biológicas conhecidas. A esfingosina é o exemplo mais representativo desta classe de compostos. Este trabalho está relacionado com a síntese de um análogo cíclico da esfingosina. A primeira parte esteve relacionada com a preparação do hidroxilactol F. Inicialmente o enecarbamato B foi preparado a partir da 2-pirrolidinona A através de 2 metodologias; a mais eficiente realizada em "one-pot" com rendimento global de 60%. A funcionalização da dupla endociclica do enecarbamato B foi efetuada com sucesso a partir da reação de cicloadição do tipo [2+2] com dicloroceteno. Esta reação levou a formação da a, a-diclorobutanona C em excelente rendimento (90%). A remoção dos cloros de C com um liga de Zn/Cu em uma solução metanóica de NH4Cl, levou a obtenção da ciclobutanona D em moderado rendimento (50%). A irradiação ultravioleta na presença de ácido acético, seguida pela substituição do grupamento acetil, com BF3-OEt2, pelo grupamento tiofenol, e finalmente a eliminação em meio básico sob refluxo, forneceu o diidrofurano E em bom rendimento global (4 etapas em 53 %). A reação de diidroxilação do diidrofurano E com OsO4 realizada em bom rendimento (87%) completou a sintese do hidroxilactol F. A segunda parte deste trabalho esteve realizada com os estudos visandose à síntese de um análogo cíclico da esfingosina. A melhor rota encontrada foi a reação do hidroxilactol F com [Ph3PCH3]+Br- que levou a obtenção da olefina desejada em baixo rendimento. A reação de metátese com 1-octadeceno, realizada em bom rendimento (80%) e a desproteção de G com Et3SiH e CF3CO2H alcançada em bom rendimento (89%), completaram a síntese do composto. A síntese convergente do análogo cíclico da esfingosina, partindo-se da 2- pirrolidinona A foi alcançada em 14 etapas e com rendimento global de 14% / Abstract: Sphingosine are natural compounds that bear multiple known biological activities. The sphingosine molecule is representative of this class of biological compounds. The present study is related to the synthesis of a new cyclic analogue of sphingosine. The first part of this dissertation was focused on the synthesis of the hydroxy lactol F. The synthesis began with distinct methodologies: The most efficient one was realized by a ¿one-pot¿ procedure to provide the enecarbamate B in 60% overall yield. The endocyclic double bond funcionalization of B was performed with sucess employing a [2+2] cycloaddition reaction with dichloroketene. This reaction yielded the corresponding a,a-dichlorocyclobutanone C in excellent yields (90%). The removal of the chlorine atoms of C was carried out using a Zn/Cu alloy in a methanol solution of NH4Cl, to give the cyclobutanone D in moderate yieelds (50%). Ultraviolet irradiation of D in the presence of acetic acid, followed by replacement of the acethyl group by thiophenol, promoted by BF3-OEt2, and elimination in basic medium under reflux, provided the dihydrofuran intermediate E in good overall yields (53% over 4 steps). Finally, stereoselective dihydroxylation of E with OsO4 furnished the hydroxylactol F in 87% yield. In the second part of this dissertation we focused on the synthesis of the cyclic analogue of sphingosine. The best route examined involved the olefination of the intermediate hydrolactol F wiht [Ph3PCH3]+Br- to provide the desired trans olefin in low yields. Olefin metathesis of this olefin with 1-octadecene gave intermediate G in a good yield of 80%. Next, the Boc protected olefin G was deprotected with Et3SiH and CF3CO2H to provide the desired cyclic analogue of sphingosine in 89% yield. The stereocontrolled total synthesis of this new cyclic analogue of sphingosine was accomplished from 2-pyrrolidinone A in 11 steps with an overall yield of 14% / Mestrado / Quimica Organica / Mestre em Química

Desde hace casi tres décadas, los esfingolípidos han entrado a la escena de los lípidos bioactivos. Dentro de esta gran familia, son sus versiones más simples los que han sido relacionados con importantes funciones celulares. La Ceramida (Cer), y su derivado por deacilación, la esfingosina (Sph), han sido reportados como segundos mensajeros de muerte en diversos tipos celulares. Por su parte, sus derivados fosforilados, en especial la esfingosina-1-fosfato (S1P), se han identificado con funciones proliferativas y pro-supervivencia. Diversas señales mitogénicas contribuyen no solo a la proliferación sino también a la migración celular, siendo de especial interés en ciertas patologías como el cáncer. Sin embargo, los procesos celulares invasivos son relevantes también en la retina, el tejido de interés de este trabajo de tesis. Diversas patologías, como la retinopatía diabética, presentan una proliferación exacerbada de las células gliales de Müller (CGM), el principal tipo glial de la retina, proceso conocido como gliosis. En su inicio, esta respuesta es un intento fisiológico por aislar al tejido neuronal de la injuria y reparar el daño. Sin embargo, en un estado patológico, ante la continuidad de la injuria, esta proliferación celular termina convirtiéndose en una cicatriz que afecta el normal funcionamiento del tejido retiniano, llevando en última instancia a la pérdida parcial o total de la visión. Por lo expuesto, resulta relevante investigar estos procesos proliferativos de la retina a fin de encontrar nuevos tratamientos terapéuticos. La información sobre las funciones del esfingolípido mitogénico S1P a nivel de la retina es escasa. Por ejemplo, se ha reportado que la S1P promueve la proliferación y respuestas pro-fibróticas y pro-inflamatorias en células de epitelio pigmentario y que participa en la neo-vascularización retinal y coroidal. Además, los niveles retinales y plasmáticos de S1P aumentan en un modelo inflamatorio inducido por lipopolisacárido. En el primer capítulo de este trabajo de tesis, planteamos la hipótesis de que la S1P podría estar involucrada en los procesos glióticos, participando como una señal clave para inducir la migración glial. Para contrastar nuestra hipótesis, utilizamos un modelo de cultivo primario de células gliales de Müller y analizamos la migración glial mediante el “ensayo de la herida”, un método sencillo y ampliamente utilizado para este propósito. Nuestros experimentos revelaron que la adición exógena de S1P induce modificaciones en el citoesqueleto de las CGM, detectadas en las primeras 8-10 h de tratamiento, que llevan a la formación de lamelipodios y al inicio de la motilidad glial sobre el sustrato. Esta motilidad glial fue claramente superior a la de los cultivos controles luego de 18 h de estímulo. Las CGM expresaron dos de los cinco receptores transmembrana para S1P, los S1PR, específicamente los subtipos S1P1 y S1P3, siendo la activación de este último la que desencadena el proceso migratorio, tal como lo demostraron experimentos utilizando antagonistas selectivos de ambos receptores. Nuestros resultados también mostraron que rio-abajo de la activación del S1P3 se activarían dos importantes vías intracelulares, la vía de ERK/MAPK y la vía de PI3K, ya que el pre-tratamiento con inhibidores de estas vías disminuyó significativamente el estímulo migratorio inducido por S1P. Cabe destacar que esta inducción del proceso migratorio no estuvo acompañada de un incremento significativo en la proliferación glial. Por otra parte, no solo la S1P exógena tuvo un rol en la migración glial. Encontramos que la inhibición de la síntesis endógena de S1P, utilizando un inhibidor selectivo de la enzima esfingosina quinasa 1 (Sphk1) (enzima que cataliza la síntesis de S1P), el inhibidor de SphK1 2 (SphKI2), prácticamente abolió la migración glial basal, es decir aquella observada aun en ausencia de S1P. Estos resultados demuestran que la S1P tiene un rol clave en la inducción de migración glial en la retina, y su bloqueo podría ser utilizado como una estrategia terapéutica para las enfermedades proliferativas de la retina. Otro conjunto de patologías de la retina, de creciente avance y lamentablemente aun sin tratamientos efectivos, son las enfermedades neurodegenerativas. Las mismas son un conjunto de patologías, entre las que destacan la retinitis pigmentosa y la degeneración de la mácula con una variada etiología, con múltiples factores, desde genéticos hasta ambientales como desencadenantes, pero que comparten un denominador común: la muerte de los fotorreceptores (FR). Esta degeneración progresiva de los FR lleva, como consecuencia final, a la pérdida de la visión. Como se describió, Cer y Sph son importantes mediadores de muerte celular y han despertado la atención en el campo de las enfermedades neurodegenerativas. Estudios pioneros encontraron que los niveles de Cer se encontraban aumentados en retinas pos mortem de pacientes con la enfermedad de Faber, y que los niveles de este esfingolípido aumentaban frente a la degeneración neuronal inducida por desprendimiento de retina. Posteriores reportes, incluidos los de nuestro laboratorio, establecieron que la Cer es un mediador clave en la muerte de los FR en cultivo. El daño oxidativo estimula la síntesis de Cer que desencadena la muerte de los FR y este proceso es mimetizado por la adición exógena de un análogo sintético de ceramidas endógenas, la C2-Ceramida (C2Cer). Hallazgos posteriores en diversos modelos animales de neurodegeneración retiniana permiten proponer a la Cer como un mediador central en la activación de la muerte de los FR. Pese a esto, aún no se conoce en profundidad cuáles son los mecanismos moleculares involucrados en este proceso. Esta pregunta resulta relevante, ya que la regulación de los distintos blancos intracelulares de Cer podría emerger como posible terapia para las enfermedades neurodegenerativas de la retina. Ante esto, nos planteamos como objetivo en el 2º capítulo de esta Tesis, estudiar los mecanismos moleculares involucrados en la muerte de los FR mediada por C2Cer, utilizando un modelo de cultivo primario de neuronas de retina de rata. Un trabajo previo de nuestro laboratorio demostró que la C2Cer, en una concentración de 10 μM induce la muerte de los FR y las neuronas amacrinas, los tipos neuronales mayoritarios en los cultivos. Estudiando el proceso de muerte de manera cronológica, encontramos que el tratamiento de cultivos neuronales con C2Cer 10 μM durante 6 h indujo selectivamente la muerte de los FR, disminuyendo el potencial de membrana mitocondrial y aumentando la formación de especies reactivas de oxígeno (ROS). En contraste, las neuronas amacrinas preservaron su viabilidad. Cabe destacar que la cantidad de células marcadas con TUNEL y de FR que expresaron caspasa-3 clivada, forma activa de la caspasa-3, permanecieron constantes y que el pretratamiento con un pan inhibidor de caspasas no evitó la muerte inducida por C2Cer. C2Cer provocó la sobre activación de la poli-ADP ribosil polimerasa-1 (PARP-1). La inhibición de PARP-1 disminuyó la muerte de los FR inducida por C2Cer. A su vez, C2Cer aumentó los niveles de polímero de poli-ADP ribosa (PAR), cuya síntesis es catalizada por PARP-1, e indujo la translocación del factor inductor de apoptosis (AIF) de las mitocondrias al núcleo de los FR, ya que dicha translocación se previno mediante la inhibición de PARP-1. El pretratamiento con un inhibidor de calpaínas y catepsinas, y con un inhibidor de la calpaína redujo la muerte de los FR, mientras que los inhibidores selectivos de catepsinas no otorgaron protección. El pretratamiento combinado con un inhibidor de PARP-1 y un inhibidor de calpaína evidenció la misma protección que cada inhibidor por sí mismo. Esto sugiere que tanto PARP-1 como calpaínas confluyen en el mismo mecanismo de acción rio-abajo de la C2Cer. Ni la autofagia ni la necroptosis estuvieron involucradas en la muerte inducida por C2Cer; no se observó un aumento en la liberación de LDH después del tratamiento con C2Cer y el pretratamiento con inhibidores de la necroptosis y de la autofagia no rescataron a los FR. Estos resultados sugieren que la C2Cer activa la muerte de los FR por un mecanismo novedoso, independiente de caspasas, que implica la activación de PARP-1, disminución del potencial de membrana mitocondrial, activación de calpaína y translocación de AIF, todas las características bioquímicas del tipo de muerte celular denominada parthanatos. Nuestros resultados, sugieren así nuevos blancos terapéuticos, y contribuyen a los avances en la búsqueda de tratamientos efectivos para las enfermedades neurodegenerativas de la retina. / For almost three decades, sphingolipids have entered the bioactive lipid scenery. Within this large family, simple sphingolipids are the ones that have been related to important cellular functions. Ceramide (Cer), and its deacylation derivative, sphingosine (Sph), have been reported as death second messengers in several cell types. On the other hand, its phosphorylated derivatives, especially sphingosine-1-phosphate (S1P) have been related to proliferative and pro-survival functions. Several mitogenic signals contribute not only to proliferation but also to cell migration, being of special interest in certain pathologies such as cancer. However, invasive cellular processes are also relevant in the retina, the tissue of interest in this thesis work. Diverse pathologies, such as diabetic retinopathy, show an exacerbated proliferation of Müller glial cells (CGM, the main glial type of the retina), a process known as gliosis. Initially, this response is a physiological attempt to isolate neuronal tissue from injury and repair the damage. However, in a pathological state, given the continuity of the injury, this cell proliferation gives rise to a scar that affects the normal functioning of the retinal tissue, ultimately leading to a partial or total vision loss. Therefore, it is relevant to investigate these retinal proliferative processes in order to find new therapeutic treatments. There is scarce information on the functions of the mitogenic sphingolipid S1P in the retina. S1P has been reported to promote proliferation, pro-fibrotic and pro-inflammatory responses in pigment epithelial cells and it also participates in retinal and choroidal neovascularization. In addition, there is an increase in retinal and plasma levels of S1P in an inflammatory model induced by lipopolysaccharide. In the first chapter of this thesis we propose the hypothesis that S1P might be involved in gliotic processes, as a key signal in the induction of glial migration. To test our hypothesis, we used a primary culture model of Müller glial cells and evaluated cell migration by the scratch-wound assay, which is a simple and widely used method. Our experiments revealed that the exogenous addition of S1P modified the CGM cytoskeleton, in the first 8-10 hours of treatment. This addition led to the formation of lamelipodia and to the onset of glial motility. This motility was markedly enhanced compared to control conditions after 18 h of S1P addition. CGM expressed two of the five transmembrane receptors for S1P (S1PR), specifically the S1P1 and S1P3 subtypes. However, only activation of S1P3 was required to induce S1P-induced CGM migration, as evidenced by experiments performed with S1P1 and S1P3 antagonists. Analysis of the signaling pathways activated downstream of S1P3 evidenced that two well-known pathways, the ERK/MAPK and PI3K pathways were activated, since pre-treatment with specific inhibitors, significant reduced S1P-induced glial migration. Of note, this induction of the migratory process was not accompanied by a significant increase in glial proliferation. On the other hand, not only exogenous S1P played a role in glial migration. Inhibition of the endogenous synthesis of S1P, using a selective inhibitor of the enzyme sphingosine kinase 1 (Sphk1) (the enzyme that catalyzes the synthesis of S1P), the SphK1 inhibitor number 2 (SphKI2), virtually blocked the basal glial migration, i.e. the migration level observed in the absence of exogenous S1P. These results demonstrated that the S1P-S1P3 axis has a key role in the induction of migration in the retina, and its targeting might provide an interesting approach in the search of new therapeutical treatments for proliferative diseases of the retina. Among retinal pathologies, neurodegenerative diseases are a set of diseases of the retina that progressively lead to vision loss and still lack effective treatments. Pathologies such as retinitis pigmentosa and macula degeneration have very diverse etiologies, varying from genetic to environmental triggers, but which share a common hallmark: the death of photoreceptors (PhRs). This progressive degeneration of PhR leads, as a final consequence, to visual disability and blindness. As described, Cer and Sph are important mediators of cell death and have attracted special attention in the field of neurodegenerative diseases. Pioneering studies found that Cer levels were increased in post-mortem retinas of patients with Faber's disease; furthermore, the levels of this sphingolipid are increased in neuronal degeneration induced by retinal detachment. Subsequent reports, including those from our laboratory, established that Cer is a key mediator in the death of PhRs in culture. Oxidative damage stimulates the synthesis of Cer, which triggers the death of photoreceptors and this process is mimicked by the exogenous addition of a synthetic analogue of an endogenous ceramide, C2-Ceramide (C2Cer). Further work using animal models of retina degeneration support a role for Cer as a crucial signal involved in the induction of PhR death. However, the molecular mechanisms involved in this process are still unknown. Uncovering them is very relevant, since regulating the different intracellular targets of Cer might provide new therapies for neurodegenerative diseases of the retina. The purpose of the second chapter of this thesis was to study the molecular mechanisms involved in the death of PhRs induced by C2Cer, using as a model primary cultures of rat retinal neurons. Previous work from our laboratory showed that 10 μM C2Cer induces the death of PhRs and amacrine neurons, the two major neuronal types in our cultures. A chronological analysis of the death process showed that treatment with 10 μM C2Cer for 6 h selectively induced the death of photoreceptors, decreasing mitochondrial membrane potential and increasing the formation of Reactive Oxygen Species (ROS). In contrast, amacrine neurons preserved their viability. Of note, the number of cells labeled with TUNEL and of photoreceptors expressing cleaved caspase-3 remained constant and a pretreatment with a pan caspase inhibitor did not prevent C2Cer-induced neuronal death. C2Cer caused over-activation of poly-ADP ribosyl polymerase-1 (PARP-1). The inhibition of PARP-1 decreased photoreceptor death induced by C2Cer. In turn, C2Cer increased poly-ADP ribose (PAR) polymer levels, the synthesis of which is catalyzed by PARP-1, and induced the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nuclei, which was prevented by PARP-1 inhibition. Pretreatment with a calpain and cathepsin inhibitor, and with a selective calpain inhibitor, reduced photoreceptor death, whereas selective cathepsins inhibitors did not provide any protection. The combined pretreatment with a PARP-1 inhibitor and a calpain inhibitor showed the same extent of protection as each inhibitor by itself. This suggests that both PARP-1 and calpains converge downstream in the same mechanism. Neither autophagy nor necroptosis were involved in C2Cer-induced cell death; no increase in LDH release was observed after C2Cer treatment and pretreatment with necroptosis and autophagy inhibitors did not rescue photoreceptors. These results suggest that the C2Cer-induced photoreceptor death signaling by a novel caspase-independent mechanism, which involves PARP-1 activation, a decrease in mitochondrial membrane potential, activation of calpain and translocation of AIF, all of which are biochemical characteristics of the cell death type named parthanatos. In conclusion, our results identify a new therapeutic target, thus contributing to the advances in the search for effective treatments for neurodegenerative diseases of the retina.

Submitted by Ana Paula Macedo (ensino@ioc.fiocruz.br) on 2013-10-03T12:26:40Z No. of bitstreams: 1 Carolina Valença Messias.pdf: 1326403 bytes, checksum: adde2666bf9c1e45634b4ea4926b6090 (MD5) / Made available in DSpace on 2013-10-03T12:26:40Z (GMT). No. of bitstreams: 1 Carolina Valença Messias.pdf: 1326403 bytes, checksum: adde2666bf9c1e45634b4ea4926b6090 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A leucemia linfoblástica aguda de células T (LLA-T) e o linfoma linfoblástico de células T (LL-T) são proliferações malignas de precursores de células T em diferentes estágios de maturação. LLA-T e LL-T são considerados atualmente duas formas de uma mesma doença, a leucemia/linfoma linfoblástico de células T (LLL-T), por compartilharem características morfológicas, imunofenotípicas e genéticas. Como os blastos de LLL-T apresentam características similares às de timócitos normais, moléculas envolvidas na migração destas células podem também estar envolvidas na migração ou homing dos linfoblastos no curso da doença. Neste sentido, foi demonstrado recentemente que o receptor 1 de esfingosina-1-fosfato (S1P1) é essencial para a saída de timócitos do timo. No presente estudo, decidimos avaliar a expressão e o papel do S1P1 na migração de quatro linhagens celulares de LLA-T (HPB-ALL, MOLT-4, CEM e JURKAT) em resposta ao seu ligante fisiológico, a esfingosina-1-fosfato (S1P). Observamos que a linhagem HPB-ALL apresentou baixa expressão de RNAm de S1P1, enquanto MOLT-4 e JURKAT apresentaram uma expressão mediana e CEM apresentou altos níveis de expressão de RNAm para este receptor. Em ensaios funcionais de migração celular observamos que a capacidade migratória das linhagens frente a S1P foi diretamente relacionada ao nível de expressão gênica do receptor. A S1P induziu a migração das linhagens analisadas em diferentes concentrações até 100 nM, e inibiu a migração quando aplicada em altas concentrações (1000 nM). As respostas migratórias foram acompanhadas pela modulação do citoesqueleto de actina. Dependendo da concentração de S1P utilizada, observamos polimerização (menores concentrações) ou despolimerização (maiores concentrações) da actina. Além disso, o prétratamento das células com W146 (inibidor de S1P1) bloqueou a migração das linhagens frente à S1P em menores concentrações e induziu a migração frente à S1P em altas concentrações, sugerindo que a migração seja especificamente mediada por S1P1. Nossos resultados sugerem que as interações mediadas por S1P/S1P1 modulam a migração não apenas de precursores de células T normais, mas também de linfoblastos de LLL-T. Desta forma, a interação S1P/S1P1 pode ser considerada como alvo para possíveis estratégias terapêuticas frente a estas neoplasias. / T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (TLBL) are malignant proliferations of T cell precursors at different stages of normal development. T-ALL and T-LBL are presently believed to represent two different forms of a single disease, the T-cell lymphoblastic leukemia/lymphoma (T-LLL), as they share morphological, immunophenotypic and genetic features. As T-LLL lymphoblasts present similar characteristics of normal T cell precursors, molecules involved on the migration of these cells might also be associated with migration and homing of T-ALL/LBL. In this context, the sphingosine-1-phosfate receptor 1 (S1P1), has been described as essential for mouse thymocyte migration and thymic egress. Herein, we evaluated the expression and role of S1P1 in four T-ALL cell lines (HPBALL, MOLT-4, CEM e JURKAT) in response to its physiological ligand, the sphingosine-1-phophate (S1P). We observed that HPB-ALL cells presented low expression levels of S1P1 mRNA, whereas MOLT-4 and JURKAT had medium levels and CEM showed high levels of S1P1 expression. In functional migration assays, we observed that the migratory response of the cells towards S1P was directly related with their expression levels of the receptor. S1P induced the migration of the cell lines analyzed in different concentrations up to 100 nM and inhibited cell migration at higher concentrations (1000 nM). Moreover, migratory responses were accompanied by the modulation of actin cytoskeleton. Depending on S1P concentrations, we observed actin polymerization (lower concentrations) or depolymerization (higher concentrations). Pre-treatment of the cells with W146 (a S1P1 inhibitor) blocked S1Pinduced migration at lower concentrations but induced migration towards S1P at high concentrations, suggesting that the migration is specifically mediated by S1P1. Our results suggest that interactions mediated by S1P/S1P1 can modulate cell migration of T-LLL blasts similarly to their normal T cell precursor counterparts. Accordingly, immune intervention upon this ligand/receptor interaction may be envisioned as a potential therapeutic strategy for these malignancies.

Submitted by Angelo Silva (asilva@icict.fiocruz.br) on 2016-07-13T18:26:36Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 71836.pdf: 6043659 bytes, checksum: baace5b9e2738bb17390dc3dd95f041d (MD5) / Approved for entry into archive by Anderson Silva (avargas@icict.fiocruz.br) on 2016-08-24T13:34:39Z (GMT) No. of bitstreams: 2 71836.pdf: 6043659 bytes, checksum: baace5b9e2738bb17390dc3dd95f041d (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) / Made available in DSpace on 2016-08-24T13:34:39Z (GMT). No. of bitstreams: 2 71836.pdf: 6043659 bytes, checksum: baace5b9e2738bb17390dc3dd95f041d (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A leucemia/linfoma linfoblástico de células T (LLL-T) é um processo patológico resultante de proliferações malignas de precursores de células T em diferentes estágios de desenvolvimento. Como os blastos de LLL-T apresentam características de precursores de células T normais, moléculas envolvidas na migração destas células podem estar envolvidas da migração e saída de blastos de LLL-T no curso da doença. Nesse sentido, foi descrito que o receptor 1 de esfingosina-1-fosfato (S1P1) é essencial para a saída de timócitos do timo. Nós observamos anteriormente que células de pacientes com leucemia linfoblástica aguda de células T (LLA-T) e linfoma linfoblástico de células T (LL-T) expressam o receptor S1P1, assim como três linhagens celulares (MOLT-4, Jurkat e CEM) que representam esta doença. Baixas concentrações de S1P induzem a quimiotaxia destas células, mas não altas concentrações de S1P, e esta migração é dependente do S1P1 e de polimerização de actina. Entretanto nossos dados também indicam que outros receptores de S1P podem estar envolvidos nesse processo. No presente estudo, avaliamos a expressão e o papel dos receptores de S1P na migração de precursores de células T neoplásicas. Nós observamos que as linhagens celulares acima descritas expressam não só S1P1, mas outros receptores de S1P. MOLT-4 expressa preferencialmente S1P1, S1P3 e S1P5, Jurkat expressa S1P1 e S1P3, enquanto CEM expressa todos os receptores de S1P, com exceção de S1P2 Verificamos ainda que altas concentrações de S1P induzem a fugotaxia da célula CEM e que este processo é dependente do S1P1. Em contrapartida, nossos dados mostram que a migração celular induzida por baixas concentrações de S1P induz não só a quimiotaxia, mas também a quimiocinese desta célula. Além disso, observamos que o S1P3, único receptor expresso comumente por estas células, não está envolvido na quimiotaxia dos blastos de LLA-T. S1P ativa ainda as vias de sinalização de AKT e ERK, mas não de Rac1, e a ativação destas vias parece ser mediada pelo S1P1. No entanto, baixas e altas concentrações de S1P ativam estas vias de forma distinta. Altas concentrações de S1P parecem induzir uma ativação mais tardia de AKT e ERK, enquanto uma diminuição na atividade de Rac1 é observada. Nossos dados sugerem que S1P1 regula diferencialmente a migração dos blastos de LLA-T, dependendo da concentração de S1P no meio. Desta forma, a interação S1P/S1P1 deve ser considerada como possível estratégia terapêutica, visando a prevenção da disseminação dos blastos que pode ser resultante da quimiotaxia ou da fugotaxia / T-cell lymphoblastic leukemia and lymphoma (T-LLL) is a pathologic process resulting from malignant proliferations of T-cell precursors at different stages of development. As T-LLL lymphoblasts present similar characteristics of normal T-cell precursors, molecules involved on the migration of these cells might also be associated with migration and homing of T-LLL blasts. In this context, sphingosine-1- phosphate receptor 1 (S1P1) has been described as essential for thymocyte egress from the thymus. We previously observed that patient\2019s cells with T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LL) express S1P1, as well as three T-ALL cell lines (MOLT-4, Jurkat, and CEM). Low S1P concentrations induce chemotaxis of T-ALL cell lines, but high S1P concentrations do not. This migration mediated through S1P1 and actin polymerization events. However, our data suggests that other S1P receptors might be involved in this process. Herein, we evaluate the expression and role of S1P receptors in the migration of neoplastic T-cell precursors. We observed that this cell lines expressed not only S1P1, but also other S1P receptors. MOLT-4 preferentially expressed S1P1, S1P3 and S1P5 Jurkat mainly expressed S1P1 and S1P3 and CEM expressed all S1P receptors, except for S1P2. We also verified that high S1P concentrations induced CEM fugetaxis and this process is dependent on S1P1. In contrast, low S1P concentrations induced not only chemotaxis but also chemokinesis. Furthermore, S1P3, the only receptor expressed commonly by these cells lines, was not involved in chemotactic responses. Moreover, S1P activated AKT and ERK signaling pathways, but it was not able to activate Rac1, and activation of these signaling pathways seems to be S1P1-dependent. However, low and high S1P concentrations activated these signaling pathways in different ways. High S1P concentrations activated AKT and ERK later than low S1P concentrations and resulted in decreased Rac1 activity. Together, our results suggest that S1P1 regulates differentially T-ALL blast migration depending on S1P concentrations on the medium. Accordingly, immune intervention upon this ligand/receptor interaction may be envisioned as a potential therapeutic strategy for preventing the spread of blasts, which can result from chemotaxis or fugetaxis process

Introdução: A lipoproteína de baixa densidade (LDL) e suas formas oxidadas (LDLox) possuem múltiplas propriedades aterogênicas, atuando na deposição de colesterol, indução e manutenção da inflamação, disfunção endotelial, surgimento de células espumosas na parede arterial e conseqüente formação da placa de ateroma. Adicionalmente, LDLox induz apoptose de células endoteliais humanas (HMEC). A lipoproteína de alta densidade (HDL) possui inúmeras atividades antiaterogênicas, incluindo ações antioxidante, anti-inflamatória e anti-trombótica. A HDL é capaz de proteger as HMEC contra apoptose. As subfrações de HDL (sHDL) são heterogêneas em sua composição físico-química e atividades biológicas. A atividade antioxidante das sHDL aumenta com a densidade (HDL2b<HDL2a<HDL3a<HDL3b <HDL3c) e está deficiente em pacientes com SMet. Contudo, a heterogeneidade da atividade anti-apoptótica das sHDL é ainda desconhecida. Objetivos: (i) avaliar a heterogeneidade da atividade protetora das sHDL de indivíduos normolipidêmicos (n=7) e de pacientes com SMet (n=16) contra apoptose de HMEC induzida por LDLox; (ii) definir os mecanismos moleculares envolvidos nesta atividade. Métodos: Através de ultracentrifugação por gradiente de densidade, isolamos cinco diferentes sHDL. HMEC foram incubadas com LDLox (200 ?g apoB/ml) na presença ou não das sHDL (5-100 ?g proteína/ml). Os marcadores de toxicidade (MTT) e de apoptose celular (microscopia de fluorescência, marcagem com anexina V, cit c, AIF, degradação Bid, atividade caspase-3 e fragmentação do ADN) foram analisados. Resultados: Todas sHDL protegeram as HMEC contra a toxicidade e apoptose induzidas pela LDLox. Com mesma concentração de proteína, as subfrações HDL3c (60% proteção - MTT - e >100% - anexina V) e 3b (43% e 67%, respectivamente) de indivíduos normolipidêmicos apresentaram atividade anti-apoptótica mais potente do que as subfrações HDL2a (29% e 28%; p<0,01 vs. HDL3c, respectivamente) e 2b (25% e 62%; p<0,001 vs. HDL3c, respectivamente). Todas sHDL reduziram geração de espécies reativas de oxigênio (ROS) induzida pela LDLox, sendo a HDL3c (54%) mais potente do que HDL2b (21%; p<0.05 vs. HDL3c). Houve correlação positiva entre as atividades anti-apoptótica e antioxidante intracelular com conteúdo de apoA-I e esfingosina 1-fosfato (E1F) das sHDL, senda HDL3b e 3c ricas em E1F. A atividade anti-apoptótica da E1F e das sHDL parece depender da interação com as células endoteliais via apoA-I e seu receptor SR-BI. Finalmente, as HDL3c (n=5) isoladas de pacientes com SMet possuem conteúdo significativamente menor de apoA-I e reduzida atividade anti-apoptótica (60%, p<0,01), quando comparada aos controles normolipidêmicos (n=5). Houve tendência à diminuição da proteção contra a geração de ROS (SMet, n=10). Conclusão: As subfrações HDL3c protegem de forma potente as células endoteliais humanas contra toxicidade e apoptose induzidas pela LDLox, assim como contra geração de ROS. Esta atividade antiapoptótica está reduzida na SMet. / Background: Low density lipoprotein (LDL) and its oxidized forms (oxLDL) have several atherogenic properties, including cholesterol deposition, inflammation, endothelial dysfunction and foam cell formation on the arterial wall, leading to atherosclerotic plaque development. In addition, oxLDL induces human endothelial cell apoptosis (HMEC). High-density lipoprotein (HDL) has number of antiatherogenic activities, as antioxidative, anti-inflammatory and anti-thrombotic actions. HDL displays anti-apoptotic activity and is able to protect endothelial cells against oxLDL-induced apoptosis. HDL subfractions (sHDL) are highly heterogeneous in their physical and chemical composition and biological functions. Antioxidative activity of HDL subfractions increases with increment in density, HDL2b<HDL2a<HDL3a<HDL3b <HDL3c. Important, HDL subfractions from subjects with metabolic syndrome (MetS), display a significantly lower antioxidative activity as compared to healthy controls. However, the heterogeneity of their anti-apoptotic activity was not demonstrated. Objectives: (i) to evaluate the heterogeneity of antiapoptotic activity of sHDL from normolipidemic controls (n=7) and MetS patients (n=16) towards oxLDL-induced apoptosis of HMEC; (ii) to define molecular mechanisms involved in this anti-apoptotic action. Methods: Five major sHDL were fractionated by density gradient ultracentrifugation. HMEC were incubated with mildly oxLDL (200 ?g apoB/ml) in the presence or absence of each sHDL (5-100 ?g protein/ml). Markers of cellular toxicity (MTT) and apoptosis (fluorescent nucleic acid staining, annexin V binding, cytochrome c, AIF and Bid, caspase-3 activity and DNA fragmentation) were observed. Results: All HDL subfractions isolated from normolipidemic subjects protected HMEC against oxLDL-induced toxicity and apoptosis. At equal protein concentrations, HDL3c (60% protection in the MTT test; >100% in annexin V biding) and 3b subfractions (43% and 67%, respectively) were more potent against oxLDL-induced toxicity and apoptosis as compared to HDL2a (29% and 28%; p<0.01 vs. HDL3c, respectively) and 2b subfractions (25% and 62%; p<0.001 vs. HDL3c, respectively). All HDL subfractions attenuated of reactive oxygen species (ROS) generation in HMEC induced by oxLDL. Again, HDL3c (54% inhibition) were more potent as compared to HDL2b (21%; p<0.05 vs. HDL3c). The anti-apoptotic and intracellular antioxidative activities of HDL3 were positively correlated with apoA-I and sphingosine 1-phosphate (S1P) content of sHDL and, possibly, depend on their cellular interaction through apoA-I and its SR-BI receptor. The sHDL3c isolated from MetS patients (n=5) possess reduced content of apoA-I and less potent anti-apoptotic activity (-60%, p<0.01) than controls (n=5). Conclusion: Normolipidemic small dense HDL3 provide potent protection of human endothelial cells from oxLDL-induced apoptosis; this anti-apoptotic activity is reduced in the MetS.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The manipulation of oocytes enclosed in follicles Ovarian Pre-Antral, allows increasing the reproductive potential of females from the isolation and cultivation. The follicular viability can be affected negatively correlated with age, malnutrition, exposure to radio or chemotherapy and health conditions. The alternatives are to use these research for development of culture media which provides the maintenance of viability allows the activation and follicular development in vitro, for subsequent fertilization. Physiologically various growth factors are involved in the process of folliculogenesis, many of which have so far not been tested in goats. Among them the Sphingosine 1-phosphate (S1P) and Leukemia Inhibitory Factor (LIF), since, follicular development were obtained with these factors in other species, allowing follicular growth and ant apoptotic effect on ovarian tissue. The aim of this study was to evaluate the addition of different concentrations of S1P and the LIF to culture media separately, evaluating the maintenance of viability, activation and follicular development. Thus, the preantral follicle cultured with S1P in situ LIF for 7 days and follicular development as possible to maintain viability, activation and growth of preantral follicle in vitro. Concluding that the S1P 1 ng/ml LIF and 10ng/ml, were the concentration that the maintenance of viability and follicular activation of in situ and grown in vitro for 7 days improved the viability, activation and preantral follicular growth. / A Manipulação de Oócito Incluso em Folículos Ovariano Pré-Antral (MOIFOPA) possibilita o aumento do potencial reprodutivo das fêmeas a partir do isolamento e cultivo de FOPA. A viabilidade folicular pode sofrer influências negativas com a idade, desnutrição, exposição a rádio ou quimioterápicos e condições sanitárias adversas. As alternativas para aproveitamento desses FOPA são pesquisas voltadas para elaboração de meios de cultivos que proporcione o manutenção da viabilidade a possibilite a ativação e o desenvolvimento folicular in vitro, para posterior fertilização. Fisiologicamente vários fatores de crescimento estão envolvidos durante o processo da foliculogênese, muitos dos quais até o momento não foram testados em cabras. Dentre eles, a Esfingosina 1-fosfato (S1P) e o Fator Inibidor da Leucemia (LIF), uma vez que, o desenvolvimento folicular foram obtido com esses fatores em outras espécies, possibilitando crescimento folicular e efeito anti-apoptótico no tecido ovariano. O objetivo desse trabalho foi avaliar a adição de diferentes concentrações de S1P e o do LIF separadamente aos meios de cultivo, avaliando a manutenção da viabilidade, ativação e desenvolvimento folicular. Dessa forma, os FOPA cultivados in situ com S1P e LIF por 7 dias possibilitaram desenvolvimento folicular conforme a manutenção da viabilidade, ativação e crescimento dos FOPA in vitro. Concluindo que a S1P 1ng/ml e o LIF 10ng/ml, foram as concentração que condicionaram a manutenção da viabilidade e a ativação dos FOPA in situ cultivados in vitro por 7 em dias melhor mantiveram a viabilidade, ativação e crescimento folicular.

Introdução: A lipoproteína de baixa densidade (LDL) e suas formas oxidadas (LDLox) possuem múltiplas propriedades aterogênicas, atuando na deposição de colesterol, indução e manutenção da inflamação, disfunção endotelial, surgimento de células espumosas na parede arterial e conseqüente formação da placa de ateroma. Adicionalmente, LDLox induz apoptose de células endoteliais humanas (HMEC). A lipoproteína de alta densidade (HDL) possui inúmeras atividades antiaterogênicas, incluindo ações antioxidante, anti-inflamatória e anti-trombótica. A HDL é capaz de proteger as HMEC contra apoptose. As subfrações de HDL (sHDL) são heterogêneas em sua composição físico-química e atividades biológicas. A atividade antioxidante das sHDL aumenta com a densidade (HDL2b<HDL2a<HDL3a<HDL3b <HDL3c) e está deficiente em pacientes com SMet. Contudo, a heterogeneidade da atividade anti-apoptótica das sHDL é ainda desconhecida. Objetivos: (i) avaliar a heterogeneidade da atividade protetora das sHDL de indivíduos normolipidêmicos (n=7) e de pacientes com SMet (n=16) contra apoptose de HMEC induzida por LDLox; (ii) definir os mecanismos moleculares envolvidos nesta atividade. Métodos: Através de ultracentrifugação por gradiente de densidade, isolamos cinco diferentes sHDL. HMEC foram incubadas com LDLox (200 ?g apoB/ml) na presença ou não das sHDL (5-100 ?g proteína/ml). Os marcadores de toxicidade (MTT) e de apoptose celular (microscopia de fluorescência, marcagem com anexina V, cit c, AIF, degradação Bid, atividade caspase-3 e fragmentação do ADN) foram analisados. Resultados: Todas sHDL protegeram as HMEC contra a toxicidade e apoptose induzidas pela LDLox. Com mesma concentração de proteína, as subfrações HDL3c (60% proteção - MTT - e >100% - anexina V) e 3b (43% e 67%, respectivamente) de indivíduos normolipidêmicos apresentaram atividade anti-apoptótica mais potente do que as subfrações HDL2a (29% e 28%; p<0,01 vs. HDL3c, respectivamente) e 2b (25% e 62%; p<0,001 vs. HDL3c, respectivamente). Todas sHDL reduziram geração de espécies reativas de oxigênio (ROS) induzida pela LDLox, sendo a HDL3c (54%) mais potente do que HDL2b (21%; p<0.05 vs. HDL3c). Houve correlação positiva entre as atividades anti-apoptótica e antioxidante intracelular com conteúdo de apoA-I e esfingosina 1-fosfato (E1F) das sHDL, senda HDL3b e 3c ricas em E1F. A atividade anti-apoptótica da E1F e das sHDL parece depender da interação com as células endoteliais via apoA-I e seu receptor SR-BI. Finalmente, as HDL3c (n=5) isoladas de pacientes com SMet possuem conteúdo significativamente menor de apoA-I e reduzida atividade anti-apoptótica (60%, p<0,01), quando comparada aos controles normolipidêmicos (n=5). Houve tendência à diminuição da proteção contra a geração de ROS (SMet, n=10). Conclusão: As subfrações HDL3c protegem de forma potente as células endoteliais humanas contra toxicidade e apoptose induzidas pela LDLox, assim como contra geração de ROS. Esta atividade antiapoptótica está reduzida na SMet. / Background: Low density lipoprotein (LDL) and its oxidized forms (oxLDL) have several atherogenic properties, including cholesterol deposition, inflammation, endothelial dysfunction and foam cell formation on the arterial wall, leading to atherosclerotic plaque development. In addition, oxLDL induces human endothelial cell apoptosis (HMEC). High-density lipoprotein (HDL) has number of antiatherogenic activities, as antioxidative, anti-inflammatory and anti-thrombotic actions. HDL displays anti-apoptotic activity and is able to protect endothelial cells against oxLDL-induced apoptosis. HDL subfractions (sHDL) are highly heterogeneous in their physical and chemical composition and biological functions. Antioxidative activity of HDL subfractions increases with increment in density, HDL2b<HDL2a<HDL3a<HDL3b <HDL3c. Important, HDL subfractions from subjects with metabolic syndrome (MetS), display a significantly lower antioxidative activity as compared to healthy controls. However, the heterogeneity of their anti-apoptotic activity was not demonstrated. Objectives: (i) to evaluate the heterogeneity of antiapoptotic activity of sHDL from normolipidemic controls (n=7) and MetS patients (n=16) towards oxLDL-induced apoptosis of HMEC; (ii) to define molecular mechanisms involved in this anti-apoptotic action. Methods: Five major sHDL were fractionated by density gradient ultracentrifugation. HMEC were incubated with mildly oxLDL (200 ?g apoB/ml) in the presence or absence of each sHDL (5-100 ?g protein/ml). Markers of cellular toxicity (MTT) and apoptosis (fluorescent nucleic acid staining, annexin V binding, cytochrome c, AIF and Bid, caspase-3 activity and DNA fragmentation) were observed. Results: All HDL subfractions isolated from normolipidemic subjects protected HMEC against oxLDL-induced toxicity and apoptosis. At equal protein concentrations, HDL3c (60% protection in the MTT test; >100% in annexin V biding) and 3b subfractions (43% and 67%, respectively) were more potent against oxLDL-induced toxicity and apoptosis as compared to HDL2a (29% and 28%; p<0.01 vs. HDL3c, respectively) and 2b subfractions (25% and 62%; p<0.001 vs. HDL3c, respectively). All HDL subfractions attenuated of reactive oxygen species (ROS) generation in HMEC induced by oxLDL. Again, HDL3c (54% inhibition) were more potent as compared to HDL2b (21%; p<0.05 vs. HDL3c). The anti-apoptotic and intracellular antioxidative activities of HDL3 were positively correlated with apoA-I and sphingosine 1-phosphate (S1P) content of sHDL and, possibly, depend on their cellular interaction through apoA-I and its SR-BI receptor. The sHDL3c isolated from MetS patients (n=5) possess reduced content of apoA-I and less potent anti-apoptotic activity (-60%, p<0.01) than controls (n=5). Conclusion: Normolipidemic small dense HDL3 provide potent protection of human endothelial cells from oxLDL-induced apoptosis; this anti-apoptotic activity is reduced in the MetS.