Efecto de la hipoxia-reoxigenación y las radiaciones ionizantes en la captación de glucosa en líneas tumorales de seno y colon cocultivadas con células endoteliales.

La captación de glucosa y su conversión en lactato juega un papel fundamental en el metabolismo tumoral, independientemente de la concentración de oxígeno presente en el tejido (efecto Warburg). Sin embrago, dicha captación varía de un tipo tumoral a otro, y dentro del mismo tumor, situación que podría depender de las características microambientales tumorales (fluctuaciones de oxígeno, presencia de otros tipos celulares) y de factores estresores asociados a los tratamientos. Se estudió el efecto de la hipoxia-reoxigenación (HR) y las radiaciones ionizantes (RI) sobre la captación de glucosa, en cultivos de líneas tumorales MCF-7 y HT-29, cultivadas de forma aislada o en cocultivo con la línea celular EAhy296. Se encontró que la captación de glucosa en HR es diferente para lo descrito en condiciones de hipoxia permanente y que es modificada en el cocultivo. Se identificaron poblaciones celulares dentro de la misma línea celular, de alta y baja captación de glucosa, lo que implicaría una simbiosis metabólica de la célula como respuesta adaptativa a las condiciones tumorales. Se evaluó la expresión de NRF2 y la translocación nuclear de NRF2 y HIF1a, como vías de respuesta a estrés celular e hipoxia. La translocación nuclear de las proteínas evaluadas explicaría el comportamiento metabólico de las células tumorales de seno, pero no de colon, por lo cual deben existir otras vías metabólicas implicadas. Las diferencias en el comportamiento de las células tumorales en HR en relación con hipoxia permitirá realizar planeaciones dosimétricas más dinámicas, que reevalúen las condiciones de oxigenación tumoral constantemente.

A "Trojan horse" strategy to reverse drug-resistance in brain tumors

Los gliomas malignos representan una de las formas más agresivas de los tumores del sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina. Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de mecanismos asociados a la expresión de los transportadores ABC podría constituir una herramienta clave en el desarrollo de nuevas terapias anticáncer.

Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein

CSRP3 or muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with alpha-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myo filaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli. (C) 2009 Elsevier Inc. All rights reserved.

Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2

Oxidized low-density lipoproteins (LDL) play a central role in atherogenesis and induce expression of the antioxidant stress protein heme oxygenase 1 (HO-1). In the present study we investigated induction of HO-1 and adaptive increases in reduced glutathione (GSH) in human aortic smooth muscle cells (SMC) in response to moderately oxidized LDL (moxLDL, 100 mu g protein/ml, 24 h), a species containing high levels of lipid hydroperoxides. Expression and activity of HO-1 and GSH levels were elevated to a greater extent by moxLDL than highly oxidized LDL but unaffected by native or acetylated LDL. Inhibitors of protein kinase C (PKC) or mitogen-activated protein kinases (MAPK) p38(MAPK) and MEK or c-jun-NH2-terminal kinase (JNK) significantly attenuated induction of HO-1. Phosphorylation of p38(MAPK), extracellular signal-regulated kinase (ERK1/2), or JNK and nuclear translocation of the transcription factor Nrf2 were enhanced following acute exposure of SMC to rnoxLDL (100 mu g proteiri/ml, 1-2 h). Pretreatment of SMC with the antioxidant vitamin C (100 mu M, 24 h) attenuated the induction of HO-1 by moxLDL. Native and oxidized LDL did not alter basal levels of intracellular ATP, mitochondrial dehydrogenase activity, or expression of the lectin-like oxidized LDL receptor (LOX-1) in SMC. These findings demonstrate for the first time that activation of PKC, p38(MAPK), JNK, ERK1/2, and Nrf2 by oxidized LDL in human SMC leads to HO-1 induction, constituting an adaptive response against oxidative injury that can be ameliorated by vitamin C. (C) 2005 Elsevier Inc. All rights reserved.

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H2O2) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint. (c) 2006 Elsevier Inc. All rights reserved.

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis. (c) 2006 Elsevier Inc. All rights reserved.

Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration

Satellite cells represent the stem cell population of adult skeletal muscle. The molecular mechanisms that control the proliferation of satellite cells are not well understood. In this study, we show that in response to injury, myofibres activate Wnt ligand transcription and activate a reporter cell line that is sensitive to the canonical Wnt-signalling pathway. Activated satellite cells on isolated cultured myofibres show robust expression of activated-β-catenin (Act-β-Cat), a key downstream transcriptional coactivator of canonical Wnt signalling. We provide evidence that the Wnt family of secreted glycoproteins act on satellite cells in a ligand-specific manner. Overexpression of Wnt1, Wnt3a or Wnt5a protein causes a dramatic increase in satellite-cell proliferation. By contrast, exposure of satellite cells to Wnt4 or Wnt6 diminishes this process. Moreover, we show that the prolonged satellite-cell quiescence induced by inhibitory Wnt is reversible and exposing inhibited satellite cells to stimulatory Wnt signalling restores their proliferation rate. Stimulatory Wnt proteins induce premature satellite cell BrdU incorporation as well as nuclear translocation of Act-β-Cat. Finally, we provide evidence that the Act-β-Cat translocation observed in single fibres during in vitro culture also occurs in cases of acute and chronic skeletal muscle regeneration in rodents and humans. We propose that Wnt proteins may be key factors that regulate the rate of satellite-cell proliferation on adult muscle fibres during the wound-healing response.

Hsc70 is a novel interactor of NF-kappaB p65 in living hippocampal neurons

Signaling via NF-κB in neurons depends on complex formation with interactors such as dynein/dynactin motor complex and can be triggered by synaptic activation. However, so far a detailed interaction map for the neuronal NF-κB is missing. In this study we used mass spectrometry to identify novel interactors of NF-κB p65 within the brain. Hsc70 was identified as a novel neuronal interactor of NF-κB p65. In HEK293 cells, a direct physical interaction was shown by co-immunoprecipitation and verified via in situ proximity ligation in healthy rat neurons. Pharmacological blockade of Hsc70 by deoxyspergualin (DSG) strongly decreased nuclear translocation of NF-κB p65 and transcriptional activity shown by reporter gene assays in neurons after stimulation with glutamate. In addition, knock down of Hsc70 via siRNA significantly reduced neuronal NF-κB activity. Taken together these data provide evidence for Hsc70 as a novel neuronal interactor of NF-κB p65.

1,8-cineol potentiates IRF3-mediated antiviral response in human stem cells and an ex vivo model of rhinosinusitis

Common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial super-infections resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential of 1,8-cineole for treating primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates Poly(I:C)-induced activity of the anti-viral transcription factor Interferon Regulatory Factor 3, while simultaneously reducing pro-inflammatory NF-κB-activity in human cell lines, inferior turbinate stem cells (ITSCs) and ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with Poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared to Poly(I:C) alone, whereas NF-κB-activity was reduced. Accordingly, 1,8-cineole- and Poly(I:C)-treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared to the Poly(I:C)-treated approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with LPS and 1,8-cineole compared to the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on pro-inflammatory NF-κB-signalling and may thus broaden its field of application.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

An endogenous regulator of inflammation, resolvin E1, modulates osteoclast differentiation and bone resorption

Background and purpose: The inflammation-resolving lipid mediator resolvin E1 (RvE1) effectively stops inflammation-induced bone loss in vivo in experimental periodontitis. It was of interest to determine whether RvE1 has direct actions on osteoclast (OC) development and bone resorption. Experimental approach: Primary OC cultures derived from mouse bone marrow were treated with RvE1 and analysed for OC differentiation, cell survival and bone substrate resorption. Receptor binding was measured using radiolabelled RvE1. Nuclear factor (NF)-kappa B activation and Akt phosphorylation were determined with western blotting. Lipid mediator production was assessed with liquid chromatography tandem mass spectrometry. Key results: OC growth and resorption pit formation were markedly decreased in the presence of RvE1. OC differentiation was inhibited by RvE1 as demonstrated by decreased number of multinuclear OC, a delay in the time course of OC development and attenuation of receptor activator of NF-kappa B ligand-induced nuclear translocation of the p50 subunit of NF-kappa B. OC survival and apoptosis were not altered by RvE1. Messenger RNA for both receptors of RvE1, ChemR23 and BLT(1) is expressed in OC cultures. Leukotriene B(4) (LTB(4)) competed with [(3)H] RvE1 binding on OC cell membrane preparations, and the LTB(4) antagonist U75302 prevented RvE1 inhibition of OC growth, indicating that BLT(1) mediates RvE1 actions on OC. Primary OC synthesized the RvE1 precursor 18R-hydroxy-eicosapentaenoic acid and LTB(4). Co-incubation of OC with peripheral blood neutrophils resulted in transcellular RvE1 biosynthesis. Conclusions and implications: These results indicate that RvE1 inhibits OC growth and bone resorption by interfering with OC differentiation. The bone-sparing actions of RvE1 are in addition to inflammation resolution, a direct action in bone remodelling.

Insulin suppresses LPS-induced iNOS and COX-2 expression and NF-kappa B activation in alveolar macrophages

The development of septic shock is a common and frequently lethal consequence of gram-negative infection. Mediators released by lung macrophages activated by bacterial products such as lipopolysaccharide (LPS) contribute to shock symptoms. We have shown that insulin downregulates LPS-induced TNF production by alveolar macrophages (AMs). In the present study, we investigated the effect of insulin on the LPS-induced production of nitric oxide (NO) and prostaglandin (PG)-E(2), on the expression of inducible nitric oxide synthase ( iNOS) and cyclooxygenase (COX)-2, and on nuclear factor kappa B (NF-kappa B) activation in AMs. Resident AMs from male Wistar rats were stimulated with LPS (100 ng/mL) for 30 minutes. Insulin (1 mU/mL) was added 10 min before LPS. Enzymes expression, NF-kappa B p65 activation and inhibitor of kappa B (I-kappa B) a phosphorylation were assessed by immunobloting; NO by Griess reaction and PGE(2) by enzyme immunoassay (EIA). LPS induced in AMs the expression of iNOS and COX-2 proteins and production of NO and PGE(2), and, in parallel, NF-kappa B p65 activation and cytoplasmic I-kappa B alpha phosphorylation. Administration of insulin before LPS suppressed the expression of iNOS and COX-2, of NO and PGE(2) production and Nuclear NF-kappa B p65 activation. Insulin also prevented cytoplasmic I-kappa Ba phosphorylation. These results show that in AMs stimulated by LPS, insulin prevents nuclear translocation of NF-kappa B, possibly by blocking I-kappa Ba degradation, and supresses the production of NO and PGE(2), two molecules that contribute to septic shock. Copyright (C) 2008 S. Karger AG, Basel.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular basis for defining the pineal gland and pinealocytes as targets for tumor necrosis factor

The pineal gland, the gland that translates darkness into an endocrine signal by releasing melatonin at night, is now considered a key player in the mounting of an innate immune response. Tumor necrosis factor (TNF), the first pro-inflammatory cytokine to be released by an inflammatory response, suppresses the translation of the key enzyme of melatonin synthesis (arylalkylamine-N-acetyltransferase, Aanat). Here, we show that TNF receptors of the subtype 1 (TNF-R1) are expressed by astrocytes, microglia, and pinealocytes. We also show that the TNF signaling reduces the level of inhibitory nuclear factor kappa B protein subtype A (NFKBIA), leading to the nuclear translocation of two NFKB dimers, p50/p50, and p50/RelA. The lack of a transactivating domain in the p50/p50 dimer suggests that this dimer is responsible for the repression of Aanat transcription. Meanwhile, p50/RelA promotes the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide, which inhibits adrenergically induced melatonin production. Together, these data provide a mechanistic basis for considering pinealocytes a target ofTNF and reinforce the idea that the suppression of pineal melatonin is one of the mechanisms involved in mounting an innate immune response. © 2011 Carvalho-Sousa, da Silveira Cruz-Machado, Tamura, Fernandes, Pinato, Muxel, Cecon and Markus.