Patología vascular: ¿causa o efecto en la enfermedad de Alzheimer? / Vascular pathology: Cause or effect in Alzheimer disease?

Introducción: La enfermedad de Alzheimer (EA) es la principal enfermedad neurodegenerativa cortical. Su incidencia aumenta con la edad, lo que provoca importantes problemas médicos, sociales y económicos, especialmente en países con población envejecida. Objetivo: El objetivo de esta revisión es poner de manifiesto las evidencias que existen sobre el modo en que la disfunción vascular puede contribuir al deterioro cognitivo en la EA, así como las posibilidades terapéuticas que de ello podrían derivarse. Desarrollo: La hipótesis vascular ha surgido como alternativa a la hipótesis de la cascada amiloide como explicación de la fisiopatología de la EA. Esta hipótesis sitúa en los vasos sanguíneos el origen de una serie de estímulos patogénicos que llevarían a la lesión neuronal y la demencia. La destrucción de la organización de la barrera hematoencefálica, la disminución del flujo sanguíneo cerebral y el establecimiento de un contexto inflamatorio serían responsables de un consecuente daño neuronal a causa de favorecer la agregación del péptido β-amiloide en el cerebro. Las vías que relacionan la disfunción vascular con la neurodegeneración han proporcionado nuevos enfoques terapéuticos y dianas farmacológicas con las que avanzar en la búsqueda de tratamientos para la EA. Conclusiones: Resulta difícil determinar si el componente vascular de la EA es la causa o el efecto de la enfermedad, pero no cabe duda de que la enfermedad vascular tiene una relación importante con la EA. Es probable que la disfunción vascular actúe sinérgicamente con los cambios neurodegenerativos en un ciclo que agrava el deterioro cognitivo propio de la EA (AU)

Introduction: Alzheimer disease (AD) is the main cortical neurodegenerative disease. The incidence of this disease increases with age, causing significant medical, social and economic problems, especially in countries with ageing populations. Objective: This review aims to highlight existing evidence of how vascular dysfunction may contribute to cognitive impairment in AD, as well as the therapeutic possibilities that might arise from this evidence. Development: The vascular hypothesis emerged as an alternative to the amyloid cascade hypothesis as an explanation for the pathophysiology of AD. This hypothesis locates blood vessels as the origin for a variety of pathogenic pathways that lead to neuronal damage and dementia. Destruction of the organisation of the blood brain barrier, decreased cerebral blood flow, and the establishment of an inflammatory context would thus be responsible for any subsequent neuronal damage since these factors promote aggregation of Beta-amyloid peptide in the brain. The link between neurodegeneration and vascular dysfunction pathways has provided new drug targets and therapeutic approaches that will add to the treatments for AD. Conclusions: It is difficult to determine whether the vascular component in AD is the cause or the effect of the disease, but there is no doubt that vascular pathology has an important relationship with AD. Vascular dysfunction is likely to act synergistically with neurodegenerative changes in a cycle that exacerbates the cognitive impairment found in AD (AU)

Vascular pathology: Cause or effect in Alzheimer disease? / Patología vascular: ¿causa o efecto en la enfermedad de Alzheimer?

INTRODUCTION: Alzheimer disease (AD) is the main cortical neurodegenerative disease. The incidence of this disease increases with age, causing significant medical, social and economic problems, especially in countries with ageing populations. OBJECTIVE: This review aims to highlight existing evidence of how vascular dysfunction may contribute to cognitive impairment in AD, as well as the therapeutic possibilities that might arise from this evidence. DEVELOPMENT: The vascular hypothesis emerged as an alternative to the amyloid cascade hypothesis as an explanation for the pathophysiology of AD. This hypothesis locates blood vessels as the origin for a variety of pathogenic pathways that lead to neuronal damage and dementia. Destruction of the organisation of the blood brain barrier, decreased cerebral blood flow, and the establishment of an inflammatory context would thus be responsible for any subsequent neuronal damage since these factors promote aggregation of ß-amyloid peptide in the brain. The link between neurodegeneration and vascular dysfunction pathways has provided new drug targets and therapeutic approaches that will add to the treatments for AD. CONCLUSIONS: It is difficult to determine whether the vascular component in AD is the cause or the effect of the disease, but there is no doubt that vascular pathology has an important relationship with AD. Vascular dysfunction is likely to act synergistically with neurodegenerative changes in a cycle that exacerbates the cognitive impairment found in AD.

Oxidative stress triggers cytokinesis failure in hepatocytes upon isolation.

Primary hepatocytes are highly differentiated cells and proliferatively quiescent. However, the stress produced during liver digestion seems to activate cell cycle entry by proliferative/dedifferentiation programs that still remain unclear. The aim of this work was to assess whether the oxidative stress associated with hepatocyte isolation affects cell cycle and particularly cytokinesis, the final step of mitosis. Hepatocytes were isolated from C57BL/6 mice by collagenase perfusion in the absence and presence of N-acetyl cysteine (NAC). Polyploidy, cell cycle, and reactive oxygen species (ROS) were studied by flow cytometry (DNA, phospho-histone 3, and CellROX(®) Deep Red) and Western blotting (cyclins B1 and D1, and proliferating cell nuclear antigen). mRNA expression of cyclins A1, B1, B2, D1, and F by reverse transcription (RT)-PCR was also assessed. Glutathione levels were measured by mass spectrometry. Here we show that hepatocyte isolation enhanced cell cycle entry, increased hepatocyte binucleation, and caused marked glutathione oxidation. Addition of 5 mM NAC to the hepatocyte isolation media prevented glutathione depletion, partially blocked ROS production and cell cycle entry of hepatocytes, and avoided the blockade of mitosis progression, abrogating defective cytokinesis and diminishing the formation of binucleated hepatocytes during isolation. Therefore, addition of NAC to the isolation media decreased the generation of polyploid hepatocytes confirming that oxidative stress occurs during hepatocyte isolation and it is responsible, at least in part, for cytokinesis failure and hepatocyte binucleation.

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs.

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.

Manipulation of cellular spheroid composition and the effects on vascular tissue fusion.

Cellular spheroids were investigated as tissue-engineered building blocks that can be fused to form functional tissue constructs. While spheroids can be assembled using passive contacts for the fusion of complex tissues, physical forces can be used to promote active contacts to improve tissue homogeneity and accelerate tissue fusion. Understanding the mechanisms affecting the fusion of spheroids is critical to fabricating tissues. Here, manipulation of the spheroid composition was used to accelerate the fusion process mediated by magnetic forces. The Janus structure of magnetic cellular spheroids spatially controls iron oxide magnetic nanoparticles (MNPs) to form two distinct domains: cells and extracellular MNPs. Studies were performed to evaluate the influence of extracellular matrix (ECM) content and cell number on the fusion of Janus magnetic cellular spheroids (JMCSs). Results showed that the integration of iron oxide MNPs into spheroids increased the production of collagen over time when compared to spheroids without MNPs. The results also showed that ring tissues composed of JMCSs with high ECM concentrations and high cell numbers fused together, but exhibited less contraction when compared to their lower concentration counterparts. Results from spheroid fusion in capillary tubes showed that low ECM concentrations and high cell numbers experienced more fusion and cellular intermixing over time when compared to their higher counterparts. These findings indicate that cell-cell and cell-matrix interactions play an important role in regulating fusion, and this understanding sets the rationale of spheroid composition to fabricate larger and more complex tissue-engineered constructs.

p38 MAPK: a dual role in hepatocyte proliferation through reactive oxygen species.

p38 MAPKs are important mediators of signal transduction that respond to a wide range of extracellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines, and oxidative stress. The most abundant family member is p38α, which helps to couple cell proliferation and growth in response to certain damaging stimuli. In fact, increased proliferation and impaired differentiation are hallmarks of p38α-deficient cells. It has been reported that reactive oxygen species (ROS) play a critical role in cytokine-induced p38α activation. Under physiological conditions, p38α can function as a mediator of ROS signaling and either activate or suppress cell cycle progression depending on the activation stimulus. The interplay between cell proliferation, p38 MAPK activation, and ROS production plays an important role in hepatocytes. In fact, low levels of ROS seem to be needed to activate several signaling pathways in response to hepatectomy and to orchestrate liver regeneration. p38 MAPK works as a sensor of oxidative stress and cells that have developed mechanisms to uncouple p38 MAPK activation from oxidative stress are more likely to become tumorigenic. So far, p38α influences the redox balance, determining cell survival, terminal differentiation, proliferation, and senescence. Further studies would be necessary in order to clarify the precise role of p38 MAPK signaling as a redox therapeutical target.

Scalable robotic biofabrication of tissue spheroids.

Development of methods for scalable biofabrication of uniformly sized tissue spheroids is essential for tissue spheroid-based bioprinting of large size tissue and organ constructs. The most recent scalable technique for tissue spheroid fabrication employs a micromolded recessed template prepared in a non-adhesive hydrogel, wherein the cells loaded into the template self-assemble into tissue spheroids due to gravitational force. In this study, we present an improved version of this technique. A new mold was designed to enable generation of 61 microrecessions in each well of a 96-well plate. The microrecessions were seeded with cells using an EpMotion 5070 automated pipetting machine. After 48 h of incubation, tissue spheroids formed at the bottom of each microrecession. To assess the quality of constructs generated using this technology, 600 tissue spheroids made by this method were compared with 600 spheroids generated by the conventional hanging drop method. These analyses showed that tissue spheroids fabricated by the micromolded method are more uniform in diameter. Thus, use of micromolded recessions in a non-adhesive hydrogel, combined with automated cell seeding, is a reliable method for scalable robotic fabrication of uniform-sized tissue spheroids.

Human mesenchymal stem cells from adipose tissue: Differentiation into hepatic lineage.

Adipose tissue represents an accessible source of mesenchymal stem cells (ADSCs), with similar characteristics to bone marrow-derived stem cells. The aim of this work was to investigate the transdifferentiation of ADSCs into hepatic lineage cells in vitro. ADSCs were obtained from human adipose tissue from lipectomy. Cells were grown in medium containing 15% AB human serum. Cultures were serum deprived for two days and exposed to a two-step protocol with two different media using growth factors and cytokines. Hepatic differentiation was assessed by RT-PCR of liver-marker genes. ADSCs exhibited a fibroblastic morphology that changed to a cuboidal shape when cells differentiated. Expression of liver genes increased when using one of the two studied media consisting of DMEM supplemented with HGF, bFGF and nicotinamide for 14 days. The results indicate that, under certain specific inducing conditions, ADSCs can be induced to differentiate into hepatic lineage in vitro. Adipose tissue may be an ideal source of high amounts of autologous stem cells.

What is regenerative medicine? Emergence of applied stem cell and developmental biology.

Regenerative medicine is an emerging, but still poorly defined, field of biomedicine. The ongoing 'regenerative medicine revolution' is based on a series of new exciting breakthrough discoveries in the field of stem cell biology and developmental biology. The main problem of regenerative medicine is not so much stem cell differentiation, isolation and lineage diversity, although these are very important issues, but rather stem cell mobilisation, recruitment and integration into functional tissues. The key issue in enhancing tissue and organ regeneration is how to mobilise circulating stem and progenitor cells and how to provide an appropriate environment ('niche') for their tissue and organo-specific recruitment, 'homing' and complete functional integration. We need to know more about basic tissue biology, tissue regeneration and the cellular and molecular mechanisms of tissue turnover (both cellular and extracellular components) at different periods of human life and in different diseases. Systematic in silico, in vitro and in vivo research is a foundation for further progress in regenerative medicine. Regenerative medicine is a rapidly advancing field that opens new and exciting opportunities for completely revolutionary therapeutic modalities and technologies. Regenerative medicine is, at its essence, an emergence of applied stem cell and developmental biology.

Quinolonas y Streptococcus pneumoniae. Mecanismo de acción y resistencia / Quinolones and Streptococcus pneumoniae. Mechanisms of action and resistance

Streptococcus pneumoniae se considera el principal agente causal de la neumonía adquirida en la comunidad, además de estar implicado con frecuencia en exacerbaciones de bronquitis crónica, otitis media aguda, sinusitis, meningitis y otras infecciones. Durante los últimos años, en búsqueda de nuevas sustancias debido a la aparición de resistencias en este microorganismo, se han estudiado, entre otros agentes antibacterianos, las fluoroquinolonas. Asimismo, la biología molecular ha ayudado a estudiar y comprender casi todos los mecanismos bioquímicos de resistencia y las rutas para la diseminación de la información genética entre las bacterias. En esta revisión se repasa el mecanismo de acción de las quinolonas y los mecanismos causantes de la resistencia de S. pneumoniae a ellas, por su importancia clínica y epidemiológica. S. pneumoniae es un caso peculiar, puesto que en este microorganismo la actividad bactericida puede producirse a través de la girasa, la topoisomerasa IV o ambas, dependiendo de la estructura de la quinolona, lo cual implica una influencia de la estructura en el logro del éxito antimicrobiano. Es importante, pues, conocer el prototipo de resistencia para hacer una recomendación de la antibioticoterapia adecuada, cuando esté indicada. (AU)

Las quinolonas: relación estructura-efectos adversos e interacciones con otros fármacos / Quinolone antibacterials: structure-side effects relationship and interactions with other drugs

Las quinolonas en conjunto pueden considerarse como un grupo con un buen balance beneficio/riesgo pues, en general, los derivados quinolónicos son seguros y bien tolerados. Frecuentemente, los efectos adversos disminuyen con la reducción de la dosis y la duración de la terapia. Sólo es necesaria la interrupción del tratamiento en el 1-3 por ciento de los pacientes. Las reacciones adversas más frecuentes son molestias del tracto gastrointestinal, estimulación del sistema nervioso central y alergias. No obstante, algunas quinolonas como el temafloxacino, el grepafloxacino y el trovafloxacino han sido retiradas del mercado por problemas de toxicidad grave. En el presente trabajo se revisa la relación estructura-efectos adversos e interacciones con otros fármacos de estos antibacterianos. El objetivo fundamental es identificar los grupos estructurales que determinan la aparición de reacciones adversas importantes para destacar las configuraciones moleculares exentas de riesgos y que, potencialmente, pueden ser valiosos instrumentos en la terapéutica antiinfecciosa (AU)

Mecanismos de resistencia bacteriana a las quinolonas / Mechanisms of bacterial resistance to quinolones

Para ejercer su efecto citotóxico las quinolonas deben penetrar a través de la membrana bacteriana y alcanzar su diana celular, la DNA girasa o la topoisomerasa IV, e inducir la muerte de la célula. Por ello, los mecanismos de resistencia a las fluoroquinolonas incluyen mutaciones en los genes que codifican la DNA girasa y la topoisomerasa IV dando lugar a las QRDR, alteraciones en la permeabilidad de la membrana que disminuyen la penetración intracelular del fármaco y actividad de transportadores activos endógenos que provocan la expulsión de los antimicrobianos desde la membrana celular al medio exterior. Estos mecanismos de resistencia pueden manifestarse solos o en combinación, si bien parece que in vivo el aumento en el grado de resistencia a las quinolonas es producto de varios mecanismos simultáneos. En el presente trabajo se revisan estos mecanismos de resistencia y se establece, en la medida de lo posible, una relación con la estructura de la quinolona. (AU)

[Mechanisms of bacterial resistance to quinolones]. / Mecanismos de resistencia bacteriana a las quinolonas.

In order to produce their cytotoxic effect, quinolones must enter the cell through the bacterial membrane to reach their target, DNA-gyrase or topoisomerase IV, and induce cell death. The mechanisms of resistance to fluoroquinolones include: those mediated by gene mutations codifying for DNA gyrase and topoisomerase IV and leading to QRDR; those characterized by changes in the permeability of the outer membrane which decrease intracellular penetration of the drug; and those caused by active endogenous carriers responsible for drug efflux. These resistance mechanisms can occur alone or in combination; in fact, it is believed that high resistance levels to quinolones in vivo are associated with simultaneous mechanisms. This article reviews such resistance mechanisms and establishes, when possible, their relation to the structure of quinolones.

[Quinolones and Streptococcus pneumoniae. Mechanisms of action and resistance]. / Quinolonas y Streptococcus pneumoniae. Mecanismo de acción y resistencia.

Streptococcus pneumoniae is considered the most frequent bacterial cause of community-acquired pneumonia, and is involved in a significant number of cases of acute exacerbations of chronic bronchitis, acute otitis, sinusitis, meningitis and other infectious diseases. Fluoroquinolones have been extensively investigated in recent years in the search for new agents that has been prompted by the emergence of resistance in this microorganism. Furthermore, the study of resistance from a molecular biology standpoint has helped in elucidating almost all the biochemical mechanisms of resistance and the routes of dissemination of genetic information between bacteria. This short review is focused on the mechanism of action of quinolones and on the mechanisms responsible for resistance of S. pneumoniae to them, given their clinical and epidemiological relevance. S. pneumoniae is a case apart because bactericidal activity against this microorganism can be produced through gyrase, topoisomerase IV or both, depending on the quinolone structure, which shows that structure has an influence on the success of treatment. Knowledge of the resistance prototype is therefore important so that the appropriate antibiotic therapy can be recommended when indicated.

Signaling by type I and II cytokine receptors: ten years after.

Discovered during the past ten years, Janus kinases and signal transducers and activators of transcription have emerged as critical elements in cytokine signaling and immunoregulation. Recently, knockout mice for all the members of these families have been generated, with remarkably specific outcomes. Equally exciting is the discovery of a new class of inhibitors, the suppressor of cytokine signaling family. The phenotypes of mice deficient in these molecules are also striking, underscoring the importance of negative regulation in cytokine signaling.

The RFG oligomerization domain mediates kinase activation and re-localization of the RET/PTC3 oncoprotein to the plasma membrane.

The RET/PTC3 oncogene arises from the fusion between the N-terminal encoding domain of the RFG gene and the tyrosine kinase encoding domain of RET receptor. RET/PTC3 is very frequent in papillary thyroid carcinomas, especially in children exposed to the Chernobyl accident. We have studied the functional consequences of the RFG-RET fusion. Here we show that the N-terminal coiled-coil domain of RGF mediates oligomerization and activation of the kinase and of the transforming capability of RET/PTC3. In addition, the RFG coiled-coil domain mediates a physical association between RET/PTC3 and RGF proteins, rendering RFG a bona fide substrate of RET/PTC3 kinase. Finally, we show that the coiled-coil domain of RGF is essential for the distribution of the RET/PTC3 protein at the membrane/particulate cell compartment level, where also most of the RFG protein is localized. We propose that fusion to the RFG coiled-coil domain provides RET kinase with a scaffold that mediates oligomerization and re-localization of the RET/PTC3 protein, a process that may be crucial for the signalling of this specific RET/PTC variant.

Overexpression of Hoxc13 in differentiating keratinocytes results in downregulation of a novel hair keratin gene cluster and alopecia.

Studying the roles of Hox genes in normal and pathological development of skin and hair requires identification of downstream target genes in genetically defined animal models. We show that transgenic mice overexpressing Hoxc13 in differentiating keratinocytes of hair follicles develop alopecia, accompanied by a progressive pathological skin condition that resembles ichthyosis. Large-scale analysis of differential gene expression in postnatal skin of these mice identified 16 previously unknown and 13 known genes as presumptive Hoxc13 targets. The majority of these targets are downregulated and belong to a subgroup of genes that encode hair-specific keratin-associated proteins (KAPs). Genomic mapping using a mouse hamster radiation hybrid panel showed these genes to reside in a novel KAP gene cluster on mouse chromosome 16 in a region of conserved linkage with human chromosome 21q22.11. Furthermore, data obtained by Hoxc13/lacZ reporter gene analysis in mice that overexpress Hoxc13 suggest negative autoregulatory feedback control of Hoxc13 expression levels, thus providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hox gene expression. Combined, these results provide a framework for understanding molecular mechanisms of Hoxc13 function in hair growth and development.

Increased in vivo phosphorylation of ret tyrosine 1062 is a potential pathogenetic mechanism of multiple endocrine neoplasia type 2B.

Mutations of the Ret receptor tyrosine kinase are responsible for inheritance of multiple endocrine neoplasia (MEN2A and MEN2B) and familial medullary thyroid carcinoma syndromes. Although several familial medullary thyroid carcinoma and most MEN2A mutations involve substitutions of extracellular cysteine residues, in most MEN2B cases there is a methionine-to-threonine substitution at position 918 (M918T) of the Ret kinase domain. The mechanism by which the MEN2B mutation converts Ret into a potent oncogene is poorly understood. Both MEN2A and MEN2B oncoproteins exert constitutive activation of the kinase. However, the highly aggressive MEN2B phenotype is not supported by higher levels of Ret-MEN2B kinase activity compared with Ret-MEN2A. It has been proposed that Ret-MEN2B is more than just an activated Ret kinase and that the M918T mutation, by targeting the kinase domain of Ret, might alter Ret substrate specificity, thus affecting Ret autophosphorylation sites and the ability of Ret to phosphorylate intracellular substrates. We show that the Ret-MEN2B mutation causes specific potentiated phosphorylation of tyrosine 1062 (Y1062) compared with Ret-MEN2A. Phosphorylated Y1062 is part of a Ret multiple effector docking site that mediates recruitment of the Shc adapter and of phosphatidylinositol-3 kinase (PI3K). Accordingly, we show that Ret-MEN2B is more active than Ret-MEN2A in associating with She and in causing constitutive activation of the Ras/mitogen-activated protein kinase and PI3K/Akt cascades. We conclude that the MEN2B mutation specifically potentiates the ability of Ret to autophosphorylate Y1062 and consequently to couple to the Ras/mitogen-activated protein kinase and the PI3K/Akt pathways. The more efficient triggering of these pathways may account for the difference between MEN2A and MEN2B syndromes.