Efecto de los reguladores de óxido nítirco LA-419 y ONO-1714 en un modelo de infarto cerebral focal en el ratón

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Anatomía, Histología y Neurociencia. Fecha de lectura: 17-11-2014Las enfermedades cerebrovasculares son la primera causa de fallecimiento en las mujeres y la tercera en los hombres en España. A pesar de su trascendencia, actualmente sólo existe un compuesto farmacológico para su tratamiento, el r-tPA, pero la carencia de productos neuroprotectores eficaces determina que su búsqueda sea actualmente una de las prioridades de la industria farmacéutica. En este estudio se han ensayado dos fármacos dirigidos al control de la producción excesiva de Óxido Nítrico (NO), causante del estrés oxidativo, como posible mecanismo de protección contra los procesos neurodegenerativos inducidos por la isquemia y así limitar el déficit neurológico secundario. Por una parte, el compuesto LA-419 (LACER, SA), es capaz de aumentar la expresión génica de la isoforma endotelial de la NO-sintasa (eNOS) a las seis horas tras el infarto pudiendo estar mediado este proceso por reguladores como Hsp90 y Akt, produciendo un incremento de los niveles de NO a nivel vascular. Esta mayor liberación que determina vasodilatación, podría estar implicada en la disminución observada en el volumen del infarto, a la vez que en la mejoría del control motor secundario al ictus isquémico. Además, el producto LA-419 regula “in vivo” las cascadas inflamatorias y apoptóticas, a la vez que promueve la generación de nuevas células que podrían integrarse en los procesos de reparación del daño isquémico, contribuyendo por estas vías al efecto neurorreparador observado. El otro compuesto ensayado en este estudio, ONO-1714, es un conocido inhibidor del sistema NO sintasa, fundamentalmente a nivel de la isoforma inducible (iNOS). Mediante un novedoso procedimiento de síntesis de este compuesto, hemos obtenido un producto que actúa muy activamente frente a iNOS en ensayos “in vitro”. Su efectividad como tratamiento crónico en nuestro estudio ha ofrecido resultados positivos en la reducción del volumen del infarto. En conclusión, nuestros resultados demuestran que los compuestos LA-419 y ONO-1714 potencian los procesos de neurorreparación y neuroprotección, y apoyan su posible uso en la práctica clínic

Astrocytes require insulin-like growth factor I to protect neurons against oxidative injury

Oxidative stress is a proposed mechanism in brain aging, making the study of its regulatory processes an important aspect of current neurobiological research. In this regard, the role of the aging regulator insulin-like growth factor I (IGF-I) in brain responses to oxidative stress remains elusive as both beneficial and detrimental actions have been ascribed to this growth factor. Because astrocytes protect neurons against oxidative injury, we explored whether IGF-I participates in astrocyte neuroprotection and found that blockade of the IGF-I receptor in astrocytes abrogated their rescuing effect on neurons. The protection mediated by IGF-I against oxidative stress (H2O2) in astrocytes is probably needed for these cells to provide adequate neuroprotection. Indeed, in astrocytes but not in neurons, IGF-I helps decrease the pro-oxidant protein thioredoxin-interacting protein 1 and normalizes the levels of reactive oxygen species. Furthermore, IGF-I cooperates with trophic signals produced by astrocytes in response to H2O2 such as stem cell factor (SCF) to protect neurons against oxidative insult. After stroke, a condition associated with brain aging where oxidative injury affects peri-infarcted regions, a simultaneous increase in SCF and IGF-I expression was found in the cortex, suggesting that a similar cooperative response takes place in vivo. Cell-specific modulation by IGF-I of brain responses to oxidative stress may contribute in clarifying the role of IGF-I in brain aging

Astrocytes require insulin-like growth factor I to protect neurons against oxidative injury

Oxidative stress is a proposed mechanism in brain aging, making the study of its regulatory processes an important aspect of current neurobiological research. In this regard, the role of the aging regulator insulin-like growth factor I (IGF-I) in brain responses to oxidative stress remains elusive as both beneficial and detrimental actions have been ascribed to this growth factor. Because astrocytes protect neurons against oxidative injury, we explored whether IGF-I participates in astrocyte neuroprotection and found that blockade of the IGF-I receptor in astrocytes abrogated their rescuing effect on neurons. The protection mediated by IGF-I against oxidative stress (H 2O 2) in astrocytes is probably needed for these cells to provide adequate neuroprotection. Indeed, in astrocytes but not in neurons, IGF-I helps decrease the pro-oxidant protein thioredoxin-interacting protein 1 and normalizes the levels of reactive oxygen species. Furthermore, IGF-I cooperates with trophic signals produced by astrocytes in response to H 2O 2 such as stem cell factor (SCF) to protect neurons against oxidative insult. After stroke, a condition associated with brain aging where oxidative injury affects peri-infarcted regions, a simultaneous increase in SCF and IGF-I expression was found in the cortex, suggesting that a similar cooperative response takes place in vivo. Cell-specific modulation by IGF-I of brain responses to oxidative stress may contribute in clarifying the role of IGF-I in brain aging. © 2014 Genis L et al.This work was funded by grants of the Spanish Ministry of Science (SAF2010-17036) and Centro Investigacion Biomedica en red Enfermedades Neurodegenerativas (CIBERNED) to IT-A.Peer Reviewe

Lack of adrenomedullin in mouse endothelial cells results in defective angiogenesis, enhanced vascular permeability, less metastasis, and more brain damage

Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. AM plays critical roles in blood vessels, including regulation of vascular stability and permeability. To elucidate the autocrine/paracrine function of AM in endothelial cells (EC) in vivo, a conditional knockout of AM in EC (AM EC-KO) was used. The amount of vascularization of the matrigel implants was lower in AM EC-KO mice indicating a defective angiogenesis. Moreover, ablation of AM in EC revealed increased vascular permeability in comparison with wild type (WT) littermates. In addition, AM EC-KO lungs exhibited significantly less tumor growth than littermate WT mice using a syngeneic model of metastasis. Furthermore, following middle cerebral artery permanent occlusion, there was a significant infarct size decrease in animals lacking endothelial AM when compared to their WT counterparts. AM is an important regulator of EC function, angiogenesis, tumorigenesis, and brain response to ischemia. Studies of AM should bring novel approaches to the treatment of vascular diseases.Peer Reviewe

Lack of adrenomedullin in mouse endothelial cells results in defective angiogenesis, enhanced vascular permeability, less metastasis, and more brain damage

Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. AM plays critical roles in blood vessels, including regulation of vascular stability and permeability. To elucidate the autocrine/paracrine function of AM in endothelial cells (EC) in vivo, a conditional knockout of AM in EC (AM EC-KO) was used. The amount of vascularization of the matrigel implants was lower in AM EC-KO mice indicating a defective angiogenesis. Moreover, ablation of AM in EC revealed increased vascular permeability in comparison with wild type (WT) littermates. In addition, AM EC-KO lungs exhibited significantly less tumor growth than littermate WT mice using a syngeneic model of metastasis. Furthermore, following middle cerebral artery permanent occlusion, there was a significant infarct size decrease in animals lacking endothelial AM when compared to their WT counterparts. AM is an important regulator of EC function, angiogenesis, tumorigenesis, and brain response to ischemia. Studies of AM should bring novel approaches to the treatment of vascular diseases

Nitric oxide: Target for therapeutic strategies in alzheimer's disease

Alzheimeŕs disease (AD) constitutes a progressive neurodegenerative disorder and the main cause of dementia. Numerous studies have focused on the pathogenic mechanism of AD to cure or prevent this devastating disease. But, despite recent advances, our understanding on the pathophysiology of this genetically complex and heterogeneous disorder is rather limited and treatment of the disease consists of medications to control the symptoms. Acetylcholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists are the only available treatments recommended to manage the cognitive deficits caused by the disease. Therefore, the production of new drugs that may be able to cure the underlying cause of this chronic disease, not just the symptoms, is a matter of clinical interest. There is data implicating nitric oxide (NO) in the progression of the disease. The three isoforms of the NO-synthesizing enzyme (NOS) operate as central mediators of amyloid beta-peptide (Aß) action, giving rise to elevated levels of NO that contributes to the maintenance, self-perpetuation and progression of the disease. Reducing Aß production and the cholinergic deficit is a goal in the treatment of AD. In addition, a possible way to delay the progression of the illness must include a rationale design of enzyme inhibitors, subtype selective, targeting NOS isoforms implicated in damage to brain cells in AD. We are now presenting an overview regarding approved drugs for AD treatment and substances that although are not in use for the treatment of AD, including NOS inhibitors, may represent useful tools to unravel the pathophysiologic enigma of AD. © 2010 Bentham Science Publishers Ltd.Peer Reviewe

Adrenomedullin and nitric oxide: Implications for the etiology and treatment of primary brain tumors

Gliomas, defined as tumors of glial origin, represent between 2-5% of all adult cancer and comprise the majority of primary brain tumors. Infiltrating gliomas, with an incidence of more than 40% of brain tumors, are the most common and destructive primary brain tumors for which conventional therapies have not significantly improved patient outcome. In fact, patients suffering from malignant gliomas have poor prognoses and the majority have local tumor recurrence after treatment. Tumor growth and spread of tumor cells depend basically upon angiogenesis and on functional abnormalities of tumor cells in the control of apoptosis, as they are paradigmatic for their intrinsic resistance to multiple pro-apoptotic stimuli. Therefore, promising strategies for treatment of brain cancer would be directed to appropriate neutralization of angiogenesis and sensibilization of cancer cells to undergo apoptosis. However, despite advances in this field, high-grade gliomas remain incurable with survival often measured in months. Therefore there is a need to discover new and more potent cocktails of drugs to target the key molecular pathways involved in glioma angiogenesis and apoptosis. This review deals with the effects of two groups of molecules closely linked to neural tissue, which have been implicated in brain cancer: nitric oxide and peptides of the adrenomedullin family. These molecules exert vasodilatory and proangiogenic actions. Adrenomedullin also has antiapoptotic functions at appropriate concentrations. The inhibition of these functions, in the case of cancer, may provide new pharmacological strategies in the treatment of this disease. © 2011 Bentham Science Publishers.Peer Reviewe

New synthesis and promising neuroprotective role in experimental ischemic stroke of ONO-1714

In an experimental permanent stroke model, we report here the contribution of ONO-1714 to brain damage prevention. Daily drug administration, twenty-one days prior to and two days after an experimental infarct, was performed by using mini-osmotic pumps (ALZET). Infarct volumes were assessed by image analysis of sequential coronal brain 1 mm 3 sections stained following the 2,3,5-triphenyltetrazolium chloride histological staining technique. Results of this study provide evidence of a significant reduction of the brain lesion size, suggesting ONO-1714 as a potential neuroprotective agent in stroke patients. ONO-1714 was prepared in our laboratory following a procedure which resulted in the supply of the desired compound in an easy and excellent yield. © 2012 Elsevier Masson SAS. All rights reserved.Peer Reviewe

Neural differentiation of transplanted neural stem cells in a rat model of striatal lacunar infarction: light and electron microscopic observations

The increased risk and prevalence of lacunar stroke and Parkinson's disease (PD) makes the search for better experimental models an important requirement for translational research. In this study we assess ischemic damage of the nigrostriatal pathway in a model of lacunar stroke evoked by damaging the perforating arteries in the territory of the substantia nigra (SN) of the rat after stereotaxic administration of endothelin-1 (ET-1), a potent vasoconstrictor peptide. We hypothesized that transplantation of neural stem cells (NSCs) with the capacity of differentiating into diverse cell types such as neurons and glia, but with limited proliferation potential, would constitute an alternative and/or adjuvant therapy for lacunar stroke. These cells showed neuritogenic activity in vitro and a high potential for neural differentiation. Light and electron microscopy immunocytochemistry was used to characterize GFP-positive neurons derived from the transplants. 48 h after ET-1 injection, we characterized an area of selective degeneration of dopaminergic neurons within the nigrostriatal pathway characterized with tissue necrosis and glial scar formation, with subsequent behavioral signs of Parkinsonism. Light microscopy showed that grafted cells within the striatal infarction zone differentiated with a high yield into mature glial cells (GFAP-positive) and neuron types present in the normal striatum. Electron microscopy revealed that NSCs-derived neurons integrated into the host circuitry establishing synaptic contacts, mostly of the asymmetric type. Astrocytes were closely associated with normal small-sized blood vessels in the area of infarct, suggesting a possible role in the regulation of the blood brain barrier and angiogenesis. Our results encourage the use of NSCs as a cell-replacement therapy for the treatment of human vascular Parkinsonism.This study was supported by grants from the Spanish Ministry for Science and Innovation (SAF2007-60010, SAF2010-15173), Servicio de Salud de Castilla La Mancha Community (SESCAM), FISCAM (PI-2008/18), Mutua Madrileña 2010, Red Neurovascular del ISCIII (RD06/0026/1001), Fundación Alicia Koplowitz, Colciencias and LACER SA.Peer reviewedPeer Reviewe