Doctora en Farmacología / La enfermedad de Chagas es causada por Trypanosoma cruzi. Este parásito desencadena una respuesta inflamatoria que tiende a controlar la infección en el hospedero. Sin embargo, la permanencia del parásito provoca la persistencia de la inflamación que finalmente conduce al desarrollo de la Cardiopatía Chagásica Crónica (CCC). La CCC es consecuencia de alteraciones microvasculares, entre otros mecanismos. Característicamente, la expresión de moléculas de adhesión celular (ECAMs) como ICAM-1, VCAM, y E-selectina, está aumentada para favorecer el reclutamiento de células inflamatorias. Es posible mejorar el tratamiento antichagásico actual modificando algunos aspectos de la fisiopatología en el hospedero. Así, se propone que simvastatina, por sus efectos pleiotrópicos que modulan la respuesta inflamatoria vascular, podría mejorar la disfunción endotelial inducida por T. cruzi. Este efecto estaría mediado por un mecanismo que involucra la producción de un eicosanoide proresolutorio de la inflamación denominado 15-epi-lipoxina A4, que deriva de la ruta sintética de 5-lipoxigenasa, o también conocido como lipoxinas gatilladas por aspirina. Se realizó un modelo de activación endotelial, en el que células endoteliales EA.hy926 y HUVEC fueron previamente tratadas con simvastatina 5 μM o benznidazol 20 μM durante 24 horas, y posteriormente infectadas con T. cruzi por 16 horas. Se observó que ambos fármacos fueron capaces de prevenir el aumento de la expresión de ECAMs y en consecuencia, disminuyeron la adhesión celular sin afectar la viabilidad celular ni la integridad del citoesqueleto. Además, este efecto es independiente de su actividad tripanocida. Por otra parte, ambos fármacos bloquearon la activación de la cascada de NF-κB y el traslado de p65 al núcleo, pero sólo simvastatina indujo la producción de 15-epi-lipoxina A4. Es más, la 15-epi-lipoxina A4 fue capaz de disminuir, por si misma, la expresión de ECAMs. Se concluye que simvastatina o benznidazol previenen la activación endotelial, aunque sólo simvastatina actúa a través de un mecanismo dependiente de la producción de este lípido pro-resolutorio de la inflamación, 15-epi-lipoxina A4. Como la inflamación y la disfunción endotelial tienen un rol pivotal en la patogénesis de la enfermedad de Chagas, es posible que simvastatina y benznidazol puedan modular el daño microvascular de la enfermedad de Chagas. Por lo tanto, en este trabajo presentamos las bases que soportan el uso de simvastatina en el tratamiento de la Cardiopatía Chagásica / Chagas disease is caused by Trypanosoma cruzi. This parasite triggers an inflammatory response to control host's infection. As the inflammatory response persists, the patients develop Chronic Chagas Cardiomyopathy (CCC). The CCC is consequence of microvascular alterations, among others mechanisms. This process is characterized by an increased expression of Endothelial Cell Adhesion Molecules (ECAMs) like ICAM-1, VCAM, and E-selectin, allowing inflammatory cell recruitment. It is possible to improve the current treatment modifying some aspects of the pathophysiology in the host. Thus, it is proposed that simvastatin may improve vascular immune response due to their pleiotropic effects in the modulating inflammatory responses and improve endothelial dysfunction induced by T. cruzi. This effect would be mediated by a mechanism that involves the production of a novel pro-resolving lipid, the 5-lypoxygenase derivative 15-epi-lipoxin A4, which belongs to aspirin triggered lipoxins. A model of endothelial activation was performed in which EA.hy926 and HUVEC endothelial cells were pretreated with 5 μM simvastatin or 20 μM benznidazole for 24 hours, and then infected with T. cruzi parasite for 16 hours. It was observed that both drugs prevented the increase in ECAMs and in consequence, decreases cell adhesion without affecting the cell viability and cytoskeleton. Thus, the effect is independent of their trypanocidal activity. Furthermore, both drugs blocked NF-κB activation and they blocked the transfer of the nucleus p65, and in the case of simvastatin, this effect was mediated by the production of 15-epi-lipoxin A4. In the other hand, the 15-epi-lipoxin A4 was able to decrease by itself, the expression of ECAMs. In conclusion, both drugs prevent the T. cruzi-induced endothelial activation but only simvastatin acts through a mechanism dependent on the production of this pro-resolving lipid, 15-epi-lipoxin A4. As the inflammation and endothelial dysfunction have a key role in the pathogenesis of Chagas disease, simvastatin and benznidazole may modulate the microvascular damage in Chagas disease. Thus, we provide the bases that support the future use of simvastatin in the treatment of cardiac Chagas disease

Memoria para optar al Título Profesional de Médico Veterinario. Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias mención Patología Animal. / Introducción. El cáncer mamario es el tipo de tumor más prevalente en la hembra canina. Información reciente demuestra que simvastatina exhibe un efecto antitumoral in vitro e induce autofagia sobre diversas líneas celulares neoplásicas. Sin embargo, no hay evidencia de que ocurra en células de carcinoma mamario canino. La autofagia es un proceso catabólico que permite a las células sobrevivir bajo condiciones de estrés metabólico, cumpliendo un rol dual en cáncer asociado a supresión y promoción tumoral dependiendo del contexto celular. En ese contexto, la autofagia jugaría un rol en la sobrevida y muerte de células tumorales en respuesta a agentes antitumorales. El objetivo de este estudio fue analizar los efectos de simvastatina sobre la autofagia y la viabilidad de células de carcinoma mamario canino CF41.Mg. Materiales y métodos. Células CF41. Mg fueron cultivadas y tratadas con simvastatina (1 y 5 M) por 24 horas y analizadas mediante citometría de flujo utilizando naranja de acridina. Se analizó la expresión de LC3 y beclina-1 en células tratadas con simvastatina y 3-metiladenina (inhibidor de autofagia) mediante inmunocitoquímica. Adicionalmente, se realizó un ensayo de viabilidad celular mediante el método de exclusión de azul tripan con células tratadas con simvastatina en presencia de Q-VD-OPh (inhibidor de apoptosis) y 3-metiladenina. Resultados Tanto la actividad autofágica como la expresión de LC3 y beclina-1 fueron inducidas por simvastatina (p<0.05). Estos efectos fueron revertidos por 3-metiladenina. Simvastatina inhibió la viabilidad celular en forma significativa, aún en presencia de 3-metiladenina (p<0.05). Q-VD-OPh bloqueó el efecto antiproliferativo desencadenado por simvastatina. Conclusiones El efecto antiproliferativo de simvastatina sobre células de carcinoma mamario canino CF41.Mg no es dependiente de la autofagia inducida por la misma droga. La citotoxicidad de simvastatina se explica casi completamente por la activación de caspasas / Introduction Mammary cancer is the most prevalent type of tumor in the female dogs. Recent data shows that simvastatin exhibit an antitumor effect in vitro and it is able to induce autophagy in some neoplastic cell lines. However, there is still no evidence in canine mammary cancer cells. Autophagy is a mayor catabolic process that allows the cells to survive under metabolic stress, performing a dual role in cancer associated to tumor suppression/progression depending on cellular context. Under this context, the autophagy may play a role in the survival and death of tumoral cell in response to anti-tumoral agents. The object of this study was to analyze the effects of simvastatin over the autophagy and viability of canine mammary carcinoma cells CF41.Mg. Materials and methods CF41.Mg cells were treated with simvastatin (1 y 5 M) for 24 hours and analyzed by flow cytometry using acridine orange dye. The expression of LC3 and beclin-1 in cells treated with simvastatin/3-methyladenine (autophagy inhibitor) by immunocytochemistry was analyzed. Cell proliferation assays with trypan blue dye in presence of simvastatin, Q-VD-OPh hydrate (pancaspase inhibitor) and 3-methyladenine were performed. Results Both autophagy activity and LC3/beclin-1 expression were induced by simvastatin (p<0.05). These effects were reversed by 3-methyladenine. This statin inhibited the cell viability, even in the presence of 3-methyladenine (p<0.05). Q-VD-OPh hydrate blocked the anti-proliferative effect of simvastatin. Conclusions The anti-proliferative effect of simvastatin over canine mammary carcinoma cells CF41.Mg it’s non-depending of the autophagy induced by the drug. The cytotoxicity of simvastatin it’s explained almost completely by the caspases activation / Financiamiento: Proyecto Fondecyt No. 11110148.

Por meio da técnica de microperfusão estacionária in vivo, túbulos proximais (TP) de ratos Wistar foram perfundidos com solução CTRL contendo ou não Sinva 100&#956;M ou Sil 10&#956;M para determinar a reabsorção de bicarbonato (JHCO3-). A perfusão de Sil diminuiu o JHCO3- em 20%, enquanto que Sinva incrementou em 19,31% o JHCO3- comparado com o CTRL. Já a Prav diminuiu em 15% o JHCO3- . Para testar se os efeitos foram dependents de NHE3, PTs foram perfundidos com CTRL, Sil, Sinva ou Prav mais S3226, um inibidor específico de NHE3. O JHCO3- remanescente (insensível à S3226) não foi diferente entre os grupos, mostrando que NHE3 foi modulado por Sil e Sinva. Já o efeito inibitório da Prav ocorre não apenas via NHE3 mas também via H+ ATPase. Para determinar se a via Rho está envolvida nestes efeitos, os PTs foram perfundidos com Y-27632 1&#956;M, inibidor de Rho GTPase. A perfusão com Y-27632 reverteu o efeito inibitório promovido tanto pelo Sil quanto pela Prav, demonstrando que o efeito inibitório destes fármacos sobre a atividade do NHE3 ocorre via Rho. Além disso, o efeito inibitório de Sil foi completamente abolido pelo inibidor de PKG dependente de GMPc KT5823 10-6 M. Para confirmar estes resultados, animais foram mantidos em gaiolas metabólicas por 24 h e tratados via oral com 7mg/Kg/dia de Sinva e 20mg/Kg/dia de Sil. Sil diminuiu a CE Na+, indicando que outros mecanismos de transporte podem estar envolvidos em alterações no manejo de Na+ pelos rins promovidas por Sil. Já a infusão de Prav em ratos Wistar pela jugular durante 30 min promoveu aumento da CE Na+, sugerindo que o fármaco age inibindo NHE3. / By means of stationary microperfusion, PT of Wistar rats were perfused with a CTRL solution with or without 100&#956;M Simva or 10&#956;M Sil to determine bicarbonate reabsorption (JHCO3-). Perfusion of Sil decreased JHCO3- by 20% and perfusion of Simva increased JHCO3- by 19,31% compared to CTRL. Prav decreased JHCO3- by 15%. To test if these effects were NHE3-dependent, PTs were perfused with CTRL, Sil, Simva or Prav plus 2&#956; M S3226, a specific NHE3 inhibitor. The reminiscent S3226-insensitive JHCO3- was not different among groups, showing that NHE3 was modulated by Sil or Simva. To determine if Rho was involved in these effects, the PTs were perfused with the Rho GTPase inhibitor Y-27632 (1&#956;M). Perfusion with Y-27632 reversed the inhibitory effect promoted by both Sil and Prav, demonstrating that the inhibitory effect of these drugs on NHE3 activity occurs via Rho. Furthermore, the inhibitory effect of Sil was completely abolished by either the protein kinase G (PKG) inhibitor dependent of cGPM KT5823 10-6 M. To confirm our results the animals were kept for 24 hours in metabolic cages and treated orally with 7mg/kg Sim or 20mg/kg Sil. The Sil decreased the Na+ excretion load, indicating that other transport mechanisms beyond the proximal tubule involved in the changes of handling of Na+ by the treatment with Sil. On the other hand, the infusion of Prav in Wistar rats by jugular for 30 min promoted an increase in Na+ CE, suggesting that the drug acts by inhibiting NHE3.