The potential of in vitro and ex vivo models to predict the efficacy of antifibrotic drugs

Fibrosis is involved in approximately 45% of all deaths in developed countries and available pharmacological treatments are scarce, if at all. Fibrosis is the result of a chronic pathological tissue repair process which can affect different organs of the body, leading to organ failure over time. In fibrotic diseases, healthy tissue is replaced by scar tissue mainly consisting of collagen type I, due to the imbalance between the synthesis and degradation. The increased deposition of extracellular matrix is driven by the activated fibroblast, the so-called myofibroblast.Only a few antifibrotic drugs are available, which is mainly due to the discordance of translation between pre-clinical studies and human clinical trials. In this thesis, we first present an optimized format of in vitro macromolecular crowding with a reduced culture time and minimal effect on myofibroblast phenotype, and tested the efficacy of four antifibrotic drugs (Imatinib, Omipalisib, Nintedanib and Galunisertib). Furthermore, we used the combination of in vitro and ex vivo models to evaluate its predictive power regarding three antifibrotic drugs (Imatinib, Pirfenidone and Galunisertib) in the context of animal models and clinical trials of kidney fibrosis. Finally, we describe the in vitro antifibrotic effect of a repurposed FDA-approved drug (Verteporfin) in Dupuytren’s disease fibroblasts, as well as a pilot study for establishing an ex vivo model for antifibrotic drug testing for the same disease

Macromolecular Crowding as a Tool to Screen Anti-fibrotic Drugs:The Scar-in-a-Jar System Revisited

An unsolved therapeutic problem in fibrosis is the overproduction of collagen. In order to screen the effect of anti-fibrotic drugs on collagen deposition, the Scar-in-a-Jar approach has been introduced about a decade ago. With macromolecular crowding a rapid deposition of collagen is seen, resulting in a substantial decrease in culture time, but the system has never been tested in an adequate way. We therefore have compared six different macromolecular crowders [Ficoll PM 70 (Fc70), Ficoll PM 400 (Fc400), a mixture of Ficoll 70 and 400 (Fc70/400), polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 500 (DxS500), and carrageenan (CR)] under profibrotic conditions (addition of TGFβ1) with primary human adult dermal fibroblasts in the presence of 0.5 and 10% FBS. We found that (1) collagen deposition and myofibroblast formation was superior with 0.5% FBS, (2) DxS500 and CR results in an aberrant collagen deposition pattern, (3) ND670 does not increase collagen deposition, and (4) CR, DxS500, and Fc40/700 affected important phenotypical properties of the cells when cultured under pro-fibrotic conditions, whereas PVP40 and PVP360 did less or not. Because of viscosity problems with PVP360, we conclude that PVP40 is the most optimal crowder for the screening of anti-fibrotic drugs. Finally, the effect of various concentrations of Imatinib, Galunisertib, Omipalisib or Nintedanib on collagen deposition and myofibroblast formation was tested with PVP40 as the crowder

Resveratrol augments ER stress and the cytotoxic effects of glycolytic inhibition in neuroblastoma by downregulating Akt in a mechanism independent of SIRT1

Cancer cells typically display increased rates of aerobic glycolysis that are correlated with tumor aggressiveness and a poor prognosis. Targeting the glycolytic pathway has emerged as an attractive therapeutic route mainly because it should spare normal cells. Here, we evaluate the effects of combining the inhibition of glycolysis with application of the polyphenolic compound resveratrol (RSV) in neuroblastoma (NB) cancer cell lines. Inhibiting glycolysis with 2-deoxy-D-glucose (2-DG) significantly reduced NB cell viability and was associated with increased endoplasmic reticulum (ER) stress and Akt activity. Administration of 2-DG increased the expression of the ER molecular chaperones GRP78 and GRP94, the prodeath protein C/EBP homology protein (CHOP) and the phosphorylation of Akt at S473, T450 and T308. Combined treatment with both RSV and 2-DG reduced GRP78, GRP94 and Akt phosphorylation but increased CHOP and NB cell death when compared with the administration of 2-DG alone. The selective inhibition of Akt activity also decreased 2-DG-induced GRP78 and GRP94 expression and increased CHOP expression, suggesting that Akt can modulate ER stress. Protein phosphatase 1 alpha (PP1 alpha) was activated by RSV, as indicated by a reduction in PP1 alpha phosphorylation at T320. Pretreatment of cells with tautomycin, a selective PP1 alpha inhibitor, prevented the RSV-mediated decrease in Akt phosphorylation, suggesting that RSV enhances 2-DG-induced cell death by activating PP1 and downregulating Akt. The RSV-mediated inhibition of Akt in the presence of 2-DG was not prevented by the selective inhibition of SIRT1, a known target of RSV, indicating that the effects of RSV on this pathway are independent of SIRT1. We propose that RSV inhibits Akt activity by increasing PP1 alpha activity, thereby potentiating 2-DG-induced ER stress and NB cell death

Verteporfin ameliorates fibrotic aspects of Dupuytren's disease nodular fibroblasts irrespective the activation state of the cells

Dupuytren's disease is a chronic, progressive fibroproliferative condition of the hand fascia which results in digital contraction. So far, treatments do not directly interfere with the (myo)fibroblasts that are responsible for the formation of the collagen-rich cords and its contraction. Here we investigated whether verteporfin (VP) is able to inhibit the activation and subsequent differentiation of DD nodular fibroblasts into myofibroblasts. Fibroblasts were isolated from nodules of 7 Dupuytren patients. Cells are treated (1) for 48 h with 5 ng/ml transforming growth factor β1 (TGF-β1) followed by 48 h with/without 250 nM VP in the absence of TGF-β1, or treated (2) for 48 h with TGF-β1 followed by 48 h with/without VP in the presence of TGF-β1. mRNA levels were measured by means of Real-Time PCR, and proteins were visualized by means of Western blotting and/or immunofluorescence. Quantitative data were statistically analyzed with GraphPad Prism using the paired t-test. We found that fibroblasts activated for 48 h with TGF-β1 show a decrease in mRNA levels of COL1A1, COL3A1, COL4A1, PLOD2, FN1EDA, CCN2 and SERPINE1 when exposed for another 48 h with VP, whereas no decrease is seen for ACTA2, YAP1, SMAD2 and SMAD3 mRNA levels. Cells exposed for an additional 48 h with TGF-β1, but now in the presence of VP, are not further activated anymore, whereas in the absence of VP the cells continue to differentiate into myofibroblasts. Collagen type I, fibronectin-extra domain A, α-smooth muscle actin, YAP1, Smad2 and Smad3 protein levels were attenuated by both VP treatments. We conclude that VP has strong anti-fibrotic properties: it is able to halt the differentiation of fibroblasts into myofibroblasts, and is also able to reverse the activation status of fibroblasts. The decreased protein levels of YAP1, Smad2 and Smad3 in the presence of VP explain in part the strong anti-fibrotic properties of VP. Verteporfin is clinically used as a photosensitizer for photodynamic therapy to eliminate abnormal blood vessels in the eye to attenuate macular degeneration. The antifibrotic properties of VP do not rely on photo-activation, as we used the molecule in its non-photoinduced state

DAF-16/FOXO requires Protein Phosphatase 4 to initiate transcription of stress resistance and longevity promoting genes

In C. elegans, the conserved transcription factor DAF-16/FOXO is a powerful aging regulator, relaying dire conditions into expression of stress resistance and longevity promoting genes. For some of these functions, including low insulin/IGF signaling (IIS), DAF-16 depends on the protein SMK-1/SMEK, but how SMK-1 exerts this role has remained unknown. We show that SMK-1 functions as part of a specific Protein Phosphatase 4 complex (PP4(SMK-1)). Loss of PP4(SMK-1) hinders transcriptional initiation at several DAF-16-activated genes, predominantly by impairing RNA polymerase II recruitment to their promoters. Search for the relevant substrate of PP4(SMK-1) by phosphoproteomics identified the conserved transcriptional regulator SPT-5/SUPT5H, whose knockdown phenocopies the loss of PP4(SMK-1). Phosphoregulation of SPT-5 is known to control transcriptional events such as elongation and termination. Here we also show that transcription initiating events are influenced by the phosphorylation status of SPT-5, particularly at DAF-16 target genes where transcriptional initiation appears rate limiting, rendering PP4(SMK-1) crucial for many of DAF-16's physiological roles

Incidencia social, familiar, biológica y académica del trastorno oposicionista desafiante

This article arises as a result of the research conducted on the knowledge of the causes, effects and behaviors deepen the understanding of the causes, effects and behaviors that the individual acquires to suffer Disorder Opposing Challenging (DOC), which is diagnosed incorrectly, since it gives no follow a protocol level Neuropsychological. The DOC treatment must be based on psychological interventions; which the family and the school on the characteristics of the disorder. Orient the intervention should be complemented with the use of drugs. A wrong treatment leads to develop conduct disorder in adolescence. (Rigau, García, Artigas, 2006).DOC introduces comorbidity with other disorders for this the importance of investigating aspects of family structure or the context in which the individual is located, to obtain a proper diagnosis or the cause of the triggering of such disorder.Keywords: disorder opposing challenging, children, negative conduct, hostile conduct, medicines.Este articulo surge como resultado de la investigación realizada sobre el conocimiento de las causas, efectos y comportamientos de profundizar en el conocimiento de las causas, efectos y comportamientos que adquiere el individuo al padecer Trastorno Oposicionista Desafiante (TOD), el cual es diagnosticado de manera errónea, porque se da sin seguir un protocolo en el ámbito neuropsicológico. El tratamiento para el TOD debe ser basado en intervenciones psicológicas que orienten la familia y la escuela sobre las características del trastorno. La intervención debe ser complementada con el uso de medicamentos. Un tratamiento erróneo conlleva a desarrollar un trastorno de conducta en la adolescencia. (Rigau, García, Artigas, 2006). El TOD presenta comorbilidad con otros trastornos, por esto la importancia de indagar aspectos sobre la estructura familiar o el contexto en el que se encuentra el individuo para obtener un diagnóstico adecuado o la causa del desencadenante de dicho trastorno.Palabras clave: Trastorno oposicional desafiante, niños, conducta negativa, conducta hostil, medicamentos

Predictive Value of Precision-Cut Kidney Slices as an Ex Vivo Screening Platform for Therapeutics in Human Renal Fibrosis

Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGFβ or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation; (2) expression of markers of TGFβ and PDGF signaling; and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis

Dislipidemias y estilos de vida de jóvenes

PublishedLa preocupación por estas alteraciones en el estilo de vida ha llevado a los entes responsables del bienestar biopsicosocial de la población, a generar estrategias en los diferentes campos del desarrollo humano, siendo una de las prioridades las instituciones educativas, como las universidades. Para esto, los lineamientos nacionales dados por el Ministerio de Salud buscan promover entornos saludables, debido a que los estudiantes universitarios son considerados la población adulto joven clave, para la promoción y prevención de la salud para las futuras generaciones

Abstract 3214: Resveratrol potentiates glycolytic inhibitor-induced neuroblastoma cell death independent of sirt activity

Abstract Introduction: Low survival rates and severe side effects of current neuroblastoma treatments underline the need for more effective less toxic therapies. Compared to normal cells, cancer cells demonstrate high glucose consumption even under aerobic conditions, therefore; targeting the glycolytic pathway in cancer cells as a potential therapy is currently an area of intense investigation. The polyphenolic compound resveratrol which can be found in grapes, peanuts, red wine and purple grape juice, has demonstrated antitumor activities both in vitro and in vivo. Here we investigate the potential of inhibiting glycolysis in combination with resveratrol treatment. Methods: To inhibit glycolysis we used the glucose analog, 2-Deoxy-D-glucose (2-DG). N-MYC amplified neuroblastoma cells (NB1691) were exposed to low dose 2-DG (0.5 and 2mM) and resveratrol 10uM. Cell viability was determined at 72hr using MTS assay. Western blotting was used to determine cell signaling pathways and apoptotic protein levels. The role of the NAD+ dependent HDAC sirtuin 1 (sirt1) was determined using 50uM of the inhibitor sirtinol. Results: While 2-DG did reduce cell viability (2mM:82+4.4% of control), the combination of 2-DG and resveratrol significantly reduced cell viability (20+2.2%) compared to resveratrol alone (69+2.8%). Western blot analysis indicated a robust activation of AKT with 2-DG with treatment, however this activation was blocked with 10uM resveratrol. Furthermore, western blot analysis revealed that this was caspase dependent cell death. PI3K/Akt inhibitors wortmannin and LY-294002 (10uM each) also reduced cell viability in combination with 2-DG to approximately 40% compared to non-treated, confirming the protective role of AKT signaling in response to 2-DG treatment. To determine if resveratrol, a well-known activator of sirt1, inhibited AKT via sirt1, cells were pretreated with sirtinol. Inhibiting sirt1 did not prevent resveratrol's inhibition of AKT. Conclusions: The oncoprotein AKT mediates signaling processes involved in evading apoptosis and promoting cell proliferation and recently has been linked to glycolysis. Resveratrol significantly increased 2-DG-induced cell death by inhibiting the 2-DG induced increase in AKT activity independent of sirt1. Here we show that combining resveratrol with 2-DG, two relatively non-toxic agents can significantly reduce neuroblastoma cell viability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3214. doi:1538-7445.AM2012-3214</jats:p