Browsing by Author "Tapia, Pablo"
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Item c-Abl Inhibition Activates TFEB and Promotes Cellular Clearance in a Lysosomal Disorder(Cell Press, 2020) Contreras, Pablo; Tapia, Pablo; González-Hodar, Lila; Peluso, Ivana; Soldati, Chiara; Napoiotano, Gennaro; Matarese, María; Las Heras, Macarena; Valls, Cristian; Martínez, Alexis; Balboa, Elisa; Castro, Juan; Nancy, Leal; Platt, Frances; Sobota, Andrzej; Winter, Dominic; Klein, Andrés; Medina, Diego; Ballabio, Andrea; Alvarez, Alejandra; Zanlungo, SilvanaThe transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB. Here, we show that under pharmacological and genetic c-Abl inhibition, TFEB translocates into the nucleus promoting the expression of its target genes independently of its well-known regulator, mammalian target of rapamycin complex 1. Active c-Abl induces TFEB phosphorylation on tyrosine and the inhibition of this kinase promotes lysosomal biogenesis, autophagy, and exocytosis. c-Abl inhibition in Niemann-Pick type C (NPC) models, a neurodegenerative disease characterized by cholesterol accumulation in lysosomes, promotes a cholesterol-lowering effect in a TFEB-dependent manner. Thus, c-Abl is a TFEB regulator that mediates its tyrosine phosphorylation, and the inhibition of c-Abl activates TFEB promoting cholesterol clearance in NPC models.Item Extracorporeal membrane oxygenation improves survival in a novel 24-hour pig model of severe acute respiratory distress syndrome(e-Century Pub. Corp, 2016) Araos, Joaquín; Alegría, Leyla; García, Patricio; Damiani, Felipe; Tapia, Pablo; Soto, Dagoberto; Salomón, Tatiana; Rodríguez, Felipe; Amthauer, Macarena; Erranz, Benjamín; Castro, Gabriel; Carreño, Pamela; Medina, Tania; Retamal, Jaime; Cruces, Pablo; Bugedo, Guillermo; Bruhn, AlejandroExtracorporeal membrane oxygenation (ECMO) is increasingly being used to treat severe acute respiratory distress syndrome (ARDS). However, there is limited clinical evidence about how to optimize the technique. Experimental research can provide an alternative to fill the actual knowledge gap. The purpose of the present study was to develop and validate an animal model of acute lung injury (ALI) which resembled severe ARDS, and which could be successfully supported with ECMO. Eighteen pigs were randomly allocated into three groups: sham, ALI, and ALI + ECMO. ALI was induced by a double-hit consisting in repeated saline lavage followed by a 2-hour period of injurious ventilation. All animals were followed up to 24 hours while being ventilated with conventional ventilation (tidal volume 10 ml/kg). The lung injury model resulted in severe hypoxemia, increased airway pressures, pulmonary hypertension, and altered alveolar membrane barrier function, as indicated by an increased protein concentration in bronchoalveolar fluid, and increased wet/dry lung weight ratio. Histologic examination revealed severe diffuse alveolar damage, characteristic of ARDS. Veno-venous ECMO was started at the end of lung injury induction with a flow > 60 ml/kg/min resulting in rapid reversal of hypoxemia and pulmonary hypertension. Mortality was 0, 66.6 and 16.6% in the SHAM, ALI and ALI + ECMO groups, respectively (p < 0.05). This is a novel clinically relevant animal model that can be used to optimize the approach to ECMO and foster translational research in extracorporeal lung support.Publication Spheroids derived from the stromal vascular fraction of adipose tissue self-organize in complex adipose organoids and secrete leptin(2023) Robledo, Fermín; González, Lila; Tapia, Pablo; Figueroa, Ana; Ezquer, Fernando; Cortés, VíctorBackground: Adipose tissue-derived stromal vascular fraction (SVF) harbors multipotent cells with potential therapeutic relevance. We developed a method to form adipose spheroids (AS) from the SVF with complex organoid structure and enhanced leptin secretion upon insulin stimulation. Methods: SVF was generated from the interscapular brown adipose tissue of newborn mice. Immunophenotype and stemness of cultured SVF were determined by flow cytometry and in vitro differentiation, respectively. Spheroids were generated in hanging drops and non-adherent plates and compared by morphometric methods. The adipogenic potential was compared between preadipocyte monolayers and spheroids. Extracellular leptin was quantified by immunoassay. Lipolysis was stimulated with isoprenaline and quantified by colorimetric methods. AS viability and ultrastructure were determined by confocal and transmission electron microscopy analyses. Results: Cultured SVF contained Sca1 + CD29 + CD44 + CD11b- CD45- CD90- cells with adipogenic and chondrogenic but no osteogenic potential. Culture on non-adherent plates yielded the highest quantity and biggest size of spheroids. Differentiation of AS for 15 days in a culture medium supplemented with insulin and rosiglitazone resulted in greater Pparg, Plin1, and Lep expression compared to differentiated adipocytes monolayers. AS were viable and maintained leptin secretion even in the absence of adipogenic stimulation. Glycerol release after isoprenaline stimulation was higher in AS compared to adipocytes in monolayers. AS were composed of outer layers of unilocular mature adipocytes and an inner structure composed of preadipocytes, immature adipocytes and an abundant loose extracellular matrix. Conclusion: Newborn mice adipose SVF can be efficiently differentiated into leptin-secreting AS. Prolonged stimulation with insulin and rosiglitazone allows the formation of structurally complex adipose organoids able to respond to adrenergic lipolytic stimulation.