Los microARNs angiogénicos de grasa tímica humana como fuente complementaria de angiogénesis y regeneración de tejidos isquémicos en cirugía coronaria

Introducción: Las enfermedades cardiovasculares crónicas representan un importante problema de salud. La cirugía de revascularización coronaria es la primera opción de tratamiento. Pero hay un 12% de pacientes que no están aptos para ello. Actualmente, la terapia génica con varios factores de crecimiento (VEGF, FGF, HGF) han mejorado el manejo de la enfermedad, pero un 30% de estos pacientes no pueden tratarse con éxito y solo muestran aumento de la vascularización sin otros resultados con importancia clínica. En los últimos años, se ha visto que los miARNs forman parte de la red regulatoria de la expresión génica (en algunos tejidos pueden disminuir su producción/secreción con el envejecimiento). Entre ellas, regulan la respuesta angiogénica y pueden tener una potencial aplicación clínica. Además, se sabe que el timo humano adulto degenera en tejido graso (TAT) y como durante la cirugía cardiovascular parte del TAT se descarta, éste tejido puede ser utilizado como una fuente de miARNs angiogénicos para regenerar tejidos isquémicos. Métodos: Análisis bioinformáticos fueron utilizados para resaltar miARNs con sólida posibilidad de regular VEGFA o con sitios de unión fuertemente conservados y determinar qué otros genes podrían estar regulados por esos miARNs en el proceso de angiogénesis. Los niveles de expresión de miR-21-5p, miR-16-5p y miR-29b-3p fueron medidos con qPCR en TAT de pacientes (n=18) con isquemia cardíaca en dos grupos: Mediana edad (44-64 años) y > 70 años. Resultados: En nuestro estudio la edad no estuvo correlacionada con el nivel de expresión de los miARNs estudiados. Así, este trabajo muestra por primera vez el perfil de expresión de miR-21-5p, miR-16-5p y miR-29b-3p en TAT de sujetos con isquemia cardíaca y su posible red de interacción en el proceso de angiogénesis. Conclusión: El TAT es una fuente de miARNs angiogénicos que podrían tener un papel relevante en la terapia angiogénica y regeneración de tejidos isquémicos en cirugía coronaria.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Myocardial ischemic subject's thymus fat: A novel source of multipotent stromal cells

Objective Adipose Tissue Stromal Cells (ASCs) have important clinical applications in the regenerative medicine, cell replacement and gene therapies. Subcutaneous Adipose Tissue (SAT) is the most common source of these cells. The adult human thymus degenerates into adipose tissue (TAT). However, it has never been studied before as a source of stem cells. Material and Methods We performed a comparative characterization of TAT-ASCs and SAT-ASCs from myocardial ischemic subjects (n = 32) according to the age of the subjects. Results TAT-ASCs and SAT-ASCs showed similar features regarding their adherence, morphology and in their capacity to form CFU-F. Moreover, they have the capacity to differentiate into osteocyte and adipocyte lineages; and they present a surface marker profile corresponding with stem cells derived from AT; CD73+CD90+CD105+CD14-CD19-CD45-HLA-DR. Interestingly, and in opposition to SAT-ASCs, TAT-ASCs have CD14+CD34+CD133+CD45- cells. Moreover, TAT-ASCs from elderly subjects showed higher adipogenic and osteogenic capacities compared to middle aged subjects, indicating that, rather than impairing; aging seems to increase adipogenic and osteogenic capacities of TAT-ASCs. Conclusions This study describes the human TAT as a source of mesenchymal stem cells, which may have an enormous potential for regenerative medicine.Instituto de Salud Carlos III/FEDER, EU (PI10/01947, PI13/02628), CTS-7895 from the Consejer?a de Econom?a e Innovaci?n, Ciencia y Empleo, Junta de Andaluc?a/FEDER, EU. R. El Bekay is supported by fellowships from the ISCIII/FEDER, EU "Miguel Servet II" (CPII13/00041). AV-R is under a contract Proyectos de I+D+i para j?venes investigadores from the Ministerio de Econom?a y Competitividad (SAF2014-60649-JIN) and co-funded by Fondo Europeo de Desarrollo Regional-FEDER.Scopu

Au@16-pH-16/miR-21 mimic nanosystem: An efficient treatment for obesity through browning and thermogenesis induction

Despite the abundance of registered clinical trials worldwide, the availability of effective drugs for obesity treatment is limited due to their associated side effects. Thus, there is growing interest in therapies that stimulate energy expenditure in white adipose tissue. Recently, we demonstrated that the delivery of a miR-21 mimic using JetPEI effectively inhibits weight gain in an obese mouse model by promoting metabolism, browning, and thermogenesis, suggesting the potential of miR-21 mimic as a treatment for obesity. Despite these promising results, the implementation of more advanced delivery system techniques for miR-21 mimic would greatly enhance the advancement of safe and efficient treatment approaches for individuals with obesity in the future. Our objective is to explore whether a new delivery system based on gold nanoparticles and Gemini surfactants (Au@16-ph-16) can replicate the favorable effects of the miR-21 mimic on weight gain, browning, and thermogenesis. We found that dosages as low as 0.2 μg miR-21 mimic /animal significantly inhibited weight gain and induced browning and thermogenic parameters. This was evidenced by the upregulation of specific genes and proteins associated with these processes, as well as the biogenesis of beige adipocytes and mitochondria. Significant increases in miR-21 levels were observed in adipose tissue but not in other tissue types. Our data indicates that Au@16-ph-16 could serve as an effective delivery system for miRNA mimics, suggesting its potential suitability for the development of future clinical treatments against obesity.Instituto de Salud Carlos III (ISCIII) PI21/01924, PI18/00785European Union (UE) PI21/01924, PI18/00785European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER) PI20-01274Universidad de Sevilla 2021/00001297Junta de Andalucía PI20-01274, PI-0235-2021, DOC-01138, RC-0006-2020, C-0060-2018, PI-0092–2017Universidad de Málaga RC-0001-2021Ministerio de Economía y Competitividad (MINECO). España SAF2014-60649-JI

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.Instituto de Salud Carlos III (PI10/01947, PI13/02628) with Fondos FEDER and the Consejería de Economía e Innovación, Ciencia y Empleo, Junta de Andalucía (CTS-7895) with Fondos FEDER. R. El Bekay is under a contract Miguel Servet type II (CPII13/00041) from the Instituto de Salud Carlos III. F-JB-S is a recipient of a "Miguel Servet II" research contract (CPII13/00042) and also belongs to the regional "Nicolás Monardes" research program of the Consejería de Salud (C-0070-2012; Junta de Andalucía, Spain). This work was supported by the FIS-Thematic Networks and Co-Operative Research Centres RIRAAF (RD07-0064). JM is under the Programa de Intensificación de la Actividad Investigadora del Sistema Nacional de Salud. AV-R is under a contract Proyectos de I+D+i para jóvenes investigadores from the Ministerio de Economía y Competitividad (SAF2014-60649-JIN). S-YR-Z is recipient of a post-doctoral contract from Consejería de Salud de la Junta de Andalucía (RH-0070-2013)

Serum vascular endothelial growth factor b and metabolic syndrome incidence in the population based cohort [email protected] study

Background/Objectives Although vascular endothelial growth factor b (VEGFb) might have an impact on the development of obesity, diabetes and related disorders, the possible relationship between VEGFb serum levels and the incidence of these metabolic complications in humans is still unknown. The aim of our study was to evaluate the association between VEGFb serum levels and the new-onset of metabolic syndrome (MS) and its components in the Spanish adult population after 7.5 years of follow-up. Subjects/Methods A total of 908 subjects from the [email protected] cohort study without MS at cross-sectional stage according to International Diabetes Federation (IDF) or Adult Treatment Panel III (ATP-III) criteria were included. Additionally, five sub-populations were grouped according to the absence of each MS component at baseline. Socio-demographic, anthropometric and clinical data were recorded. The Short Form of International Physical Activity Questionnaire (SF-IPAQ) was used to estimate physical activity. A fasting blood extraction and an oral glucose tolerance test were performed. Serum determinations of glucose, lipids, hsCRP and insulin were made. VEGFb levels were determined and categorized according to the 75th percentile of the variable. New cases of MS and its components were defined according to ATPIII and IDF criteria. Results A total of 181 or 146 people developed MS defined by IDF or ATP-III criteria respectively. Serum triglyceride levels, hs-CRP and systolic blood pressure at the baseline study were significantly different according to the VEGFb categories. Adjusted logistic regression analysis showed that the likelihood of developing MS and abdominal obesity was statistically reduced in subjects included in the higher VEGFb category. Conclusion Low serum levels of VEGFb may be considered as early indicators of incident MS and abdominal obesity in the Spanish adult population free of MS, independently of other important predictor variables.This investigation has been supported by CIBERDEM (Ministerio de Economia, Industria y Competitividad-ISCIII), Ministerio de Sanidad, Servicios Sociales e IgualdadISCIII, Instituto de Salud Carlos III (research grants PI20/01322, PI18/01165, PI17/02136, PI14/00710) and cofunding by the European Regional Development Fund (ERDF) "A way to build Europe". LifeScan Espana (Madrid, Spain) kindly donated the glucometers and test strips for capillary glucose measurements. Cristina MaldonadoAraque is a researcher in the `Rio Hortega' program (CM19/00186) financed by the Instituto de Salud Carlos III. Natalia Colomo is a member of the regional "Accion B para clinicos investigadores" research program of the Consejeria de Salud of the Junta de Andalucia, Spain (B-0002-17). Gemma Rojo-Martinez belongs to the Nicolas Monardes research program of the Consejeria de Salud (C-0060-2012; Junta de Andalucia, Spain)

Au@16-pH-16/miR-21 mimic nanosystem: An efficient treatment for obesity through browning and thermogenesis induction

Despite the abundance of registered clinical trials worldwide, the availability of effective drugs for obesity treatment is limited due to their associated side effects. Thus, there is growing interest in therapies that stimulate energy expenditure in white adipose tissue. Recently, we demonstrated that the delivery of a miR-21 mimic using JetPEI effectively inhibits weight gain in an obese mouse model by promoting metabolism, browning, and thermogenesis, suggesting the potential of miR-21 mimic as a treatment for obesity. Despite these promising results, the implementation of more advanced delivery system techniques for miR-21 mimic would greatly enhance the advancement of safe and efficient treatment approaches for individuals with obesity in the future. Our objective is to explore whether a new delivery system based on gold nanoparticles and Gemini surfactants (Au@16-ph-16) can replicate the favorable effects of the miR-21 mimic on weight gain, browning, and thermogenesis. We found that dosages as low as 0.2 μg miR-21 mimic /animal significantly inhibited weight gain and induced browning and thermogenic parameters. This was evidenced by the upregulation of specific genes and proteins associated with these processes, as well as the biogenesis of beige adipocytes and mitochondria. Significant increases in miR-21 levels were observed in adipose tissue but not in other tissue types. Our data indicates that Au@16-ph-16 could serve as an effective delivery system for miRNA mimics, suggesting its potential suitability for the development of future clinical treatments against obesity.This research was supported by the following grants: This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project PI21/01924 and PI18/00785 and co-funded by the European Union, by the Consejería de Transformación Económica, Industria, Conocimiento y Universidades-Junta de Andalucía and ERDF-EU (PI20-01274) and by University of Sevilla VI PP USO SSGG (2021/00001297). PI-0092–2017 and PI-0235–2021 from Consejeria de Salud (Junta de Andalucia), Spain. S.L. is a recipient of a Plan Andaluz de Investigación, Desarrollo e Innovación post-doctoral grant from the Consejería de Economía, Conocimiento, Empresas y Universidades (DOC-01138). A.M.G. was a recipient of a Plan Propio de Investigación, Transferencia y Divulgación Científica postdoctoral grant from University of Málaga. FJBS, and REBAV-R are under contract with the ‘Nicolas Monardes’ program from the Servicio Andaluz de Salud, Consejería de Salud y Consumo-Junta de Andalucía (RC-0001-2021, RC-0006-2020 and C-0060-2018, respectively. A.V-R. was supported by a grant from the Ministerio de Economía y Competitividad (Proyectos I+D+i para Jóvenes Investigadores, SAF2014-60649-JIN).Peer reviewe

Papel de miR-21 en la activación del browning. Una posible estrategia para combatir la obesidad y la resistencia a la insulina.

El tratamiento in vivo e in vitro con mimic miR-21 aumentó significativamente la expresión de los genes termorreguladores y activadores del browning, Ucp1, Pgc-1α, PparƔ, Prdm 16, Cidea, Vegf-A y Fgf21 en la grasa marrón y la grasa beige. Además, el tratamiento con mimic miR-21 aumentó la expresión del marcador de la grasa beige Tmem 26 en la grasa beige y la grasa blanca. En 3T3-L1, el tratamiento con mimic miR-21 reguló la expresión de los factores angiogénicos, adipogénicos, apoptoticos, tales como las isoformas VEGFs, las angiopoyetinas, Timp-3, Bcl-2, Casp3, Bid, PparƔ, Cebpα, Fasn y Irs-1. Conclusión: Este estudio muestra el potencial papel que puede jugar el miR-21 en la activación de la termogénesis y el browning, mediante la activación del Ucp1, Pgc-1α, Prdm 16, PparƔ, Fgf21, Cidea, Vegf-A y Tmem 26, induciendo así la reducción de la ganancia de peso y mejorando la sensibilidad a la insulina.Objetivos del estudio: La regulación de la expansión del tejido adiposo (TA) está relacionada con el desarrollo de la obesidad y las comorbidades asociadas a ella, como la resistencia a la insulina. Es conocido que el microARN miR-21 juega un papel relevante en la regulación de varios procesos fisiológicos controlando el crecimiento y la proliferación celular. Sin embargo, hasta la fecha, el papel de este miARN en la regulación de la funcionalidad del TA en relación con la obesidad y la resistencia a la insulina, no está bien definido. El objetivo del presente estudio fue estudiar la implicación del miR-21 en la regulación de la funcionalidad del TA y su relación con la obesidad y la resistencia a la insulina. Materiales y métodos: Los niveles de expresión de miR-21 fueron medidos mediante qPCR en el TA visceral y subcutáneo de sujetos delgados sanos (n=10), obesos mórbidos sanos (n=10) y de ratones (C57BL/6J) sometidos a dieta estándar (DE, 10% kcal en grasa, n=10) y ratones obesos con dieta alta en grasa (DAG, 45% kcal en grasa, n=10). En el estudio in vivo los ratones obesos fueron tratados con mimic miR-21 (0,5µg) durante 8 semanas. Los ratones se pesaron 2 veces por semana. En estudio in vitro, los explantes de tejidos interescapulares procedentes de ratones delgados y las células 3T3-L1 fueron tratados con mimic miR-21. Los factores angiogénicos, adipogénicos, apoptoticos, termorreguladores y implicados en el browning fueron medidos mediante qPCR. Se usó la prueba t de student para el análisis estadístico. Resultados: La expresión de miR-21 aumentó significativamente con la obesidad en ambos tejidos TAV y TAS de humano y de ratón. In vivo, el tratamiento con mimic miR-21 redujo la ganancia de peso y mejoro la sensibilidad a la insulina en comparación con el control. No se notó ninguna diferencia en la actividad locomotora y la ingesta entre los dos grupos de ratones

Adipogenic Impairment of Adipose Tissue-Derived Mesenchymal Stem Cells in Subjects With Metabolic Syndrome: Possible Protective Role of FGF2.

The decreased expansion capacity of adipose tissue plays a crucial role in the onset of disorders associated with metabolic syndrome. The aim of this study was to examine the state of adipose tissue-derived mesenchymal stem cells (ASCs) from obese subjects with different metabolic profiles. This was a 2-year study to enroll subjects who underwent bariatric surgery or cholecystectomy. University Hospital. Patients who underwent either bariatric surgery (20 morbidly obese) or cholecystectomy (40 subjects) participated in the study. ASCs were obtained from both visceral and subcutaneous adipose tissue. Adipogenic, fibrotic gene expression was quantified by quantitative polymerase chain reaction; Smad7 and fibroblast growth factor 2 were quantified by western blotting and enzyme-linked immunosorbent assay, respectively. The susceptibility of ASCs to apoptosis, their population doubling time, and their clonogenic potential were evaluated. The worsening metabolic profile of the patients was accompanied by a decrease in the intrinsic levels of adipogenic gene expression, reduced proliferation rate, clonogenic potential, and exportation of fibroblast growth factor 2 to the cell surface of the ASCs derived from both tissues. In addition, the ASCs from patients without metabolic syndrome showed differences in susceptibility to apoptosis and expression of TGFβ-signaling inhibitory protein Smad7 with respect to the ASCs from patients with metabolic syndrome. Our results suggest that the decrease in adipogenic-gene mRNA and clonogenic potential, as well as the accumulation of fibrotic proteins with metabolic alterations, could be a relevant mechanism controlling the number and size of neogenerated adipocytes and involved in alteration of adipose-tissue expansion

Nanosystem based on microRNA for treating obesity

La presente invención se refiere a composiciones, métodos y usos, consistentes en un nanosistema funcionalizado y basado en miARN para el tratamiento de la obesidad, la pérdida de peso y/o la reducción de grasa localizada, que comprenden miR-21 u otros compuestos derivados o equivalentes como polinucleótidos modificados, mimético sintético y/o isomiR de miR-21; y un vehículo que comprende una nanopartícula optimizada para unir efectivamente oligonucleótidos, conformando un nanosistema adecuado para la transfección in vivo, y en particular para la liberación in vivo de genes en el tejido adiposo.The present invention relates to compositions, methods and uses, consisting of a functionalised miRNA-based nanosystem for treating obesity, weight loss and/or reduction of localised fat, which comprises miR-21 or other derived or equivalent compounds such as modified polynucleotides, synthetic mimetic and/or isomiR of miR-21; and a carrier which comprises an optimised nanoparticle for effectively binding oligonucleotides, forming an adequate nanosystem for in vivo transfection and, in particular, for the in vivo release of genes in fatty tissue.Españ

The cannabinoid CB1 receptor and mTORC1 signalling pathways interact to modulate glucose homeostasis in mice

The endocannabinoid system (ECS) is an intercellular signalling mechanism that is present in the islets of Langerhans and plays a role in the modulation of insulin secretion and expansion of the β-cell mass. The downstream signalling pathways mediating these effects are poorly understood. Mammalian target of rapamycin complex 1 (mTORC1) signalling is a key intracellular pathway involved in energy homeostasis and is known to importantly affect the physiology of pancreatic islets. We investigated the possible relationship between cannabinoid type 1 (CB1) receptor signalling and the mTORC1 pathway in the endocrine pancreas of mice by using pharmacological analysis as well as mice genetically lacking the CB1 receptor or the downstream target of mTORC1, the kinase p70S6K1. In vitro static secretion experiments on islets, western blotting, and in vivo glucose and insulin tolerance tests were performed. The CB1 receptor antagonist rimonabant decreased glucose-stimulated insulin secretion (GSIS) at 0.1 µM while increasing phosphorylation of p70S6K1 and ribosomal protein S6 (rpS6) within the islets. Specific pharmacological blockade of mTORC1 by 3 nM rapamycin, as well as genetic deletion of p70S6K1, impaired the CB1-antagonist-mediated decrease in GSIS. In vivo experiments showed that 3 mg/kg body weight rimonabant decreased insulin levels and induced glucose intolerance in lean mice without altering peripheral insulin sensitivity; this effect was prevented by peripheral administration of low doses of rapamycin (0.1 mg/kg body weight), which increased insulin sensitivity. These findings suggest a functional interaction between the ECS and the mTORC1 pathway within the endocrine pancreas and at the whole-organism level, which could have implications for the development of new therapeutic approaches for pancreatic β-cell diseases