MiARNs angiogénicos en la regulación del tejido adiposo y su relación con el desarrolo de la resistencia a la insulina asociada a la obesidad

Para llevar a cabo estos objetivos se utilizaron muestras humanas de TA visceral y subcutáneo y sistemas celulares in vitro e in vivo. In vitro, utilizamos células de una línea celular de preadipocitos, las células 3T3-L1. Los experimentos in vivo fueron realizados en ratones de la cepa C57BL-6J los cuales fueron alimentados con dieta alta en grasa para obtener un modelo de ratón con obesidad inducida por dieta. Los resultados nos han permitido determinar que en TA visceral y subcutáneo humano, la expresión de miR-15b, miR-20a, miR-20b, miR-27b, miR-126, miR-130a, miR-27, miR-221, miR-222, miR-296 y Let-7f están influenciados por obesidad y diabetes, cuando estos factores se presentan conjuntamente en un modo específico de depósito de grasa, Además, la interacción entre obesidad, TA, DMT2 y un grupo de microARNs formado por miR-20b, miR-296 y Let-7f, podrían estar involucrados en las vías de señalización de angiogénesis y Wnt por la regulación de seis genes diana: CDKN1A, CX3CL1, HIF1A, PPP2R1B, STAT3 y VEGFA. miR-221 y miR-222 parecen estar regulando DVL2 e IL1RAP en relación a la obesidad y la resistencia a la insulina. Además, las modificaciones que afectan la metilación de los genes CPS1, ELMO1, STEAP3, IQCE y MGDA1 parecen estar asociadas a la DMT2 en el estado de obesidad. Fecha de lectura de Tesis: 27 Septiembre 2018Según los últimos datos emitidos por la Organización Mundial de la Salud, la obesidad ha alcanzado proporciones epidémicas a nivel mundial. El incremento del índice de masa corporal puede conducir al desarrollo de otras enfermedades, y aumentar el riesgo de desarrollar resistencia a la insulina (RI) y diabetes mellitus tipo 2 (DMT2). La obesidad se caracteriza por un aumento excesivo de grasa como resultado de un balance energético positivo y por la presencia de inflamación de bajo grado. La relación entre estas patologías está bien establecida, sin embargo, los mecanismos que conducen a ellas no están totalmente definidos. El crecimiento del tejido adiposo (TA) puede producirse por hiperplasia o por hipertrofia, dos mecanismos complejos cuyos principales factores son adipogénesis, apoptosis y angiogénesis. Estudios recientes sugieren que una alteración en la capacidad de expansión o funcionalidad del TA puede conducir a que el exceso de grasa se deposite en otros órganos y esto aumentar el riesgo al desarrollo de comorbilidades asociadas a la obesidad. Los objetivos de este estudio fueron 1) Analizar el nivel de expresión de 11 miARNs angiogénicos en tejido adiposo humano en relación con obesidad y DMT2, 2) realizar un análisis bioinformático para determinar los genes diana de estos miARNs, clasificarlos para saber en qué procesos biológicos intervienen y crear una red de interacción para relacionar a los microARNs con sus genes diana y las vías de señalización en las cuales están involucrados, 3) estudiar el efecto que ejercen estos microARNs sobre sus genes dianas, incluyendo un análisis de metilación de ADN

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

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)

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

Inflammatory gene expression in adipose tissue according to diagnosis of anxiety and mood disorders in obese and non-obese subjects

Abstract Psychiatric disorders have been widely reported to be associated with systemic inflammation upregulation and adiposity. However, there are no data that link adipose tissue inflammation to these mental disorders. The analysis of adipokines and inflammation-related markers in adipose tissue could help to elucidate the potential association between obesity and mental health. An observational study was conducted in samples of patients consisting of non-obese and obese subjects, who were diagnosed with anxiety or mood disorders. Gene expression of adiponectin (ADIPOQ), leptin (LEP) and inflammatory markers (IL6, IL1B, TNF, CCL2, CSF3, ITGAM, and PLAUR) were determined in visceral (VAT) and subcutaneous (SAT) adipose tissues. Our results showed that the gene expression of adipokines and inflammation-related markers was higher in the VAT and SAT of obese subjects compared with non-obese subjects. Regarding mental disorders, all the inflammatory genes in the VAT were significantly higher in non-obese subjects with anxiety or mood disorders than in subjects without mental disorders, except for TNF and ITGAM. Additionally, IL6 expression was significantly lower in SAT. In contrast, obese patients diagnosed with anxiety or mood disorders only showed significantly lower expression levels of IL1B in VAT and ADIPOQ in SAT when compared with obese subjects without mental disorders. These data suggest the potential involvement of VAT inflammation in anxiety and mood disorders, involving complex mechanisms which are strongly affected by obesity

miR-20b, miR-296, and Let-7f Expression in Human Adipose Tissue is Related to Obesity and Type 2 Diabetes.

This study aimed to analyze the potential association of different microRNA (miRNA) molecules with both type 2 diabetes (T2D) and obesity and determine their target genes. Quantitative PCR was used to analyze the miR-20b, miR-296, and Let-7f levels in human visceral and subcutaneous adipose tissues (ATs) in relation to obesity and T2D, miRTarBase 4.0 was used for validation of target genes, and the Protein Analysis Through Evolutionary Relationships (PANTHER) Classification System and the Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to annotate the biological processes of the predicted targets. In AT, miR-20b, miR-296, and Let-7f levels were significantly different between normoglycemic subjects and those with T2D. In visceral adipose tissue, miRNA levels were higher in normoglycemic/obesity samples than in T2D/obesity samples. miR-20b-miR-296 and Let-7f target genes that showed significant differences in both ATs in relation to obesity and T2D were CDKN1A, CX3CL1, HIF1A, PPP2R1B, STAT3, and VEGFA. These genes are known to be principally involved in the vascular endothelial growth factor (VEGF) and WNT pathways. This study provides experimental evidence of the possible correlation between AT miR-20b-miR-296-Let-7f with obesity and T2D, which might involve vascular endothelial growth factor and WNT-dependent pathways that are regulated by six different genes, suggesting a novel signaling pathway that could be important for understanding the mechanisms underlying the AT dysfunction associated with obesity and T2D.This work was cofunded by the European Union through the European Regional Development Fund (FEDER) and was supported by grants from the Ministry of Economy and Competitiveness, Institute of Health Carlos III (PI15/01114, PI13/02628; PI12/02355), Ministry of Economy and Knowledge (PI‐CTS‐08181/2011; CTS‐7895/2011), and Consejeria de Salud, Junta de Andalucia (PI‐0092‐2017). RE is under a contract of a “Nicolas Monarde” program from the Servicio Andaluz de Salud

Osteogenic differentiation of human thymus and subcutaneous adipose tissues derived stromal cells (TAT-ASC and SAT-ASC).

<p>Representative photomicrographs of cells obtained from thymus fat (TAT-ASCs) (A) and from subcutaneous adipose tissue (SAT-ASCs) (B) (n = 26) grown in osteogenic medium for 14 days. Osteogenic differentiation of ASCs was confirmed by detecting calcium deposition by Alizarin Red S staining. (C) Osteogenic process confirmation was carried out by mRNA expression analysis of the osteogenic marker ALPL. mRNAs were normalized to cyclophilin expression levels. Each sample from each subject was analyzed separately and in triplicate. Data represent the mean ± SEM of the values obtained from all samples. The relationship between control and osteogenic differentiation was analyzed by range test with Wilcoxon sign. ^<b>*</b>p<0.05. Image magnification is 20X.</p

miR-21 mimic blocks obesity in mice: A novel therapeutic option

MicroRNAs (miRNAs) are promising drug targets for obesity and metabolic disorders. Recently, miRNA mimics are providing a unique mechanism of action that guides the process for drug development and sets out the context of their therapeutic application. miRNA (miR)-21 expression in white adipose tissue (WAT) has been associated with obesity. We aimed to analyze miR-21 expression levels in relation to diabetes and obesity to determine the effect that miR-21 mimic has on processes involved in WAT functionality, to dissect the underlying molecular mechanisms, and to study the potential therapeutic application of the miR-21 mimic against obesity. We found higher miR-21 levels in WAT from non-diabetic obese compared to normoweight humans and mice. Moreover, in 3T3-L1 adipocytes, miR-21 mimic affect genes involved in WAT functionality regulation and significantly increase the expression of genes involved in browning and thermogenesis. Interestingly, treatment with the miR-21 mimic blocked weight gain induced by a high-fat diet in obese mice, without modifying food intake or physical activity. This was associated with metabolic enhancement, WAT browning, and brown adipose tissue (AT) thermogenic programming through vascular endothelial growth factor A (VEGF-A), p53, and transforming growth factor β1 (TGF-β1) signaling pathways. Our findings suggest that miR-21 mimic-based therapy may provide a new opportunity to therapeutically manage obesity and consequently, its associated alterations

Comparative analysis of adipogenic and osteogenic marker mRNA expression levels between differentiated ASCs from elderly and middle aged myocardial ischemic subjects.

<p>FASN, ADRP, CEBPα and ALPL mRNA expression levels were analyzed in human adult thymus fat and subcutaneous adipose tissues (TAT-ASCs and SAT-ASCs) from middle age (45–65 years, n = 8) and elderly (> 70 years, n = 18) subjects. Each sample from each subject was analyzed separately and in triplicate. Data represent the mean ± SEM of the values obtained from all samples. The comparison between the two groups were carried out with Mann-Whitney U-Test.*p<0.05.</p