Tejido adiposo tímico como fuente de angiogénesis

El mayor reto de la ingeniería tisular por el momento es la inducción de una adecuada vascularización. Numerosos métodos se han utilizado para ello, pero con resultados insatisfactorios (Lovett M. y col.; 2009). El Tejido Adiposo, órgano endocrino activo con densa vasculatura, secreta una amplia cantidad de factores angiogénicos y adipogénicos y parece constituir una atractiva fuente de dichos factores bioactivos muy a considerar para las aplicaciones de ingeniería tisular. El tejido adiposo de timo (TAT) y sus células derivadas (células madre mesenquimales y progenitoras endoteliales derivadas del TAT), gracias a su potencial de diferenciación y su capacidad proangiogénica y antiapoptótica, pueden servir como fuente de angiogénesis de gran utilidad, en comparación con un tejido adiposo comúnmente usado para ese tipo de terapias como es el tejido adiposo subcutáneo (TAS), para ayudar en la neo-formación de los vasos sanguíneos y regeneración de los tejidos isquémicos en pacientes de edad avanzada con isquemia cardíaca. La grasa tímica podría ser una novedosa fuente de factores con gran capacidad para inducir la angiogénesis y un tejido del que obtener células madre mesenquimales y progenitoras endoteliales que ayudasen a la neoformación de vasos sanguíneos y a la regeneración de tejidos isquémicos. Desarrollo En sujetos de mediana (45-65 años) y tercera (>70 años) edad con cardiopatía isquémica (n=35) se realizó un análisis comparativo de los niveles de expresión génica (análisis de PCR cuantitativa a tiempo real) y proteica (inmunodetección por Western Blot) de los parámetros angiogénicos (VEGF-A, VEGF-B, VEGF-C y VEGF-D), endoteliales (VEGF-R1, VEGF-R2 y VEGF-R3) y adipogénicos (PPARɣ2, ADRP y FABP4) del TAT y el TAS. Además, se llevó a cabo un análisis de citometría de flujo para cuantificar la expresión de marcadores endoteliales de superficie celular (Thy/CD90, CD29, CD49a/VLA1) en las células de la fracción vascular estromal de ambos tejidos adiposos en relación con el incremento de la edad. Para determinar el efecto fisiológico que poseen el TAT y el TAS para inducir la angiogénesis se utilizó un ensayo in vivo en la membrana corioalantoidea de codorniz (CAM). Por otro lado se llevó a cabo la caracterización de las células madre mesenquimales procedentes de TAT y TAS en relación con el incremento de la edad mediante ensayos de la capacidad de proliferación celular (curvas de crecimiento celular), ensayos de unidades formadoras de colonias fibroblastoides (CFU-assay), caracterización inmunofenotípica por citometría de flujo y evaluación cualitativa (tinciones específicas) y cuantitativa (análisis de PCR cuantitativa a tiempo real) del potencial de diferenciación hacia células de linaje adipogénico, osteogénico y endotelial. Finalmente se realizó un análisis de la capacidad de formación de túbulos de células endoteliales procedentes de la vena safena humana (HSaVEC) en medio condicionado generado por cultivos de células procedentes de la fracción del estroma vascular del TAT y TAS. Resultados En el TAT de sujetos de edad avanzada, los niveles de expresión génica de marcadores angiogénicos, endoteliales y adipogénicos se incrementaron en comparación con los sujetos de mediana edad, mientras que en el TAS estos parámetros decayeron; además, la expresión de CD31 y los VEGFs correlacionó significativa y positivamente con la edad en el TAT, correlación que fue negativa en el TAS. Por otro lado, la capacidad de inducción angiogénica mostrada por el TAT fue mayor que la del TAS con independencia de la edad del tejido implantado. Por ello, TAT constituye una potente, atractiva y prometedora fuente de actividad angiogénica en comparación con el TAS que no ve alterada sus características en función de la edad del paciente donador. A partir de Tejido Adiposo, tanto de timo como subcutáneo, es posible aislar colonias individuales con células adherentes de morfología fibroblastoide que pueden ser expandidas en cultivo y diferenciadas hacia células de linaje adipogénico, osteogénico y endotelial. La edad del paciente donador no afectó a la capacidad de diferenciación de las células madre mesenquimales de TAT. Estas células madre mesenquimales presentan una cinética de crecimiento celular normal equiparable al de células madre mesenquimales obtenidas a partir de otras fuentes. En el TAT, el doblaje poblacional no pareció verse afectado por el efecto de la edad del paciente, hecho que sí se intuyó en el TAS. Respecto a su caracterización inmunofenotípica, las células madre mesenquimales de nuestro estudio cumplieron con el criterio establecido por la ISCT y con otros aplicados para caracterizar colonias de células madre mesenquimales derivadas de Tejido Adiposo; además, en TAT encontramos presencia de marcadores de células progenitoras endoteliales lo que las podría dotar de mayor predisposición para su diferenciación hacia células endoteliales. Conclusión La glándula tímica puede perder su función inmunogénica con la vejez, pero esta involución podría consistir en el reemplazamiento de su composición inmunogénica por otra función, la de tejido adiposo. El TAT constituye una fuente de factores angiogénicos y de células madre mesenquimales con capacidad para diferenciarse en células endoteliales y es, probablemente, el candidato más adecuado para ser utilizado en terapia celular para sujetos con isquemia cardiaca

Ethylene is involved in strawberry fruit ripening in an organ-specific manner

The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.Facultad de Ciencias ExactasInstituto de Fisiología Vegeta

Ethylene is involved in strawberry fruit ripening in an organ-specific manner

The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.Facultad de Ciencias ExactasInstituto de Fisiología Vegeta

Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry (Fragaria × ananassa)

RNA-seq has been used to perform global expression analysis of the achene and the receptacle at four stages of fruit ripening, and of the roots and leaves of strawberry (Fragaria × ananassa). About 967 million reads and 191 Gb of sequence were produced, using Illumina sequencing. Mapping the reads in the related genome of the wild diploid Fragaria vesca revealed differences between the achene and receptacle development program, and reinforced the role played by ethylene in the ripening receptacle. For the strawberry transcriptome assembly, a de novo strategy was followed, generating separate assemblies for each of the ten tissues and stages sampled. The Trinity program was used for these assemblies, resulting in over 1.4 M isoforms. Filtering by a threshold of 0.3 FPKM, and doing Blastx (E-value < 1 e-30) against the UniProt database of plants reduced the number to 472,476 isoforms. Their assembly with the MIRA program (90% homology) resulted in 26,087 contigs. From these, 91.34 percent showed high homology to Fragaria vesca genes and 87.30 percent Fragaria iinumae (BlastN E-value < 1 e-100). Mapping back the reads on the MIRA contigs identified polymorphisms at nucleotide level, using FREEBAYES, as well as estimate their relative abundance in each sample

Differences in firing efficiency, chromatin and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins

Eukaryotic genome replication depends on thousands of DNA replication origins (ORIs). A major challenge is to learn ORI biology in multicellular organisms in the context of growing organs to understand their developmental plasticity. We have identified a set of ORIs of Arabidopsis thaliana and their chromatin landscape at two stages of post-embryonic development. ORIs associate with multiple chromatin signatures including transcription start sites (TSS) but also proximal and distal regulatory regions and heterochromatin, where ORIs colocalize with retrotransposons. In addition, quantitative analysis of ORI activity led us to conclude that strong ORIs have high GC content and clusters of GGN trinucleotides. Development primarily influences ORI firing strength rather than ORI location. ORIs that preferentially fire at early developmental stages colocalize with GC-rich heterochromatin, but at later stages with transcribed genes, perhaps as a consequence of changes in chromatin features associated with developmental processes. Our study provides the set of ORIs active in an organism at the post-embryo stage that should allow us to study ORI biology in response to development, environment, and mutations with a quantitative approach. In a wider scope, the computational strategies developed here can be transferred to other eukaryotic systems

Munc18c in Adipose Tissue Is Downregulated in Obesity and Is Associated with Insulin

OBJECTIVE: Munc18c is associated with glucose metabolism and could play a relevant role in obesity. However, little is known about the regulation of Munc18c expression. We analyzed Munc18c gene expression in human visceral (VAT) and subcutaneous (SAT) adipose tissue and its relationship with obesity and insulin. MATERIALS AND METHODS: We evaluated 70 subjects distributed in 12 non-obese lean subjects, 23 overweight subjects, 12 obese subjects and 23 nondiabetic morbidly obese patients (11 with low insulin resistance and 12 with high insulin resistance). RESULTS: The lean, overweight and obese persons had a greater Munc18c gene expression in adipose tissue than the morbidly obese patients (p<0.001). VAT Munc18c gene expression was predicted by the body mass index (B = −0.001, p = 0.009). In SAT, no associations were found by different multiple regression analysis models. SAT Munc18c gene expression was the main determinant of the improvement in the HOMA-IR index 15 days after bariatric surgery (B = −2148.4, p = 0.038). SAT explant cultures showed that insulin produced a significant down-regulation of Munc18c gene expression (p = 0.048). This decrease was also obtained when explants were incubated with liver X receptor alpha (LXRα) agonist, either without (p = 0.038) or with insulin (p = 0.050). However, Munc18c gene expression was not affected when explants were incubated with insulin plus a sterol regulatory element-binding protein-1c (SREBP-1c) inhibitor (p = 0.504). CONCLUSIONS: Munc18c gene expression in human adipose tissue is down-regulated in morbid obesity. Insulin may have an effect on the Munc18c expression, probably through LXRα and SREBP-1c