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

Obesity-associated insulin resistance is correlated to adipose tissue vascular endothelial growth factors and metalloproteinase levels

<p>Abstract</p> <p>Background</p> <p>The expansion of adipose tissue is linked to the development of its vasculature, which appears to have the potential to regulate the onset of obesity. However, at present, there are no studies highlighting the relationship between human adipose tissue angiogenesis and obesity-associated insulin resistance (IR).</p> <p>Results</p> <p>Our aim was to analyze and compare angiogenic factor expression levels in both subcutaneous (SC) and omentum (OM) adipose tissues from morbidly obese patients (n = 26) with low (OB/L-IR) (healthy obese) and high (OB/H-IR) degrees of IR, and lean controls (n = 17). Another objective was to examine angiogenic factor correlations with obesity and IR.</p> <p>Here we found that <it>VEGF-A </it>was the isoform with higher expression in both OM and SC adipose tissues, and was up-regulated 3-fold, together with <it>MMP9 </it>in OB/L-IR as compared to leans. This up-regulation decreased by 23% in OB/-H-IR compared to OB/L-IR. On the contrary, <it>VEGF-B</it>, <it>VEGF-C </it>and <it>VEGF-D</it>, together with <it>MMP15 </it>was down-regulated in both OB/H-IR and OB/L-IR compared to lean patients. Moreover, <it>MMP9 </it>correlated positively and <it>VEGF-C</it>, <it>VEGF-D </it>and <it>MMP15 </it>correlated negatively with HOMA-IR, in both SC and OM.</p> <p>Conclusion</p> <p>We hereby propose that the alteration in <it>MMP15</it>, <it>VEGF-B</it>, <it>VEGF-C </it>and <it>VEGF-D </it>gene expression may be caused by one of the relevant adipose tissue processes related to the development of IR, and the up-regulation of <it>VEGF-A </it>in adipose tissue could have a relationship with the prevention of this pathology.</p

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)

Munc18c in adipose tissue is downregulated in obesity and is associated with insulin.

Journal Article;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.This work was supported in part by grants from Instituto de Salud Carlos III [CP04/00133, PS09/01060, PS09/00997], Servicio Andaluz de Salud [PI0255/2007]. L. Garrido-Sánchez is supported by a fellowship from the Programa Juan de la Cierva [JCI-2009-04086]. E. Garcia-Fuentes is supported by the Research Stabilization Program of the Instituto de Salud Carlos III (ISCIII). R. El Bekay is supported by fellowships from the Fondo de Investigación Sanitaria (FIS) "Miguel Servet" [CP07/00288].Ye

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

Munc18c in adipose tissue is downregulated in obesity and is associated with insulin.

Journal Article;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.This work was supported in part by grants from Instituto de Salud Carlos III [CP04/00133, PS09/01060, PS09/00997], Servicio Andaluz de Salud [PI0255/2007]. L. Garrido-Sánchez is supported by a fellowship from the Programa Juan de la Cierva [JCI-2009-04086]. E. Garcia-Fuentes is supported by the Research Stabilization Program of the Instituto de Salud Carlos III (ISCIII). R. El Bekay is supported by fellowships from the Fondo de Investigación Sanitaria (FIS) "Miguel Servet" [CP07/00288].Ye

Differences in the neovascular potential of thymus versus subcutaneous adipose-derived stem cells from patients with myocardial ischemia.

Adipose tissue-derived multipotent mesenchymal cells (ASCs) participate in the formation of blood vessels under hypoxic conditions. It is probable that the susceptibility of ASCs to the influence of age and aging-associated pathologies compromises their therapeutic effectiveness depending on the adipose tissue (AT) depot. Our aim was to examine the neovascular potential under hypoxic conditions of ASCs-derived from thymic (thymASCs) and subcutaneous (subASCs) AT from 39 subjects with and without type 2 diabetes mellitus (T2DM) and of different ages who were undergoing coronary bypass surgery (CBS). We confirmed a significant decrease in the percentage of CD34+ CD31- CD45- subASCs in the cell yield of subASCs and in the survival of cultured endothelial cells in the medium conditioned by the hypox-subASCs with increasing patient age, which was not observed in thymASCs. While the length of the tubules generated by hypox-subASCs tended to correlate negatively with patient age, tubule formation capacity of the hypoxic thymASCs increased significantly. Compared with subASCs, thymASCs from subjects over age 65 and without T2DM showed higher cell yield, tubule formation capacity, VEGF secretion levels and the ability to promote endothelial cell survival in their conditioned medium. Deterioration in subASCs neovascular potential relative to thymASCs derived from these subjects was accompanied by higher expression levels of NOX4 mRNA and fibrotic proteins. Our results indicate that thymASCs from patients over age 65 and without T2DM have a higher angiogenic potential than those from the other patient groups, suggesting they may be a good candidate for angiogenic therapy in subjects undergoing CBS.Ministry of Economy and Knowledge, Grant/Award Number: CTS‐7895/2011; This workwas co‐funded by the European Unionthrough the European Regional DevelopmentFund (FEDER) and supported by grants fromthe Ministry of Economy and Competitiveness,Institute of Health Carlos III, Grant/AwardNumber: PI15/01114, PI13/02628 and PI12/0235

Hypoxia is associated with a lower expression of genes involved in lipogenesis in visceral adipose tissue.

BACKGROUND: A key role for HIF-1α in the promotion and maintenance of dietary obesity has been proposed. We analyzed the association between hypoxia and de novo lipogenesis in human adipose tissue. METHODS: We studied HIF-1α mRNA and protein expression in fasting status in visceral adipose tissue (VAT) from non-obese and morbidly obese subjects, and in VAT from wild-type and ob/ob C57BL6J mice in both fasting and feeding status. We also analyzed the effect of hypoxia on the VAT mRNA expression of genes involved in lipogenesis. RESULTS: HIF-1α was increased in VAT from morbidly obese subjects. In fasting status, C57BL6J ob/ob mice had a higher VAT HIF-1α mRNA expression than C57BL6J wild-type mice. In feeding status, VAT HIF-1α mRNA expression significantly increased in C57BL6J wild-type, but not in C57BL6J ob/ob mice. In humans, HIF-1α mRNA expression correlated positively with body mass index and insulin resistance. VAT HIF-1α mRNA expression correlated negatively with ACC1, PDHB and SIRT3 mRNA expression, and positively with PPAR-γ. VAT explants incubated in hypoxia showed reduced SIRT3 and increased PPAR-γ, SREBP-1c, ACLY, ACC1 and FASN mRNA expression. CONCLUSIONS: Morbidly obese subjects have a higher level of VAT HIF-1α. Postprandial status is associated with an increase in HIF-1α mRNA expression in C57BL6J wild-type mice. Hypoxia alters the mRNA expression of genes involved in de novo lipogenesis in human VAT.This work was supported in part by grants from Instituto de Salud Carlos III (PS09/01060, PS09/00997 and CP13/00188) and Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía (CTS-08081) (Spain). This study has been co-funded by FEDER funds.Ye

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

Anthropometric and metabolic characteristics of the study participants.

<p>Anthropometric and metabolic characteristics of the study participants.</p