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

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

Context: The decreased expansion capacity of adipose tissue plays a crucial role in the onset of disorders associated with metabolic syndrome. Objetive: 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. Design: This was a 2-year study to enroll subjects who underwent bariatric surgery or cholecystectomy. Setting: University Hospital Patients and Intervention: Patients who underwent either bariatric surgery (20 morbidly obese) or cholecystectomy (40 subjects) participated in the study. Main Outcome Measures: ASCs were obtained from both visceral and subcutaneous adipose tissue. Adipogenic, fibrotic genes expression was quantified by qPCR; Smad7 and fibroblast growth factor 2 (FGF2) were quantified by western blotting and ELISA respectively. The susceptibility of ASCs to apoptosis, their population doubling time and clonogenic potential were evaluated Results The worsening metabolic profile of the subjects was accompanied by a decrease in the intrinsic levels of adipogenic genes expression, reduced proliferation rate, clonogenic potential and exportation of FGF2 to the cell surface of the ASCs derived from both tissues. In addition, the ASCs from NonMS subjects showed differences in susceptibility to apoptosis and expression of TGFß signaling inhibitory protein Smad7 with respect to the ASCs from MS subjects. Conclusions/Interpretation Our results suggest that the decrease in adipogenic genes 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 adipose tissue expansion alteration.This work was cofunded by the European Union through the European Regional Development Fund and supported by grants from the Ministry of Economy and Competitiveness, ISCII (Grants PI13/02628, PI12/02355, FIS PI14/00696, and PI12/ 01373), and the Ministry of Economy and Knowledge (Grants PI-CTS-08181/2011, CTS-7895/2011, and CTS-656). R. E.-B. and M. B.-L. are supported by a fellowship from the ISCIII “MiguelServet II” (CP13/00041) and “Miguel Servet I”(CP15/ 00028)

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

Effect of acute and chronic red wine consumption on lipopolysaccharide concentrations

Background: Chronic red wine (RW) consumption has been associated with decreased cardiovascular disease risk, mainly attributed to an improvement in lipid profile. RW intake is also able to change the composition of gut microbiota. High fat intake has recently been reported to increase metabolic endotoxemia. The gut microbiota has been proposed as the main resource of plasma lipopolysaccharides (LPSs) in metabolic endotoxemia. Objective: We analyzed the effect on LPS concentrations of chronic RW consumption and acute RW intake in relation to high fat intake in middle-aged men. Design: For the chronic study, 10 middle-aged male volunteers were randomly assigned in a crossover trial, and after a washout period, all subjects received RW, dealcoholized red wine (DRW), or gin for 20 d. Serum endotoxin and LPS-binding protein (LBP) concentrations were determined after the washout period and after each of the treatments, and changes in fecal microbiota were quantified. For the acute study, 5 adult men underwent a fat overload or a fat overload together with the consumption of RW, DRW, or gin. Baseline and postprandial serum LPS and LBP concentrations and postprandial chylomicron LPS concentrations were measured. Results: There were no significant differences in the change in LPS or LBP concentrations between chronic RW, DRW, and gin consumption. Bifidobacterium and Prevotella amounts were significantly increased by RW and correlated negatively with LPS concentrations. There were no differences in postprandial serum LPS, LBP, or chylomicron LPS concentrations between acute RW, DRW, or gin intake together with a fatty meal. Conclusion: Chronic RW consumption increases Bifidobacterium and Prevotella amounts, which may have beneficial effects by leading to lower LPS concentrations. This trial was registered at controlled-trials.CIBER, CB06/03/0018 of the Instituto de Salud Carlos III (ISCIII), the ISCIII FIS PS09/00997, “Consejeria de Innovación (Junta de Andalucia)” CTS04369, “Consejeria de Salud” (Junta de Andalucia) PI696/2010; in part by the INGENIO-CONSOLIDER Program, Fun-C-Food CSD2007-063 and AGL2006-14228-C03-02 from the Spanish Ministry; and by Programa de Formación de Profesorado Universitario (FPU) grants from Education Ministry, Madrid, Spain [AP2009-4537 (to MC-P) and AP2008-01922 (MB-O)]. Torres SA provided the red wine and dealcoholized red wine used in the study, and Gin Xoriguer provided the gin used in the study

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)

Metabolic endotoxemia promotes adipose dysfunction and inflammation in human obesity

Impaired adipose tissue (AT) lipid handling and inflammation is associated with obesity-related metabolic diseases. Circulating lipopolysaccharides (LPSs) from gut microbiota (metabolic endotoxemia), proposed as a triggering factor for the low-grade inflammation in obesity, might also be responsible for AT dysfunction. Nevertheless, this hypothesis has not been explored in human obesity. To analyze the relationship between metabolic endotoxemia and AT markers for lipogenesis, lipid handling, and inflammation in human obesity, 33 patients with obesity scheduled for surgery were recruited and classified according to their LPS levels. Visceral and subcutaneous AT gene and protein expression were analyzed and adipocyte and AT in vitro assays performed. Subjects with obesity with a high degree of metabolic endotoxemia had lower expression of key genes for AT function and lipogenesis (SREBP1, FABP4, FASN, and LEP) but higher expression of inflammatory genes in visceral and subcutaneous AT than subjects with low LPS levels. In vitro experiments corroborated that LPS are responsible for adipocyte and AT inflammation and downregulation of PPARG, SCD, FABP4, and LEP expression and LEP secretion. Thus, metabolic endotoxemia influences AT physiology in human obesity by decreasing the expression of factors involved in AT lipid handling and function as well as by increasing inflammation.M. Clemente-Postigo was a recipient of an Formación de Profesorado Universitario (FPU) grant (AP2009-4537) from the Ministry of Education (Madrid, Spain), B. Ramos-Molina was supported by the Sara Borrell program (CD16/00034) from the Instituto de Salud Carlos III (ISCIII; Madrid, Spain), and F. Cardona and R. El Bekay were supported by the Nicolas Monardes program (C-0032-2016 and C-0030-2016) from the Consejería de Salud (Junta de Andalucía, Spain) and cofunded by the Fondo Europeo de Desarrollo Regional (FEDER). This study was supported by Centro de Investigación Biomédica En Red (CIBER, CB06/03/0018) of the ISCIII, Madrid (Spain); P11-CTS-08181 from the Consejería de Economía, Innovación, Ciencia y Empleo (Junta de Andalucía); and PI14/00082, PI15/01114, and PIE14/00031 from the ISCIII; and cofunded by the Fondo Europeo de Desarrollo Regional (FEDER)

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

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

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