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

Genetic Epidemiology of Glucose-6-Dehydrogenase Deficiency in the Arab World

A systematic search was implemented using four literature databases (PubMed, Embase, Science Direct and Web of Science) to capture all the causative mutations of Glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDD) in the 22 Arab countries. Our search yielded 43 studies that captured 33 mutations (23 missense, one silent, two deletions, and seven intronic mutations), in 3,430 Arab patients with G6PDD. The 23 missense mutations were then subjected to phenotypic classification using in silico prediction tools, which were compared to the WHO pathogenicity scale as a reference. These in silico tools were tested for their predicting efficiency using rigorous statistical analyses. Of the 23 missense mutations, p.S188F, p.I48T, p.N126D, and p.V68M, were identified as the most common mutations among Arab populations, but were not unique to the Arab world, interestingly, our search strategy found four other mutations (p.N135T, p.S179N, p.R246L, and p.Q307P) that are unique to Arabs. These mutations were exposed to structural analysis and molecular dynamics simulation analysis (MDSA), which predicting these mutant forms as potentially affect the enzyme function. The combination of the MDSA, structural analysis, and in silico predictions and statistical tools we used will provide a platform for future prediction accuracy for the pathogenicity of genetic mutations.QUST-CAS-FALL-15/16-24 gran

An Abnormal Nitric Oxide Metabolism Contributes to Brain Oxidative Stress in the Mouse Model for the Fragile X Syndrome, a Possible Role in Intellectual Disability

Background. Fragile X syndrome is the most common genetic cause of mental disability. Although many research has been performed, the mechanism underlying the pathogenesis is unclear and needs further investigation. Oxidative stress played major roles in the syndrome. The aim was to investigate the nitric oxide metabolism, protein nitration level, the expression of NOS isoforms, and furthermore the activation of the nuclear factor NF-κB-p65 subunit in different brain areas on the fragile X mouse model. Methods. This study involved adult male Fmr1-knockout and wild-type mice as controls. We detected nitric oxide metabolism and the activation of the nuclear factor NF-κBp65 subunit, comparing the mRNA expression and protein content of the three NOS isoforms in different brain areas. Results. Fmr1-KO mice showed an abnormal nitric oxide metabolism and increased levels of protein tyrosine nitrosylation. Besides that, nuclear factor NF-κB-p65 and inducible nitric oxide synthase appeared significantly increased in the Fmr1-knockout mice. mRNA and protein levels of the neuronal nitric oxide synthase appeared significantly decreased in the knockout mice. However, the epithelial nitric oxide synthase isoform displayed no significant changes. Conclusions. These data suggest the potential involvement of an abnormal nitric oxide metabolism in the pathogenesis of the fragile X syndrome

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

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

VEGF Gene Expression in Adult Human Thymus Fat: A Correlative Study with Hypoxic Induced Factor and Cyclooxigenase-2

UNLABELLED:It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT) produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties. DESIGN:Adult thymus fat and subcutaneous adipose tissue specimens were obtained from 28 patients undergoing cardiac surgery, making this tissue readily available as a prime source of adipose tissue. We focused our investigation on determining VEGF gene expression and characterizing the different genes, mediators of inflammation and adipogenesis, and which are known to play a relevant role in angiogenesis regulation. RESULTS:We found that VEGF-A was the isoform most expressed in TAT. This expression was accompanied by an upregulation of HIF-1alpha, COX-2 and HO-1 proteins, and by increased HIF-1 DNA binding activity, compared to SAT. Furthermore, we observed that TAT contains a high percentage of mature adipocytes, 0.25% of macrophage cells, 15% of endothelial cells and a very low percentage of thymocyte cells, suggesting the cellular variability of TAT, which could explain the differences in gene expression observed in TAT. Subsequently, we showed that the expression of genes known as adipogenic mediators, including PPARgamma1/gamma2, FABP-4 and adiponectin was similar in both TAT and SAT. Moreover the expression of these latter genes presented a significantly positive correlation with VEGF, suggesting the potential association between VEGF and the generation of adipose tissue in adult thymus. CONCLUSION:Here we suggest that this fat has a potential angiogenic function related to ongoing adipogenesis, which substitutes immune functions within the adult thymus. The expression of VEGF seems to be associated with COX-2, HO-1 and adipogenesis related genes, suggesting the importance that this new fat has acquired in research in relation to adipogenesis and angiogenesis

Rac2 GTPase activation by angiotensin II is modulated by Ca2+/calcineurin and mitogen-activated protein kinases in human neutrophils

Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca2+ elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca2+/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.This work was financed by grants from the Ministerio de Educación y Ciencia (BFU2006-13802), and the Consejería de Innovación, Ciencia y Empresa (P06-CTS-1936), Junta de Andalucía, Spain, awarded to F S

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)

Fast, ultrasensitive detection of reactive oxygen species using a carbon nanotube based-electrocatalytic intracellular sensor

Herein, we report a highly sensitive electrocatalytic sensor-cell construct that can electrochemically communicate with the internal environment of immune cells (e.g., macrophages) via the selective monitoring of a particular reactive oxygen species (ROS), hydrogen peroxide. The sensor, which is based on vertically aligned single-walled carbon nanotubes functionalized with an osmium electrocatalyst, enabled the unprecedented detection of a local intracellular “pulse” of ROS on a short second time scale in response to bacterial endotoxin (lipopolysaccharide-LPS) stimulation. Our studies have shown that this initial pulse of ROS is dependent on NADPH oxidase (NOX) and toll like receptor 4 (TLR4). The results suggest that bacteria can induce a rapid intracellular pulse of ROS in macrophages that initiates the classical innate immune response of these cells to infection

Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity

AIMS: Obesity is characterized by a low-grade systemic inflammatory state and adipose tissue (AT) dysfunction, which predispose individuals to the development of insulin resistance (IR) and metabolic disease. However, a subset of obese individuals, referred to as metabolically healthy obese (MHO) individuals, are protected from obesity-associated metabolic abnormalities. Here, we aim at identifying molecular factors and pathways in adipocytes that are responsible for the progression from the insulin-sensitive to the insulin-resistant, metabolically unhealthy obese (MUHO) phenotype. RESULTS: Proteomic analysis of paired samples of adipocytes from subcutaneous (SC) and omental (OM) human AT revealed that both types of cells are altered in the MUHO state. Specifically, the glutathione redox cycle and other antioxidant defense systems as well as the protein-folding machinery were dysregulated and endoplasmic reticulum stress was increased in adipocytes from IR subjects. Moreover, proteasome activity was also compromised in adipocytes of MUHO individuals, which was associated with enhanced accumulation of oxidized and ubiquitinated proteins in these cells. Proteasome activity was also impaired in adipocytes of diet-induced obese mice and in 3T3-L1 adipocytes exposed to palmitate. In line with these data, proteasome inhibition significantly impaired insulin signaling in 3T3-L1 adipocytes. INNOVATION: This study provides the first evidence of the occurrence of protein homeostasis deregulation in adipocytes in human obesity, which, together with oxidative damage, interferes with insulin signaling in these cells. CONCLUSION: Our results suggest that proteasomal dysfunction and impaired proteostasis in adipocytes, resulting from protein oxidation and/or misfolding, constitute major pathogenic mechanisms in the development of IR in obesity.IMIBIC/Universidad de Córdoba-SCAI (ProteoRed, PRB2-ISCIII)MINECO/FEDERJunta de Andalucía/FEDERCIBERobn(Instituto de Salud Carlos III