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Calamia V.,Osteoarticular and Aging Research Laboratory
Molecular & cellular proteomics : MCP | Year: 2012

Chondroitin sulfate (CS) is a symptomatic slow acting drug for osteoarthritis (OA) widely used for the treatment of this highly prevalent disease, characterized by articular cartilage degradation. However, little is known about its mechanism of action, and recent large scale clinical trials have reported variable results on OA symptoms. Herein, we aimed to study the modulations in the intracellular proteome and the secretome of human articular cartilage cells (chondrocytes) treated with three different CS compounds, with different origin or purity, by two complementary proteomic approaches. Osteoarthritic cells were treated with 200 μg/ml of each brand of CS. Quantitative proteomics experiments were carried out by the DIGE and stable isotope labeling with amino acids in cell culture (SILAC) techniques, followed by LC-MALDI-MS/MS analysis. The DIGE study, carried out on chondrocyte whole cell extracts, led to the detection of 46 spots that were differential between conditions in our study: 27 were modulated by CS1, 4 were modulated by CS2, and 15 were modulated by CS3. The SILAC experiment, carried out on the subset of chondrocyte-secreted proteins, allowed us to identify 104 different proteins. Most of them were extracellular matrix components, and 21 were modulated by CS1, 13 were modulated by CS2, and 9 were modulated by CS3. Each of the studied compounds induces a characteristic protein profile in OA chondrocytes. CS1 displayed the widest effect but increased the mitochondrial superoxide dismutase, the cartilage oligomeric matrix protein, and some catabolic or inflammatory factors like interstitial collagenase, stromelysin-1, and pentraxin-related protein. CS2 and CS3, on the other hand, increased a number of structural proteins, growth factors, and extracellular matrix proteins. Our study shows how, from the three CS compounds tested, CS1 induces the activation of inflammatory and catabolic pathways, whereas CS2 and CS3 induce an anti-inflammatory and anabolic response. The data presented emphasize the importance of employing high quality CS compounds, supported by controlled clinical trials, in the therapy of OA. Finally, the present work exemplifies the usefulness of proteomic approaches in pharmacological studies. Source


Arufe M.C.,Health Science University | Arufe M.C.,Universitario run A | De La Fuente A.,Osteoarticular and Aging Research Laboratory | Mateos J.,Universitario run A | And 5 more authors.
Stem Cells and Development | Year: 2011

Mesenchymal stem cells (MSCs) from umbilical cord stroma were isolated by plastic adherence and characterized by flow cytometry, looking for cells positive for OCT3/4 and SSEA-4 as well as the classic MSC markers CD44, CD73, CD90, Ki67, CD105, and CD106 and negative for CD34 and CD45. Quantitative reverse transcriptase-polymerase chain reaction analysis of the genes ALP, MEF2C, MyoD, LPL, FAB4, and AMP, characteristic for the differentiated lineages, were used to evaluate early and late differentiation of 3 germ lines. Direct chondrogenic differentiation was achieved through spheroid formation by MSCs in a chondrogenic medium and the presence of chondrogenic markers at 4, 7, 14, 28, and 46 days of culture was tested. Immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analyses were utilized to assess the expression of collagen type I, collagen type II, and collagen type X throughout the time studied. We found expression of all the markers as early as 4 days of chondrogenic differentiation culture, with their expression increasing with time, except for collagen type I, which decreased in expression in the formed spheroids after 4 days of differentiation. The signaling role of Wnt during chondrogenic differentiation was studied by western blot. We observed that β-catenin expression decreased during the chondrogenic process. Further, a secretome study to validate our model of differentiation in vitro was performed on spheroids formed during the chondrogenesis process. Our results indicate the multipotential capacity of this source of human cells; their chondrogenic capacity could be useful for future cell therapy in articular diseases. © 2011 Mary Ann Liebert, Inc. Source


Calamia V.,Osteoarticular and Aging Research Laboratory | Calamia V.,University of La Coruna | Fernandez-Puente P.,Osteoarticular and Aging Research Laboratory | Mateos J.,Osteoarticular and Aging Research Laboratory | And 9 more authors.
Molecular and Cellular Proteomics | Year: 2012

Chondroitin sulfate (CS) is a symptomatic slow acting drug for osteoarthritis (OA) widely used for the treatment of this highly prevalent disease, characterized by articular cartilage degradation. However, little is known about its mechanism of action, and recent large scale clinical trials have reported variable results on OA symptoms. Herein, we aimed to study the modulations in the intracellular proteome and the secretome of human articular cartilage cells (chondrocytes) treated with three different CS compounds, with different origin or purity, by two complementary proteomic approaches. Osteoarthritic cells were treated with 200 μg/ml of each brand of CS. Quantitative proteomics experiments were carried out by the DIGE and stable isotope labeling with amino acids in cell culture (SILAC) techniques, followed by LC-MALDI-MS/MS analysis. The DIGE study, carried out on chondrocyte whole cell extracts, led to the detection of 46 spots that were differential between conditions in our study: 27 were modulated by CS1, 4 were modulated by CS2, and 15 were modulated by CS3. The SILAC experiment, carried out on the subset of chondrocyte-secreted proteins, allowed us to identify 104 different proteins. Most of them were extracellular matrix components, and 21 were modulated by CS1, 13 were modulated by CS2, and 9 were modulated by CS3. Each of the studied compounds induces a characteristic protein profile in OA chondrocytes. CS1 displayed the widest effect but increased the mitochondrial superoxide dismutase, the cartilage oligomeric matrix protein, and some catabolic or inflammatory factors like interstitial collagenase, stromelysin-1, and pentraxin-related protein. CS2 and CS3, on the other hand, increased a number of structural proteins, growth factors, and extracellular matrix proteins. Our study shows how, from the three CS compounds tested, CS1 induces the activation of inflammatory and catabolic pathways, whereas CS2 and CS3 induce an anti-inflammatory and anabolic response. The data presented emphasize the importance of employing high quality CS compounds, supported by controlled clinical trials, in the therapy of OA. Finally, the present work exemplifies the usefulness of proteomic approaches in pharmacological studies. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Segura V.,University of Navarra | Medina-Aunon J.A.,CSIC - National Center for Biotechnology | Guruceaga E.,University of Navarra | Gharbi S.I.,CSIC - National Center for Biotechnology | And 19 more authors.
Journal of Proteome Research | Year: 2013

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed. © 2012 American Chemical Society. Source


Rodriguez-Lorenzo A.,Uppsala University Hospital | Arufe M.C.,Osteoarticular and Aging Research Laboratory | Arufe M.C.,University of La Coruna | De La Fuente A.,Osteoarticular and Aging Research Laboratory | And 2 more authors.
Annals of Plastic Surgery | Year: 2014

BACKGROUND: In this article, the authors investigated whether the prefabrication of an autologous pedicled flap by isolation from the surrounding with artificial skin substitutes would increase mesenchymal stem cell (MSC) seeding. METHODS: Mesenchymal stem cells were isolated from human umbilical cords and were cultured and characterized by fluorescence-activated cell sorting. Oxacarbocyanine and its green fluorescence emission were used to label the MSCs population.Sixteen adult Wistar rats were randomized in 4 groups (n = 4 animals per group). In group 1, a prefabricated groin flap (GF) with skin substitutes was harvested without cell injection; in group 2, 1 million MSCs were injected subcutaneously in the area corresponding to the GF without flap harvesting; in Group 3, a prefabricated GF with skin substitutes was harvested and 1 million MSCs were injected subcutaneously; and in Group 4, a prefabricated GF with skin substitutes was harvested and 2 million MSCs were injected subcutaneously. All procedures were performed bilaterally in each animal. Animals were sacrificed 2 weeks after the surgery. Flap viability was then assessed by clinical inspection and histology, and seeding of MSCs was observed. RESULTS: All flaps survived 2 weeks after the surgery. Oxacarbocyanine-labeled cells were found in all prefabricated flaps injected (Groups 3 and 4) in higher number in comparison with the group where subcutaneous injection without flap harvesting was performed (Group 2). This difference was statistically significant (P < 0.05). CONCLUSIONS: Prefabricated skin flaps with skin substitutes may provide a useful vehicle for the implantation of MSCs to serve as an autologous microvascular bioscaffold.Copyright © 2013 by Lippincott Williams & Wilkins. Source

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