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  • Overexpression of four and a half LIM domains protein 2 promotes epithelial-mesenchymal transition-like phenotype in fish pre-osteoblasts
    Publication . Rafael, Marta S.; Laizé, Vincent; Florindo, C.; Ferraresso, Serena; Bargelloni, Luca; Cancela, Leonor
    FHL2 is a multifunctional protein involved in gene transcription regulation and cytoarchitecture modulation that has been recently associated with epithelial-mesenchymal transition (EMT) in colon cancer. Overexpression of FHL2 in a fish pre-osteoblastic cell line promoted cell dedifferentiation and impaired its extracellular matrix mineralization capacity. Cell cultures also acquired a novel threedimensional structure organization, their proliferation rate was enhanced and gene expression profile was altered in agreement with an EMT-like phenotype upon overexpression of FHL2. Altogether, our results provide additional support to the relevance of FHL2 for cell differentiation and its association with hallmarks of cancer phenotype.
  • Desenvolvimento de sistemas celulares de peixe adequados ao estudo da mineralização in vitro
    Publication . Marques, C. L.; Rafael, Marta S.; Tiago, Daniel; Cancela, Leonor; Laizé, Vincent
    Os peixes foram recentemente reconhecidos como organismos modelo apropriados para o estudo da biologia de vertebrados, particularmente de processos relacionados com a mineralização tecidular e o desenvolvimento do esqueleto. Apesar de existirem alguns estudos in vivo, a identificação de mecanismos associados à calcificação em peixes tem sido prejudicada pelo facto de não existirem sistemas celulares para estudos in vitro. Este artigo descreve um protocolo simples e de baixo custo adequado ao desenvolvimento de culturas celulares mineralogénicas, derivadas de tecidos calcificados de peixes.
  • Gla-rich protein is involved in the cross-talk between calcification and inflammation in osteoarthritis
    Publication . Cavaco, Sofia; S B Viegas, Carla; Rafael, Marta S.; Ramos, Acacio; Magalhes, Joana; Blanco, Francisco J.; Vermeer, Cees; Simes, D
    Osteoarthritis (OA) is a whole-joint disease characterized by articular cartilage loss, tissue inflammation, abnormal bone formation and extracellular matrix (ECM) mineralization. Disease-modifying treatments are not yet available and a better understanding of osteoarthritis pathophysiology should lead to the discovery of more effective treatments. Gla-rich protein (GRP) has been proposed to act as a mineralization inhibitor and was recently shown to be associated with OA in vivo. Here, we further investigated the association of GRP with OA mineralization-inflammation processes. Using a synoviocyte and chondrocyte OA cell system, we showed that GRP expression was up-regulated following cell differentiation throughout ECM calcification, and that inflammatory stimulation with IL-1 beta results in an increased expression of COX2 and MMP13 and up-regulation of GRP. Importantly, while treatment of articular cells with gamma-carboxylated GRP inhibited ECM calcification, treatment with either GRP or GRP-coated basic calcium phosphate (BCP) crystals resulted in the down-regulation of inflammatory cytokines and mediators of inflammation, independently of its gamma-carboxylation status. Our results strengthen the calcification inhibitory function of GRP and strongly suggest GRP as a novel anti-inflammatory agent, with potential beneficial effects on the main processes responsible for osteoarthritis progression. In conclusion, GRP is a strong candidate target to develop new therapeutic approaches.
  • Use of an innovative system and nanotechnology-based strategy for therapeutic applications of Gla-rich protein (GRP)
    Publication . Viegas, Carla; Edelweiss, Evelina; Schneider, Justine; Schaeffer-Reiss, Christine; Poterszman, Arnaud; Rafael, Marta S.; Araujo, Nuna C. P.; Macedo, Anjos; Alves de Matos, António; Simes, Dina
    Introduction: Gla-rich protein (GRP) is a vitamin K-dependent protein (VKDP) acting as a calcification inhibitor and anti-inflammatory agent in cardiovascular and articular systems, and THP1 monocyte/macrophage cells [1,2]. Calcification and inflammation processes are known to be involved in the etiology of several calcification-related chronic inflammatory diseases such as atherosclerosis, CKD and osteoarthritis, in a complex bi-directional interplay that drives disease progression. Here, we developed an innovative system to produce human c-carboxylated GRP (cGRP), and a nanotechnology strategy based on GRP loading into extracellular vesicles (EVs) as a gold standard delivery system for GRP in therapeutic applications. Materials and methods: Human GRP protein was co-expressed with c-carboxylase enzyme (GGCX), vitamin K oxidoreductase (GGCX) and furin, in the insect cell baculovirus system in the presence of vitamin K. GRP released in the cell culture media was characterized by mass spectrometry based techniques and Western blot analysis. EVs released by the insect cells overexpressing GRP were isolated by ultracentrifugation, and characterized for GRP content through TEM-immunogold staining, Western blot, ELISA, qPCR. Functional assays using isolated EVs containing GRP were performed in primary vascular smooth muscle cells (VSMCs) and THP1 monocyte/macrophage cells, for anti-mineralizing and anti-inflammatory screening.Results: GRP released in the cell culture media when co-expressed with GGCX, VKOR and furin in the presence of vitamin K, is processed at the pro-peptide and contain Gla residues. EVs released by the insect cells in this system were shown to be loaded with GRP protein and mRNA, and capable of reducing ECM calcium deposition of calcifying VSMCs and the production of TNFa in THP1 monocyte/macrophage cells stimulated with LPS. Discussion and conclusions: While the successful production of human cGRP constitutes a major achievement, this innovative methodology will open new opportunities for the production of other biological active VKDPs. Furthermore, EVs loaded with GRP were shown to have anti-mineralizing and anti-inflammatory properties, with promising therapeutic potentialities for calcification-related chronic inflammatory diseases.
  • Establishment of primary cell cultures from fish calcified tissues
    Publication . Marques, C. L.; Rafael, Marta S.; Cancela, Leonor; Laizé, Vincent
    Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case.
  • Role of the transcriptional regulator FHL2: identification of fish target genes and regulatory mechanisms
    Publication . Rafael, Marta S.; Cancela, Leonor; Laizé, Vincent; Schüle, R.
    Four and a half LIM domains protein 2 (FHL2) is a multifunctional molecule, recognized for its involvement in central cellular regulatory mechanisms, including gene transcription, signal transduction and modulation of cytoskeleton architecture. Physiologically, FHL2 was shown to be associated with bone metabolism, skin wound healing response and muscle regeneration process. Despite many studies to unveil mechanisms of FHL2 action, all of them using mammalian models, available data are still insufficient to fully understand its physiological role, particularly during early development. This work presents original insights on FHL2 tissue patterning and mechanisms of action using the teleost gilthead seabream (S. aurata, L.) as a vertebrate model. First, we collected evidence that FHL2 transcript is strongly associated with development of fish craniofacial musculature and it is specific for red/slow muscle cells never described before. Then, and to better characterise the relevance of FHL2 on tissue mineralization, we engineered seabream VSa16 pre‐osteoblastic cell line to overexpress the corresponding protein. Surprisingly, extracellular matrix mineralization capacity was impaired and cells dedifferentiated through an epithelialmesenchymal transition‐like mechanism, known to be involved in progression of cells towards a cancer phenotype and their capacity of migration. This finding is extremely relevant since FHL2 has been repeatedly related with various cancer types. Finally, and although experimental data is still missing, we also present human transcriptional data towards a possible link between FHL2 gene expression and cancerous tissues. In addition, protein structure and phylogenetic studies indicate that FHL2 protein is remarkably conserved among vertebrate species. We propose that protein function has been conserved from fish to human and that fish is therefore a suitable vertebrate model to further study mechanisms of FHL2 action.
  • Fish bone-derived cell lines: An alternative in vitro cell system to study bone biology
    Publication . Rafael, Marta S.; Marques, C. L.; Parameswaran, V.; Cancela, Leonor; Laizé, Vincent
    Human bone diseases represent a major health problem worldwide and effective therapies have still to be developed. Despite numerous studies using mammalian systems, cellular and molecular processes governing bone and cartilage homeostasis in vertebrates are still not fully understood. Recently, fish have emerged as a suitable model and a promising alternative to the classical mammalian systems to study vertebrate development, in particular skeletogenesis. To complement in vivo developmental studies and identify signalling pathways involved in development processes, fish cell lines have been developed, in particular bone-derived cells. This work intends to review what is presently known about fish bone-derived cell lines, focusing on their relevance for bone biology studies.
  • Four-and-a-half LIM domains protein 2 (FHL2) is associated with the development of craniofacial musculature in the teleost fish Sparus aurata
    Publication . Rafael, Marta S.; Laizé, Vincent; Bensimon-Brito, A.; Leite, Ricardo; Schüle, R.; Cancela, Leonor
    Four-and-a-half LIM domains protein 2 (FHL2) is involved in major cellular mechanisms such as regulation of gene transcription and cytoskeleton modulation, participating in physiological control of cardiogenesis and osteogenesis. Knowledge on underlying mechanisms is, however, limited. We present here new data on FHL2 protein and its role during vertebrate development using a marine teleost fish, the gilthead seabream (Sparus aurata L.). In silico comparison of vertebrate protein sequences and prediction of LIM domain three-dimensional structure revealed a high degree of conservation, suggesting a conserved function throughout evolution. Determination of sites and levels of FHL2 gene expression in seabream indicated a central role for FHL2 in the development of heart and craniofacial musculature, and a potential role in tissue calcification. Our data confirmed the key role of FHL2 protein during vertebrate development and gave new insights into its particular involvement in craniofacial muscle development and specificity for slow fibers.
  • Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling
    Publication . Willems, Brecht A.; Furmanik, Malgorzata; Caron, Marjolein M. J.; Chatrou, Martijn L. L.; Kusters, Dennis H. M.; Welting, Tim J. M.; Stock, Michael; Rafael, Marta S.; Viegas, Carla S. B.; Simes, Dina C.; Vermeer, Cees; Reutelingsperger, Chris P. M.; Schurgers, Leon J.
    Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP(-/-) mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, beta-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.
  • Identification of Sparus aurata bone morphogenetic protein 2: Molecular cloning, gene expression and in silico analysis of protein conserved features in vertebrates
    Publication . Rafael, Marta S.; Laizé, Vincent; Cancela, Leonor
    Bone morphogenetic protein 2 (BMP-2) is a secreted signaling molecule that acts as an inducer of bone formation and a regulator of embryonic development. The objectives of this work were as follows: (1) to clone the full-length cDNA of BMP-2 in a marine fish model, (2) analyze its gene expression during development, in adult tissues and in cell lines, and (3) identify protein conserved features of vertebrate BMP-2. Using a combination of RT- and 5′-RACE-PCR, a 1653-bp fragment corresponding to Sparus aurata BMP-2 cDNA (SaBMP-2) was amplified. Levels of SaBMP-2 gene expression were estimated using quantitative real-time PCR and shown to be strongly increased (150-fold induction) at gastrulation, thus suggesting a key role for BMP-2 in fish development. Tissue distribution of SaBMP-2 mRNA revealed highest levels in the calcified tissues bone, caudal fin and scales and in liver. BMP-2 was also found to be highly expressed in S. aurata bone-derived cell lines VSa13 and VSa16 and to be up-regulated (more than 10-fold induction) in mineralized VSa13 chondrocyte-like cells. Using bioinformatic tools and all vertebrate protein sequences available, conserved features of BMP-2 were characterized. The mature protein was shown to be highly conserved across 20 species indicating that BMP-2 function has been conserved throughout evolution, a finding that is in agreement with the widely accepted view of the important role played by BMPs in vertebrate development.