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- Effect of variable levels of dietary cholesterol and plant sterols on the growth performance and bone metabolism in gilthead seabream (Sparus aurata) juvenilesPublication . Dias, J.; Colen, Rita; Rodrigues, V.; Aragão, C.; Engrola, S.; Viegas, Michael; Laizé, Vincent; Gavaia, Paulo J.; Cancela, LeonorCholesterol is found in all animal tissues and is an important component of biological cell membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients by plant proteins and vegetable oils tends to reduce dietary cholesterol levels.
- Polyunsaturated fatty acids regulate cell proliferation, extracellular matrix mineralization and gene expression in a gilthead seabream skeletal cell linePublication . Viegas, Michael; Dias, J.; Cancela, Leonor; Laizé, VincentPolyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are defining components of farmed fish feeds. They are known effectors of bone metabolism and may reduce the occurrence of skeleton deformities in farmed fish, given that dietary levels are optimized. Underlying mechanisms of bone-related effects of PUFAs are however poorly understood. An in vitro cell system developed from gilthead seabream vertebra and capable of mineralization was used to assess the effect of AA, EPA and DHA on cell proliferation, extracellular matrix (ECM) mineralization and expression of a selection of marker genes. While all fatty acids promoted morphological changes and stimulated cell proliferation, AA and EPA inhibited ECM mineralization and DHA had the opposite effect. When fatty acids were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. qPCR data indicated that, while expression of COL1A1, ON, MGP and RUNX2 genes remained unaltered, that of OP, BMP2, TNAP and COX2 genes was significantly regulated upon exposure to individual PUFAs. The concomitant increase in BMP2 and TNAP gene expression in DHA-treated cells and the strong decrease in OP transcript in AA- and EPA-treated cells may explain the pro- and anti-mineralogenic effects of these fatty acids, respectively. An increase in COX2 gene expression was also observed in EPA- and DHA-treated cells but the biological significance of this result remains ambiguous. This work also demonstrated the suitability of our in vitro cell systems to get insights into bone-related effects of PUFAs and to investigate the intracellular pathways involved in their proliferative and mineralogenic effects.
- Anti-osteogenic activity of cadmium in zebrafishPublication . Tarasco, Marco; Cardeira Da Silva, João; Viegas, Michael; Caria, Joana; Martins, Gil; Gavaia, Paulo; Cancela, M. Leonor; Laizé, VincentAmong the many anthropogenic chemicals that end up in the aquatic ecosystem, heavy metals, in particular cadmium, are hazardous compounds that have been shown to affect developmental, reproductive, hepatic, hematological, and immunological functions in teleost fish. There is also evidence that cadmium disturbs bone formation and skeletal development, but data is scarce. In this work, zebrafish was used to further characterize the anti-osteogenic/osteotoxic effects of cadmium and gain insights into underlying mechanisms. Upon exposure to cadmium, a reduction of the opercular bone growth was observed in 6-days post-fertilization (dpf) larvae and an increase in the incidence of skeletal deformities was evidenced in 20-dpf post-larvae. The extent and stiffness of newly formed bone was also affected in adult zebrafish exposed to cadmium while regenerating their caudal fin. A pathway reporter assay revealed a possible role of the MTF-1 and cAMP/PKA signaling pathways in mechanisms of cadmium osteotoxicity, while the expression of genes involved in osteoblast differentiation and matrix production was strongly reduced in cadmium-exposed post-larvae. This work not only confirmed cadmium anti-osteogenic activity and identified targeted pathways and genes, but it also suggested that cadmium may affect biomechanical properties of bone.
- Dietary lipid quality regulates bone composition and metabolism in gilthead seabream (Sparus aurata) juvenilesPublication . Dias, J.; Rodrigues, V.; Colen, Rita; Rosa, Joana; Viegas, Michael; Cardeira Da Silva, João; Cancela, Leonor; Gavaia, Paulo J.; Laizé, VincentReplacement of significant amounts of marine fish oils by vegetable oils is a major trend in the aquaculture feed industry. However, knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is extremely scarce in fish. We speculate that changes in the dietary ratio of fatty acids may modulate tissue eicosanoids production and affect bone formation in fastgrowing gilthead seabream, an important fish species for aquaculture in the Mediterranean region.
- Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectivesPublication . Marques, Cátia L.; Fernandez, Ignacio; Viegas, Michael; Cox, C. J.; Martel, Paulo; Rosa, Joana; Cancela, Leonor; Laizé, VincentBMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage-and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to downregulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events.