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- Gla portein localization and histological characterization of bone stuctures in relevant aquaculture fishPublication . Roberto, Vania Palma; Cancela, Maria Leonor; Gavaia, PauloOsteocalcin (Oc) and Matrix Gia Protein (MGP) are vitamin K-dependente proteins known for their Ca2" binding capacity. Considering the problem of skeletal malformations in Mediterranean fish aquaculture, we investigated a possible correlation between vertebral deformities and changes of Oc and Mgp accumulation sites. ln this work we proposed to characterize bony and cartilaginous tissues from important fish for aquaculture, using molecular tools. The sites of gene expression and accumulation of osteocalcin and Mgp were investigated throughout Northern blot analysis and immunohistochemistry, following the cloning of those cDNAs not yet available, together with histological analysis. ln Scophthalmus maximus, oc mRNA was detected in vertebrae and branchial arches while mgp mRNA was highly expressed in branchial arches, followed by vertebrae, kidney and heart. ln S. maximus and Diplodus sargus, Mgp accumulated in chondrocytes from cartilages, in the vertebral mineralizing fronts and in notochord cells. Mgp was also found in scales of D. sargus. Ocaccumulated mainly in bone tissues, like the ceratobranchial bone and vertebral bone matrix. ln cultured Sparus aurata, Mgp accumulated mainly in vertebrae growth zones and in notochord cells while Oc was immunodetected in bone matrix of vertebrae and vertebral arches, both in non-deformed and in deformed vertebrae. We also identified Oc in the non-calcified notochord cells of deformed vertebrae, suggesting that abnormal skeletal development resulted in modifications on sites of osteocalcin expression and/or accumulation. Accordingly, histological analysis of normal and deformed vertebrae revealed calcification in blood vessel walls and pathological formation of chondroid bone, in the affected area of deformed vertebrae.
- Mgp expression and accumulation in heart and kidney of turbot (Scophthalmus maximus)Publication . Roberto, Vania Palma; Cavaco, S.; Simes, D; Gavaia, Paulo J.; Cancela, LeonorMatrix γ-carboxyglutamic acid (Gla) protein (Mgp) is a vitamin K-dependent protein normally found associated with the organic matrix of cartilage and bone in vivo. After the discovery of Mgp in various soft tissues, this protein was proposed to act as a local inhibitor of mineralization although its molecular mechanisms of action remain incompletely understood.
- Matrix gla protein in turbot (Scophthalmus maximus): gene expression analysis and identification of sites of protein accumulationPublication . Roberto, Vania Palma; Cavaco, S.; S B Viegas, Carla; Simes, D; Ortiz-Delgado, J. B.; Sarasquete, C.; Gavaia, Paulo J.; Cancela, LeonorMatrix Gla protein (Mgp) is a secreted vitamin K-dependent extracellular matrix protein and a physiological inhibitor of calcification whose gene structure, amino acid sequence and tissue distribution have been conserved throughout evolution. In the present work, the turbot (Scophthalmus maximus) mgp cDNA was cloned and the sequence of the deduced protein compared to that of other vertebrates. As expected, it was closer to teleosts than to other vertebrate groups but there was a strict conservation of amino-acids thought to be important for protein function. Analysis of mgp gene expression indicated branchial arches as the site with higher levels of expression, followed by heart, vertebra and kidney. These results were confirmed by in situ hybridization with a strong mgp expression in branchial arch chondrocytes. Mgp was found to accumulate in gills where it appeared to be restricted to chondrocytes from branchial filaments, while in vertebrae it was localized in vertebral end plates, in growth zones, in vertebral arches and spines and in notochord cells. In the soft tissues analysed, Mgp was mainly detected in kidney and heart, consistent with previous data and providing further evidence for a role of Mgp as a calcification inhibitor and a modulator of the mineralization process. Our studies provide evidence that turbot, an important new species for aquaculture, is also a useful model to study function and expression of Mgp.
- Evidence for the conservation of miR-223 in zebrafish (Danio rerio): implications for functionPublication . Roberto, Vania Palma; Tiago, Daniel; Gautvik, K.; Cancela, M. LeonorMicroRNAs (miRNAs) are an abundant and conserved class of small RNAs, which play important regulatory functions by interacting with the 3' untranslated region (UTR) of target mRNAs. Through this mechanism, miR-223 was shown to regulate genes involved in mammalian haematopoiesis, both in physiological and pathological contexts. MiR-223 is essential for normal myelopoiesis in mammals, promoting granulocyte, osteoclast and megakaryocyte differentiation and suppressing erythropoiesis. However, there is a general lack of knowledge regarding miR-223 function in other vertebrates, which could help to clarify its role in other processes, such as development. In this work, we explored the functional conservation of miR-223 using zebrafish as a model. We show that miR-223 gene structure and genomic context have been maintained between human and zebrafish. In addition, we identified 22 novel sequences of miR-223 precursor and demonstrate that it contains domains highly conserved among vertebrates, suggesting function preservation throughout evolution. Furthermore, collected evidences show that miR-223 expression is highly correlated with haematopoietic events and osteoclastogenesis throughout zebrafish development. In adults, expression of miR-223 in zebrafish tissues mimics the distribution in mice, with high levels found in the major fish haematopoietic organ, the head kidney. These results suggest a conservation of miR-223 role in haematopoiesis, and osteoclastogenesis between zebrafish and human. Accordingly, validated targets of miR-223 in mammalian models were investigated and defined as putative targets in zebrafish, by in silico and gene expression analysis. Our data compiles critical evidence showing that miR-223, a highly conserved miRNA, appears to have kept similar regulatory functions throughout evolution.
- Identification and molecular characterization of bone-related micrornas: functional implicationsPublication . Roberto, Vania Palma; Cancela, Leonor; Tiago, Daniel; Gautvik, KaareMicroRNAs (miRNAs) are a conserved class of small RNAs providing a post-transcriptional mechanism for fine-tuning of intricate physiological and pathological cellular processes, such as those affecting development. Skeletogenesis however, was so far poorly investigated and mainly focused on mammalian models, with a general lack of knowledge concerning other vertebrates. We aimed at the identification of bone-related miRNAs and their characterization from an evolutionary perspective, using fish (mostly zebrafish) as model, in comparison to mammalian systems. First, we focused on miR-223, a miRNA that was associated with bone remodelling. We demonstrated that miR-223 genomic organization/context and primary/secondary structures are largely maintained between human and zebrafish. As in mammals, miR-223 expression in zebrafish was highly correlated with hematopoietic events and osteoclastogenesis. Finally, miR-223 targets identified in mammals were also predicted in zebrafish, supporting a functional conservation of this miRNA. In a second set of experiments, we studied the biological role of miR-29a, a bone-related miRNA that was fairly investigated in mammals, but with no mineralogenic effects yet demonstrated. We took advantage of our fish bone-derived systems to explore miR-29a mineralogenic effects through gain-of-function experiments. We demonstrated a strong stimulation of this process through a mechanism probably involving the canonical Wnt signalling. Once more, through bioinformatics analysis, patterns of expression and target prediction/validation, we provided evidences for miR-29 conservation throughout evolution. Finally, we explored miR-214 putative roles on skeleton formation in vertebrates. Although our initial hypothesis of miR-214 involvement in osteogenesis was recently demonstrated by Wang et al. (2013), we proceeded with our investigation and finally showed that miR-214 is also associated with chondrogenesis. Overexpression of miR-214 in ATDC5 cells mitigated differentiation and down-regulated Mgp and Osteocalcin, probably by targeting Atf4. This work provides novel evidence that some miRNAs have conserved functions across vertebrates and, probably, conserved regulatory mechanisms of action.
- Evidences for a new role of miR-214 in chondrogenesisPublication . Roberto, Vania Palma; Gavaia, Paulo; Nunes, Maria Joao; Rodrigues, Elsa; M. Leonor Cancela; Tiago, DanielmiR-214 is known to play a role in mammalian skeletal development through inhibition of osteogenesis and stimulation of osteoclastogenesis, but data regarding other vertebrates, as well as a possible role in chondrogenesis, remain unknown. Here, we show that miR-214 expression is detected in bone and cartilage of zebrafish skeleton, and is downregulated during murine ATDC5 chondrocyte differentiation. Additionally, we observed a conservation of the transcriptional regulation of miR-214 primary transcript Dnm3os in vertebrates, being regulated by Ets1 in ATDC5 chondrogenic cells. Moreover, overexpression of miR-214 in vitro and in vivo mitigated chondrocyte differentiation probably by targeting activating transcription factor 4 (Atf4). Indeed, miR-214 overexpression in vivo hampered cranial cartilage formation of zebrafish and coincided with downregulation of atf4 and of the key chondrogenic players sox9 and col2a1. We show that miR-214 overexpression exerts a negative role in chondrogenesis by impacting on chondrocyte differentiation possibly through conserved mechanisms.
- Antioxidant, mineralogenic and osteogenic activities of Spartina alterniflora and Salicornia fragilis extracts rich in polyphenolsPublication . Roberto, Vania Palma; Surget, Gwladys; Le Lann, Klervi; Mira, Sara; Tarasco, Marco; Guérard, Fabienne; Poupart, Nathalie; Laizé, Vincent; Stiger-Pouvreau, Valérie; Cancela, M. LeonorOsteoporosis is an aging-related disease and a worldwide health issue. Current therapeutics have failed to reduce the prevalence of osteoporosis in the human population, thus the discovery of compounds with bone anabolic properties that could be the basis of next generation drugs is a priority. Marine plants contain a wide range of bioactive compounds and the presence of osteoactive phytochemicals was investigated in two halophytes collected in Brittany (France): the invasive Spartina alterniflora and the native Salicornia fragilis. Two semi-purified fractions, prepared through liquid-liquid extraction, were assessed for phenolic and flavonoid contents, and for the presence of antioxidant, mineralogenic and osteogenic bioactivities. Ethyl acetate fraction (EAF) wasrich in phenolic compounds and exhibited the highest antioxidant activity. While S. fragilis EAF only triggered a weak proliferative effect in vitro, S. alterniflora EAF potently induced extracellular matrix mineralization (7-fold at 250µg/mL). A strong osteogenic effect was also observed in vivo using zebrafish operculum assay (2.5-fold at 10µg/mL in 9-dpf larvae). Results indicate that polyphenol rich EAF of S. alterniflora has both antioxidant and bone anabolic activities. As an invasive species, this marine plant may represent a sustainable source of molecules for therapeutic applications in bone disorders.
- The xenobiotic sensor PXR in a marine flatfish species (Solea senegalensis): Gene expression patterns and its regulation under different physiological conditionsPublication . Marques, Carlos; Roberto, Vania Palma; Granadeiro, Luis; Trindade, Marlene; Gavaia, Paulo; Laizé, Vincent; Leonor Cancela, M.; Fernandez, IgnacioThe pregnane X receptor (PXR) is a nuclear receptor belonging to the NR1I sub-family and a known master regulator of xenobiotic metabolism. New roles have been recently proposed in mammals through its activation by vitamin K (VK) such as regulation of glucose metabolism, bone homeostasis, reproduction, neuronal development and cognitive capacities. In marine fish species little is known about PXR and its potential roles. Here, expression patterns of pxr transcripts and conservation of protein domains were determined in the Senegalese sole (Solea senegalensis), a marine flatfish model species in aquatic ecotoxicology. In addition to a full coding sequence transcript (sspxrl), two variants lacking DNA and/or ligand binding domains (sspxr2 and sspxr3) were also identified. The expression of sspxrl during early development and in adult tissues was ubiquitous, but highest levels were observed in liver, intestine and skin. Expression was also detected by in situ hybridization in chondrocytes and cells from the granular and inner nuclear layers in three month old fish. Finally, sspxrl expression was shown to be differentially regulated under physiological conditions related with fasting, VK and warfarin metabolism. The present work provides new and basic knowledge regarding pxr sequence and expression patterns in a marine flatfish species to unveil the potential impact of xenobiotics on marine fish physiology, and will allow a better and more ecosystemic environmental risk assessment of different pollutants over the marine environments with the development of reporter assays using PXR sequences from evolutionary distantly marine species (such as vertebrate and invertebrate marine species). (C) 2017 Elsevier Ltd. All rights reserved.
- Vertebral deformities and local accumulation of gla proteins in sparus aurataPublication . Roberto, Vania Palma; Gavaia, Paulo J.; Cancela, LeonorDevelopment of skeletal deformities is a frequent problem in Mediterranean fish aquaculture. Investigation of proteins involved in tissue calcification may help us uncover a possible correlation between the appearance of skeletal deformities and a defect in the site of expression and/or accumulation of a particular gene or protein. The purpose of this study was to investigate if the presence of skeletal deformities was related with any changes in the sites of osteocalcin (Oc or Bgp – Bone Gla Protein) and matrix Gla protein (Mgp) accumulation using immunohistochemistry methods.
- Reprogramming iPSCs to study age-related diseases: models, therapeutics, and clinical trialsPublication . Esteves, Filipa; Brito, David; Rajado, Ana Teresa; Silva, Nádia; Apolónio, Joana; Roberto, Vania Palma; Araújo, Inês Maria; Nóbrega, Clévio; Castelo-Branco, Pedro; Bragança, José; P. Andrade, Raquel; M. Calado, Sofia; Faleiro, L; Matos, Carlos A; Marques, Nuno; Marreiros, Ana; Nzwalo, Hipólito; Pais, Sandra; Palmeirim, Isabel; S, Simão; Joaquim, Natércia; Miranda, Rui; Pêgas, António; Raposo, Daniela Marques; Sardo, AnaThe unprecedented rise in life expectancy observed in the last decades is leading to a global increase in the ageing population, and age-associated diseases became an increasing societal, economic, and medical burden. This has boosted major efforts in the scientific and medical research communities to develop and improve therapies to delay ageing and age-associated functional decline and diseases, and to expand health span. The establishment of induced pluripotent stem cells (iPSCs) by reprogramming human somatic cells has revolutionised the modelling and understanding of human diseases. iPSCs have a major advantage relative to other human pluripotent stem cells as their obtention does not require the destruction of embryos like embryonic stem cells do, and do not have a limited proliferation or differentiation potential as adult stem cells. Besides, iPSCs can be generated from somatic cells from healthy individuals or patients, which makes iPSC technology a promising approach to model and decipher the mechanisms underlying the ageing process and age-associated diseases, study drug effects, and develop new therapeutic approaches. This review discusses the advances made in the last decade using iPSC technology to study the most common age-associated diseases, including age-related macular degeneration (AMD), neurodegenerative and cardiovascular diseases, brain stroke, cancer, diabetes, and osteoarthritis.