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- Metallothioneins in the clam Ruditapes decussatus: an overviewPublication . Bebianno, Maria João; Serafim, MA; Simes, DinaThe clam Ruditapes decussatus is a suspension-feeding bivalve mollusc widely distributed in European waters and in the Mediterranean. Due to is economic importance it is heavily harvested in many countries, and particularly in Portugal. Its ability to accumulate high metal concentrations along with its economic importance was the main reason for its selection as a bioindicator. Metallothionein (MT) concentrations in the clams R. decussatus followed by gel filtration chromatography, differential pulse polarography and SDS-PAGE, after Cd exposure, revealed that MT is induced in different tissues (whole soft tissues, gills, digestive gland and remaining tissues) but the level of MT induction is tissue dependent. MT from the gills and the digestive gland give a more sensitive response to assess the effects of metal exposure directly from the water or from the food than in the whole soft tissues. MT levels were also measured in the gills, digestive gland and remaining tissues of R. decussatus collected in the Ria Formosa lagoon (Portugal) from areas of different metal load and during the period of sexual differentiation of the clam. Data revealed that there were significant differences of MT concentrations among sites and season but not among sex. Purification of MT from the digestive gland of R. decussatus revealed four MT isoforms. The molecular weight of one of these isoforms, determined by SDS-PAGE, was of the same order of magnitude as that of MT from other bivalve species. Similarly the amino acid sequence of the beta domain of the MT of the digestive gland of the clam also shows some degree of similarity with the similar MT sequence from mussels and oysters. It is, therefore suggested that there is some degree of similarity in the MT structure among these species.
- 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.
- Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: implications for calcification-related chronic inflammatory diseasesPublication . Viegas, Carla; Costa, Ruben M.; Santos, Lúcia; Videira, Paula A.; Silva, Zelia; Araujo, Nuna C. P.; Macedo, Anjos L.; Matos, Antonio P.; Vermeer, Cees; Simes, DinaCalcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and gamma-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein gamma-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNF alpha, IL-1 beta and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.
- Amentadione from the alga Cystoseira usneoides as a novel osteoarthritis trotective agent in an ex vivo co-culture OA ModelPublication . Araujo, Nuna C. P.; Viegas, Carla; Zubía, Eva; Magalhães, Joana; Ramos, Acácio; Carvalho, Maria M.; Cruz, Henrique; Sousa, João Paulo; Blanco, Francisco J.; Vermeer, Cees; Simes, DinaOsteoarthritis (OA) remains a prevalent chronic disease without effective prevention and treatment. Amentadione (YP), a meroditerpenoid purified from the alga Cystoseira usneoides, has demonstrated anti-inflammatory activity. Here, we investigated the YP anti-osteoarthritic potential, by using a novel OA preclinical drug development pipeline designed to evaluate the anti-inflammatory and anti-mineralizing activities of potential OA-protective compounds. The workflow was based on in vitro primary cell cultures followed by human cartilage explants assays and a new OA co-culture model, combining cartilage explants with synoviocytes under interleukin-1β (IL-1β) or hydroxyapatite (HAP) stimulation. A combination of gene expression analysis and measurement of inflammatory mediators showed that the proposed model mimicked early disease stages, while YP counteracted inflammatory responses by downregulation of COX-2 and IL-6, improved cartilage homeostasis by downregulation of MMP3 and the chondrocytes hypertrophic differentiation factors Col10 and Runx2. Importantly, YP downregulated NF-κB gene expression and decreased phosphorylated IkBα/total IkBα ratio in chondrocytes. These results indicate the co-culture as a relevant pre-clinical OA model, and strongly suggest YP as a cartilage protective factor by inhibiting inflammatory, mineralizing, catabolic and differentiation processes during OA development, through inhibition of NF-κB signaling pathways, with high therapeutic potential.
- Nanoencapsulation of Gla-Rich Protein (GRP) as a novel approach to target inflammationPublication . Viegas, Carla; Araújo, Nuna; Carreira, Joana; Pontes, Jorge Filipe; Macedo, Anjos L.; Vinhas, Maurícia; Moreira, Ana S.; Faria, Tiago Q.; Grenha, Ana; de Matos, António A.; Schurgers, Leon; Vermeer, Cees; Simes, DinaChronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP’s therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG’s anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1β, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.
- Gla-rich protein (GRP) as an early and novel marker of vascular calcification and kidney dysfunction in diabetic patients with CKD: a pilot cross-sectional studyPublication . Silva, Ana P.; Viegas, Carla; Mendes, Filipa; Macedo, Ana; Guilherme, Patrícia; Tavares, Nelson; Dias, Carolina; Rato, Fátima; Santos, Nélio; Faísca, Marília; Almeida, Edgar de; Neves, Pedro Leão; Simes, DinaVascular calcification (VC) is one of the strongest predictors of cardiovascular risk in chronic kidney disease (CKD) patients. New diagnostic/prognostic tools are required for early detection of VC allowing interventional strategies. Gla-rich protein (GRP) is a cardiovascular calcification inhibitor, whose clinical utility is here highlighted. The present study explores, for the first time, correlations between levels of GRP in serum with CKD developmental stage, mineral metabolism markers, VC and pulse pressure (PP), in a cohort of 80 diabetic patients with mild to moderate CKD (stages 2-4). Spearman's correlation analysis revealed a positive association of GRP serum levels with estimated glomerular filtration rate (eGFR) and α-Klotho, while a negative correlation with phosphate (P), fibroblast growth factor 23 (FGF-23), vascular calcification score (VCS), PP, calcium (x) phosphate (CaxP) and interleukin 6 (IL-6). Serum GRP levels were found to progressively decrease from stage 2 to stage 4 CKD. Multivariate analysis identified low levels of eGFR and GRP, and high levels of FGF-23 associated with both the VCS and PP. These results indicate an association between GRP, renal dysfunction and CKD-mineral and bone disorder. The relationship between low levels of GRP and vascular calcifications suggests a future, potential utility for GRP as an early marker of vascular damage in CKD.
- 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.
- Nanoencapsulation as a novel delivery approach for therapeutic applications of gla-rich protein (GRP)Publication . Araújo, Nuna; Viegas, Carla; Pontes, Jorge Filipe; Marreiros, Catarina; Raimundo, Pedro; Macedo, Anjos L.; Alves de Matos, António; Grenha, Ana; Vermeer, Cees; Simes, DinaGla rich protein (GRP) is a vitamin K dependent protein, shown to function as an inhibitor of pathological calcification and as an anti-inflammatory agent, with potential therapeutic use for age-related diseases such as osteoarthritis (OA) [1,2]. OA is a leading cause of disability and morbidity in the older population and constitutes a major world wide challenge for our health system. Presently, there are no drugs approved that can prevent, stop, or even restrain progression of OA. GRP has been shown to be able to lower inflammation and mineralisation processes in the articular tissue. Chitosan/tripolyphosphate (TPP) nanoparticles were selected for this study due to their biocompatibility, biodegradability and capacity to overcome the problem of low solubility of GRP in physiological conditions. This study aims to produce and characterise chitosan/TPP nanoparticles as GRP-delivery vehicles and test its anti-inflammatory potential in human macrophages.
- Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological CalcificationsPublication . Viegas, Carla; Cavaco, Sofia; Neves, Pedro L.; Ferreira, Ana; Joao, Alexandre; Williamson, Matthew K.; Price, Paul A.; Cancela, M. Leonor; Simes, DinaMineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization. (Am J Pathol 2009, 175:2288-2298; DOI; 10.2353/ajpath.2009.090474)
- Gla-Rich protein, magnesium and phosphate associate with mitral and aortic valves calcification in Didabetic patients with moderate CKDPublication . Silva, Ana P.; Viegas, Carla; Guilherme, Patrícia; Tavares, Nelson; Dias, Carolina; Rato, Fátima; Santos, Nélio; Faísca, Marília; de Almeida, Edgar; Neves, Pedro L.; Simes, Dina C.Accelerated and premature cardiovascular calcification is a hallmark of chronic kidney disease (CKD) patients. Valvular calcification (VC) is a critical indicator of cardiovascular disease and all-cause mortality in this population, lacking validated biomarkers for early diagnosis. Gla-rich protein (GRP) is a cardiovascular calcification inhibitor recently associated with vascular calcification, pulse pressure, mineral metabolism markers and kidney function. Here, we examined the association between GRP serum levels and mitral and aortic valves calcification in a cohort of 80 diabetic patients with CKD stages 2–4. Mitral and aortic valves calcification were detected in 36.2% and 34.4% of the patients and associated with lower GRP levels, even after adjustments for age and gender. In this pilot study, univariate, multivariate and Poisson regression analysis, show that low levels of GRP and magnesium (Mg), and high levels of phosphate (P) are associated with mitral and aortic valves calcification. Receiver operating characteristic (ROC) curves showed that the area under the curve (AUC) values of GRP for mitral (0.762) and aortic (0.802) valves calcification were higher than those of Mg and P. These results suggest that low levels of GRP and Mg, and high levels of P, are independent and cumulative risk factors for VC in this population; the GRP diagnostic value might be potentially useful in cardiovascular risk assessment.