Percorrer por autor "Simes, Dina"
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- Amentadione from the Alga Cystoseira usneoides as a Novel Osteoarthritis Protective 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.
- 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.
- Amentadione is a new modulating agent for osteoarthritis in an ex-vivo co-culture preclinical assayPublication . Araujo, Nuna C. P.; Viegas, Carla; Perrolas, Inês; Costa, Rúben; Magalhães, Joana; Blanco, Francisco; Ramos, Acácio; Miguel, Maria; Vermeer, Cees; Zubía, Eva; Simes, DinaOsteoarthritis (OA) is a whole-joint disease where inflammation interplays with extracellular matrix mineralization in a cycle that leads to its degradation. The lack of effective preventing treatments and disease modifying agents, demands new therapeutic targets and development of effective drugs.
- An extremely acidic environment microalga from Tinto River as a novel source of anti-inflammatory activityPublication . Robles, María; Viegas, Carla; Torronteras, Rafael; Garbayo, Inés; Vega, José M.; Simes, Dina; Vílchez, CarlosCoccomyxa onubensis (C. onubensis) is an acidotolerant microalga isolated from the extremely acidic Tinto River (Huelva), which contains high levels of metals in solution, mainly Fe and Cu. This makes C. onubensis an adequate candidate to easily cope with high levels of oxidative stress by increasing the levels of certain molecules and enzymes, which helps them elicit an adequate antioxidant biochemical response. Thus, C. onubensis is a promising source of bioactive compounds which exhibit in vitro anti-inflammatory activity, including fatty acids, (poly)phenolic compounds and carotenoids. In this study, the correlations between the antioxidant response and anti-inflammatory activity of cell extracts obtained from Fe (III)-stressed microalgal cultures were analyzed. The results suggested a direct relationship between the antioxidant capacity of the microalgal extracts and Fe (III) concentration in the culture medium. Consequently, the production of some of the target antioxidant molecules, including carotenes, xanthophylls and (poly)phenols, increased. The levels of these molecules increased the most in cell extracts obtained from microalgal cultures at 0.25 mM of Fe (III), which was correlated with a 50 % increase in the anti-inflammatory activity of the microalgal extracts in THP-1 differentiated human macrophages. Fe (III)-modulated oxidative stress allowed us to define culture conditions that can enhance the antiinflammatory activity of C. onubensis extracts, which are enriched in valuable antioxidant molecules. Overall, this study highlighted the utility of a microalgal species from a highly acidic environment as a novel, natural source of anti-inflammatory agents, based on its ability to cope with the oxidative conditions of its habitat.
- First evidence of dermo-protective activity of marine sulfur-containing histidine compoundsPublication . Brancaccio, Mariarita; Milito, Alfonsina; S B Viegas, Carla; Palumbo, Anna; Simes, Dina; Castellano, ImmacolataAmong natural products, ovothiol (ovo), produced by marine invertebrates, bacteria, and microalgae, is receiving increasing interest for its unique antioxidant properties. Recently, ovo has been shown to exhibit anti-inflammatory activity in an in vitro model of endothelial dysfunction and in an in vivo model of liver fibrosis.The aim of this study was to evaluate the effect of ovo and its precursor 5-thiohistidine (5-thio) in comparison with ergothioneine (erg), in human skin cells and tissues upon inflammation. We used both an in vitro and ex vivo model of human skin, represented by a keratinocytes cell line (HaCaT) and skin biopsies, respectively. We observed that ovo, 5-thio, and erg were not cytotoxic in HaCaT cells, but instead exerted a protective function against TNF-alpha-induced inflammation. In order to get insights on their mechanism of action, we performed western blot analysis of ERK and JNK, as well as sub-cellular localization of Nrf2, a key mediator of the anti-inflammatory response. The results indicated that the pre-treatment with ovo, 5-thio, and erg differently affected the phosphorylation of ERK and JNK. However, all the three molecules promoted the accumulation of Nrf2 in the nucleus of HaCaT cells. In addition, gene expression analysis by RTqPCR and ELISA assays performed in ex vivo human skin tissues pre-treated with thiohistidines and then inflamed with IL-1 beta revealed a significant downregulation of IL-8, TNF-alpha and COX-2 genes and a concomitant significant decrease in the cytokines IL-6, IL-8 and TNF-alpha production. Moreover, the protective action of ovo and 5-thio resulted to be stronger when compared with dexamethasone, a corticosteroid drug currently used to treat skin inflammatory conditions.Our findings suggest that ovo and 5-thio can ameliorate skin damage and may be used to develop natural skin care products to prevent the inflammatory status induced by environmental stressors and aging.
- Gla rich protein (GRP) mediates vascular smooth muscle cell (VSMC) osteogenic differentiation, extracellular vesicle (EV) calcification propensity, and immunomodulatory propertiesPublication . Maia, Teresa M.; Macedo, Anjos L.; Matos, António P.; Neves, José; Viegas, Carla; Camilo Carreira, Joana Sofia; Simes, DinaVascular calcification (VC) is a complex process involving vascular smooth muscle cell (VSMC) osteogenic differentiation, inflammation, and extracellular vesicle (EV) calcification and communication networks. Gla rich protein (GRP) is a calcification inhibitor involved in most of these processes. However, the molecular mechanism of GRP in VC and the specific characteristics, cargo, and functionality of calcifying EVs require further elucidation. Here, we use a combination of human ex vivo aortic fragments and primary vascular smooth muscle cell (VSMC) models to obtain new information on GRP function in VC and EVs released by VSMCs. We demonstrate that GRP inhibits VSMC osteogenic differentiation through downregulation of bone-related proteins and upregulation of mineralization inhibitors, with decreased mineral crystallinity in EVs deposited into the tissue extracellular matrix (ECM). EVs isolated by ultracentrifugation at 30K and 100K from the cell media (CM) and deposited in the ECM from control (CTR) and mineralizing (MM) VSMCs were biochemically, physically, and proteomically characterized. Four different EV populations were identified with shared markers commonly present in all EVs but with unique protein cargo and specific molecular profiles. Comparative proteomics identified several regulated proteins specifically loaded into MM EV populations associated with multiple processes involved in VC. Functional analysis demonstrated that 30K and 100K ECM-MM EVs with higher calcium and lower GRP levels induced macrophage inflammation. Our findings reinforce the functional relevance of GRP in multiple VC processes and suggest that ECM EVs released under calcification stress function as a new signaling axis on the calcification-inflammation cycle.
- 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.
- 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.
- 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 is associated with vascular calcification, inflammation, and mineral markers in peritoneal dialysis patientsPublication . MARREIROS, CATARINA; Viegas, Carla; Malho Guedes, Anabela; Silva, Ana Paula de Andrade; Águas, Ana Catarina; Faísca, Marília; Schurgers, Leon; Simes, DinaVascular calcification (VC) is a crucial risk factor for cardiovascular diseases (CVD), particularly in chronic kidney disease (CKD) populations. However, the specific relationship between VC and end-stage renal disease (ESRD) patients undergoing peritoneal dialysis (PD) remains to be fully understood. The identification of new biomarkers to improve VC diagnosis and monitoring would significantly impact cardiovascular risk management in these high-risk patients. Gla-rich protein (GRP) is a VC inhibitor and an anti-inflammatory agent and thus is a potential VC marker in CKD. Here we explored the potential role of GRP as a marker for CVD and investigated the impact of VC in 101 PD patients. Methods: Circulating total Gla-rich protein (tGRP) was quantified in serum and in 24 h dialysate samples. VC score (VCS) was determined using the Adragão method. Results: Serum tGRP was negatively associated with VCS, serum calcium (Ca), phosphate (P), and high-sensitivity C-reactive protein (hsCRP), while it was positively associated with magnesium (Mg). A total of 35.6% of PD patients presented with extensive calcifications (VCS ≥ 3), and the lowest tGRP serum levels were present in this group (419.4 ± 198.5 pg/mL). tGRP in the 24 h dialysate was also negatively associated with VCS and with serum Ca and P. Moreover, serum Ca, P, and VCS were identified as independent determinants of serum tGRP levels. Conclusions: The association of serum tGRP with VC, mineral, and inflammation markers reinforces its potential use as a novel VC biomarker in CKD patients undergoing PD.
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