Interplay between mineralization and inflammation in vascular calcification and osteoarthritis: extracellular vesicles and marine bioactive compounds as a therapeutic approach

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Amentadione from the Alga Cystoseira usneoides as a Novel Osteoarthritis Protective Agent in an Ex Vivo Co-Culture OA Model

Publication . 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, Dina

Osteoarthritis (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.

2020-12-07Journal article

Open access

Nanoencapsulation of Gla-Rich Protein (GRP) as a novel approach to target inflammation

Publication . 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, Dina

Chronic 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.

2022-04-27Journal article

Open access

Interplay between mineralization and inflammation in osteoarthritis: gla-rich protein and marine bioactive compounds as new therapeutic approaches

Publication . Araújo, Nuna Cláudia Peixoto de; Simes, Dina; Viegas, Carla; Vermeer, Cees

Osteoarthritis (OA) is a prevalent joint disorder with significant global impact, characterized by limited treatment options and challenges in early diagnosis. This research aimed to address a deeper understanding of the onset and progression of OA, while exploring novel therapeutic strategies. Pathologic calcification and inflammation, associated with degradation of cartilage extracellular matrix, are prominent features observed in OA. Gla-Rich Protein (GRP), a vitamin K-dependent protein, has recently shown promising potential in possessing anti-inflammatory and anti-mineralization properties in articular cells, suggesting its role in the interplay between inflammation and mineralization in OA. Based on that, we developed chitosan-based nanoparticles to encapsulate GRP, aiming to enhance its bioavailability and thereby facilitating its application in functional assays. These nanoparticles effectively delivered GRP and retained its anti-inflammatory activity in human activated macrophages (THP-1 MoM) and chondrocytes. The novel nanoformulation demonstrated promising potential for therapeutic applications in chronic inflammatory diseases. Furthermore, we developed an experimental pipeline in order to evaluate potential OA-modifying compounds and their effects on inflammation and mineralization. The pipeline involved a series of activity assessments, starting from a simpler cell-based model of OA and progressing to a more complex co-culture model based on human cartilage. This progressive approach allowed us to examine potential drugs as mediators of inflammation and mineralization, mimicking the early disease stages and providing valuable insights into the complex pathological processes involved in OA progression. In the search for new active compounds to treat OA, we investigated the utilization of a marine bioactive compound, amentadione (YP), in the context of OA treatment, using the established pipeline. Amentadione exhibited strong anti-inflammatory effects, by downregulating key inflammatory mediators such as cyclooxygenase 2 (COX-2) and interleukin 6 (IL-6), and also demonstrated the ability to regulate nuclear factor κB (NF-κB) signaling pathways. By decreasing matrix metalloproteinase-3 (MMP3) levels and chondrocyte hypertrophic differentiation factors, Col10 and Runx2, YP contributed to cartilage homeostasis. These findings highlight the high therapeutic potential of amentadione as a cartilage protective factor in OA. In summary, this research highlights the therapeutic potential of both GRP and marine bioactive compound YP in the management of inflammatory responses in OA. The utilization of novel drug delivery systems, including chitosan-based nanoparticles, along with the comprehensive evaluation of compounds using the established experimental pipeline, offers the possibility of better understanding the underlying disease mechanisms and to explore new potential active compounds in OA.

2024-03-18Doctoral thesis

Embargoed access