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- Effect of marine-derived extracts on mineralogenesisPublication . Carletti, Alessio; Cancela, Leonor; Gavaia, PauloBone erosive pathologies are the leading cause of fractures worldwide and represent a pressing medical and economic burden. Diseases like osteoporosis, Paget’s disease of bone, hyperparathyroidism, and renal osteodystrophy, have different pathophysiological roots but they all share a common feature: they lead to loss of bone mineral and result into increased bone fragility. This class of disorders are also a compelling pharmaceutical challenge. Currently, there is a limited choice of therapeutic agents available to treat bone loss and they are often characterized by short-timed efficacy and severe side effects. Meanwhile, fish grown in aquaculture, the primary source of seafood for human consumption, typically suffer from skeletal abnormalities that are universally present in all culture conditions and fish species. These skeletal defects appear to be largely caused by factors intrinsically related to the condition of captivity, including the lack of an adequate nutrition. The EU-funded Marie-Curie ITN project BIOMEDAQU, in the scope of which this PhD project is framed, provided a multidisciplinary platform bringing together research in biomedicine and aquaculture (from which Biomed-Aqu) with the objective of creating new knowledge with applications in both research fields. In this context, marine-based pharmacology, the branch of pharmaceutical research focalized on the screening and characterization of marine natural compounds, can contribute to fulfill knowledge gaps and provide translational applications for both disciplines. Different groups of marine organisms have been studied as sources of “osteoactive” compounds, some of which were described for their highly promising pharmacological and nutraceutical potential. The objective of this PhD project was to screen and characterize extracts obtained from various groups of marine organisms, selected as candidates for the isolation of compounds with potential applications for the biomedical sector, in the development of drugs to treat human bone erosive pathologies, and for the aquaculture industry, to be used as nutraceuticals to be incorporated into fish feeds to ameliorate skeletal health. With this aim, we put in place a medium-scale screening project evaluating about 150 extracts and fractions obtained from different marine organisms that recent pharmacological research has identified as promising sources of bioactive compounds, including holothurians, tunicates, cyanobacteria, marine bacteria, and microalgae. As a result of this screening activity, we identified ethanolic extracts from two microalgal species, Skeletonema costatum and Tetraselmis striata (CTP4 strain), as the most promising for their pro-osteogenic activities. Then, we further characterized them by testing using in vitro bone-derived cellular models, and in vivo, in the model organism zebrafish (Danio rerio), used as platform to investigate more in-depth the molecular mechanism of actions of the two extracts. By doing this, we revealed that the ethanolic extract from the microalga Tetraselmis striata CTP4 possessed the strongest osteoanabolic properties. We then wanted to provide a proof of concept of possible applications of these two extracts in the aquaculture industry by exploring their potential to be used as dietary supplements for the commercially reared species Sparus aurata. Accordingly, we found that Tetraselmis striata CTP4 is the most promising extract in this sense, in light of its capacity to promote fish growth, bone mineralization, and reduce the incidence of skeletal anomalies in seabream juveniles. Finally, we decided to dissect the molecular mechanism behind the osteoactivity of the ethanolic extracts from Skeletonema costatum, and validate its potential application for the treatment of bone erosive disorders in human patients. We found that the extract has mainly an anti-osteoclastogenic activity, and we put it in the prospect of its previously known antiinflammatory potential. We also provide evidence that its application can limit bone loss in a medaka model of osteoporosis, and that its anti-osteoclastogenic properties are conserved in a mammalian in vitro cell model. Overall, through this PhD project, by identifying and characterizing these two microalgae extracts in the context of bone mineralization, we have provided the substrate for future research aimed at isolating compounds with potential applications in human medicine and as dietary supplements for aquaculture nutrition.
- Antioxidant and anti-inflammatory extracts from sea cucumbers and tunicates induce a pro-osteogenic effect in Zebrafish LarvaePublication . Carletti, Alessio; Cardoso, Carlos; Lobo-Arteaga, Jorge; Sales, Sabrina; Juliao, Diana; Ferreira, Inês; Chainho, Paula; Dionísio, Maria Ana; Gaudêncio, Maria J.; Afonso, Cláudia; Lourenço, Helena; Cancela, M. Leonor; Bandarra, Narcisa M.; J. Gavaia, PauloBone metabolic disorders such as osteoporosis are characterized by the loss of mineral from the bone tissue leading to its structural weakening and increased susceptibility to fractures. A growing body of evidence suggests that inflammation and oxidative stress play an important role in the pathophysiological processes involved in the rise of these conditions. As the currently available therapeutic strategies are often characterized by toxic effects associated with their long-term use, natural antioxidants and anti-inflammatory compounds such as polyphenols promise to be a valuable alternative for the prevention and treatment of these disorders. In this scope, the marine environment is becoming an important source of bioactive compounds with potential pharmacological applications. Here, we explored the bioactive potential of three species of holothurians (Echinodermata) and four species of tunicates (Chordata) as sources of antioxidant and anti-inflammatory compounds with a particular focus on polyphenolic substances. Hydroethanolic and aqueous extracts were obtained from animals' biomass and screened for their content of polyphenols and their antioxidant and anti-inflammatory properties. Hydroethanolic fractions of three species of tunicates displayed high polyphenolic content associated with strong antioxidant potential and anti-inflammatory activity. Extracts were thereafter tested for their capacity to promote bone formation and mineralization by applying an assay that uses the developing operculum of zebrafish (Danio rerio) to assess the osteogenic activity of compounds. The same three hydroethanolic fractions from tunicates were characterized by a strong in vivo osteogenic activity, which positively correlated with their anti-inflammatory potential as measured by COX-2 inhibition. This study highlights the therapeutic potential of polyphenol-rich hydroethanolic extracts obtained from three species of tunicates as a substrate for the development of novel drugs for the treatment of bone disorders correlated to oxidative stress and inflammatory processes.
- The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish modelsPublication . Carletti, Alessio; Rosa, Joana; Pes, Katia; Borges, Inês; Santos, Tamara; Barreira, Luísa; Varela, João; Pereira, Hugo; Cancela, M. Leonor; J. Gavaia, Paulo; Laizé, VincentSkeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.
- Zebrafish as a model to unveil the Pro-Osteogenic effects of Boron-Vitamin D3 synergismPublication . Sojan, Jerry Maria; Gundappa, Manu Kumar; Carletti, Alessio; Gaspar, Vasco; Gavaia, Paulo; Maradonna, Francesca; Carnevali, OlianaThe micronutrient boron (B) plays a key role during the ossification process as suggested by various in vitro and in vivo studies. To deepen our understanding of the molecular mechanism involved in the osteogenicity of B and its possible interaction with vitamin D3 (VD), wild-type AB zebrafish (Danio rerio) were used for morphometric analysis and transcriptomic analysis in addition to taking advantage of the availability of specific zebrafish osteoblast reporter lines. First, osteoactive concentrations of B, VD, and their combinations were established by morphometric analysis of the opercular bone in alizarin red-stained zebrafish larvae exposed to two selected concentrations of B (10 and 100 ng/ml), one concentration of VD (10 pg/ml), and their respective combinations. Bone formation, as measured by opercular bone growth, was significantly increased in the two combination treatments than VD alone. Subsequently, a transcriptomic approach was adopted to unveil the molecular key regulators involved in the synergy. Clustering of differentially expressed genes revealed enrichment toward bone and skeletal functions in the groups co-treated with B and VD. Downstream analysis confirmed mitogen-activated protein kinase as the most regulated pathway by the synergy groups in addition to transforming growth factor-beta signaling, focal adhesion, and calcium signaling. The best-performing synergistic treatment, B at 10 ng/ml and VD at 10 pg/ml, was applied to two zebrafish transgenic lines, Tg(sp7:mCherry) and Tg(bglap:EGFP), at multiple time points to further explore the results of the transcriptomic analysis. The synergistic treatment with B and VD induced enrichment of intermediate (sp7(+)) osteoblast at 6 and 9 days post fertilization (dpf) and of mature (bglap(+)) osteoblasts at 15 dpf. The results obtained validate the role of B in VD-dependent control over bone mineralization and can help to widen the spectrum of therapeutic approaches to alleviate pathological conditions caused by VD deficiency by using low concentrations of B as a nutritional additive.
- Metabolic bone disorders and the promise of marine osteoactive compoundsPublication . Carletti, Alessio; Gavaia, Paulo; Cancela, M. Leonor; Laizé, VincentMetabolic bone disorders and associated fragility fractures are major causes of disability and mortality worldwide and place an important financial burden on the global health systems. These disorders result from an unbalance between bone anabolic and resorptive processes and are characterized by different pathophysiological mechanisms. Drugs are available to treat bone metabolic pathologies, but they are either poorly effective or associated with undesired side effects that limit their use. The molecular mechanism underlying the most common metabolic bone disorders, and the availability, efficacy, and limitations of therapeutic options currently available are discussed here. A source for the unmet need of novel drugs to treat metabolic bone disorders is marine organisms, which produce natural osteoactive compounds of high pharmaceutical potential. In this review, we have inventoried the marine osteoactive compounds (MOCs) currently identified and spotted the groups of marine organisms with potential for MOC production. Finally, we briefly examine the availability of in vivo screening and validation tools for the study of MOCs.