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- Iron overload in a murine model of hereditary hemochromatosis is associated with accelerated progression of osteoarthritis under mechanical stressPublication . Camacho, A.; F. Simao, Marcio; Ea, H. -K.; Cohen-Solal, M.; Richette, P.; Branco, J.; Cancela, LeonorObjective: Hereditary hemochromatosis (HH) is a disease caused by mutations in the Hfe gene characterised by systemic iron overload and associated with an increased prevalence of osteoarthritis (OA) but the role of iron overload in the development of OA is still undefined. To further understand the molecular mechanisms involved we have used a murine model of HH and studied the progression of experimental OA under mechanical stress.Design: OA was surgically induced in the knee joints of 10-week-old C57BL6 (wild-type) mice and Hfe-KO mice. OA progression was assessed using histology, micro CT, gene expression and immunohistochemistry at 8 weeks after surgery.Results: Hfe-KO mice showed a systemic iron overload and an increased iron accumulation in the knee synovial membrane following surgery. The histological OA score was significantly higher in the Hfe-KO mice at 8 weeks after surgery. Micro CT study of the proximal tibia revealed increased subchondral bone volume and increased trabecular thickness. Gene expression and immunohistochemical analysis showed a significant increase in the expression of matrix metallopeptidase 3 (MMP-3) in the joints of Hfe-KO mice compared with control mice at 8 weeks after surgery.Conclusions: HH was associated with an accelerated development of OA in mice. Our findings suggest that synovial iron overload has a definite role in the progression of HH-related OA.
- Lab-it is taking molecular genetics to schoolPublication . F. Simao, Marcio; Conceição, Natércia; Imaginário, S.; Amaro, João; Leonor Cancela, M.The Molecular Genetics Mobile Lab or “Laboratório itinerante de Genética Molecular” (Lab-it) was funded in 2008 by Leonor Cancela to promote the learning of molecular genetics which had been introduced at that time into high school biology programms. The project aimed to introduce hands-on laboratory activities in molecular genetics to complement the theoretical concepts taught in school. These included the development of experimental protocols based on theoretical scenarios focusing on themes of forensics sciences, biomedical applications, diagnostic methods, and ecological research using basic molecular biology techniques, such as DNA extraction, polymerase chain reaction (PCR), electrophoresis, and restriction enzyme application. In these scenarios, the students execute all the procedures with the help of the Lab-it instructor and using the Lab-it equipment, followed by a discussion of the results with all the participants and the school teacher. These approaches help the students to consolidate the concepts of molecular biology and simultaneously promote discussions on new advances in the area and choices for university careers. In addition to practical sessions, Lab-it also promotes seminars on topics of interest to the students and teachers. Since 2008, 18 high schools have participated in the region of Algarve, averaging each year about 400 students participating in practical activities. In 2021, despite the COVID pandemic, 9 schools and 379 students were involved in Lab-it practical sessions and 99% of them considered the activity to contribute to better understanding the molecular biology methods approached in theoretical classes and expressed high interest in those sessions.
- Expression of four new ferritins from grooved carpet shell clam Ruditapes decussatus challenged with Perkinsus olseni and metals (Cd, Cu and Zn)Publication . Simão, Márcio; Leite, Ricardo B.; Cancela, M. LeonorIron has a fundamental role in life and in its biochemical reactions but, when in excess, it can promote the formation of free radicals which can lead to cell death. Therefore, managing the levels of iron is essential to regulate the production of oxidative stress related to iron, and ferritins are one of the main protein families involved in this process. Ferritins are approximate to 480 kDa multimeric proteins composed by 24 subunits, each with 19-26 kDa, which can accumulate up to 4500 iron atoms. Besides their role in managing iron bioavailability, they have also developed a role in organism immunity and defence present throughout evolution. In this work, we identified and characterized, for the first time, four different ferritin subunits in the clam Ruditapes decussatus, a bivalve commercially and ecologically important along the south Atlantic coast and in the Mediterranean basin, which is a major target of the parasitic protozoa Perkinsus olseni, considered one of the main causes of high levels of clam mortality. Following phylogenetic annotation, the four ferritins subunits identified were subdivided into two cytosolic and two secreted forms. All four subunits maintain the canonical ferritin structure with four main helices alpha (A-D) and a small helix (E), but the secreted ferritins present an additional helix in their N-terminal region (F), located after the signal peptide and with possible antimicrobial properties. Additionally, we identified in ferritin 4 an extra helix alpha (G) located between helices B and C. These alpha helix domains revealed high degree of similarity with antimicrobial peptides associated with antibacterial and antifungal activities. Analysis of the expression of these subunits showed that ferritins 1 and 2 are ubiquitously expressed while ferritins 3 and 4 are present mainly in visceral mass. Ferritin 1 lacked a putative functional iron response element (IRE) and appeared to be under a tight regulation. Ferritins 2 and 3 showed a strong response to infection by parasite Perkinsus olseni in contrast to ferritin 4, whose main response was related to exposure to a combination of metals. the synergistic effect between metals and infection promoted a general upregulation of the four ferritins. In conclusion, our results suggest that ferritins, besides their function in iron and metals detoxification, may play a determinant role in clam immune response.
- Type I Diabetes in Zebrafish reduces sperm quality and increases Insulin and Glucose transporter transcriptsPublication . Diogo, Patricia; Martins, Gil; F. Simao, Marcio; Marreiros, Ana; Eufrásio, Ana Catarina; Cabrita, Elsa; J. Gavaia, PauloType I diabetes is a prominent human pathology with increasing incidence in the population; however, its cause is still unknown. This disease promotes detrimental effects on reproduction, such as lower sperm motility and DNA integrity. Hence, the investigation of the underlying mechanisms of this metabolic disturbance in reproduction and its transgenerational consequences is of the utmost importance. The zebrafish is a useful model for this research considering its high homology with human genes as well as its fast generation and regeneration abilities. Therefore, we aimed to investigate sperm quality and genes relevant to diabetes in the spermatozoa of Tg(ins:nfsb-mCherry) zebrafish, a model for type I diabetes. Diabetic Tg(ins:nfsb-mCherry) males showed significantly higher expression of transcripts for insulin a (insa) and glucose transporter (slc2a2) compared to controls. Sperm obtained from the same treatment group showed significantly lower sperm motility, plasma membrane viability, and DNA integrity compared to that from the control group. Upon sperm cryopreservation, sperm freezability was reduced, which could be a consequence of poor initial sperm quality. Altogether, the data showed similar detrimental effects related to type I diabetes in zebrafish spermatozoa at the cellular and molecular levels. Therefore, our study validates the zebrafish model for type I diabetes research in germ cells.
- Iron-enriched diet contributes to early onset of osteoporotic phenotype in a mouse model of hereditary hemochromatosisPublication . Simão, Márcio; Camacho, António; Ostertag, Agnès; Cohen-Solal, Martine; Pinto, I. Jorge; Porto, Graça; Hang Korng, Ea; Cancela, M. LeonorOsteoporosis is associated with chronic iron overload secondary to hereditary hemochromatosis (HH), but the causative mechanisms are incompletely understood. The main objective of this study was to investigate the role of dietary iron on osteoporosis, using as biological model the Hfe-KO mice, which have a systemic iron overload. We showed that these mice show an increased susceptibility for developing a bone loss phenotype compared to WT mice, which can be exacerbated by an iron rich diet. The dietary iron overload caused an increase in inflammation and iron incorporation within the trabecular bone in both WT and Hfe-KO mice. However, the osteoporotic phenotype was only evident in Hfe-KO mice fed the iron-enriched diet. This appeared to result from an imbalance between bone formation and bone resorption driven by iron toxicity associated to Hfe-KO and confirmed by a decrease in bone microarchitecture parameters (identified by micro-CT) and osteoblast number. These findings were supported by the observed downregulation of bone metabolism markers and upregulation of ferritin heavy polypeptide 1 (Fth1) and transferrin receptor-1 (Tfrc), which are associated with iron toxicity and bone loss phenotype. In WT mice the iron rich diet was not enough to promote a bone loss phenotype, essentially due to the concomitant depression of bone resorption observed in those animals. In conclusion the dietary challenge influences the development of osteoporosis in the HH mice model thus suggesting that the iron content in the diet may influence the osteoporotic phenotype in systemic iron overload conditions.
- Musculoskeletal complications associated with pathological iron toxicity and its molecular mechanismsPublication . Simão, Márcio; Cancela, M. LeonorIron is fundamental for several biological functions, but when in excess can lead to the development of toxic events. Some tissues and cells are more susceptible than others, but systemic iron levels can be controlled by treating patients with iron-chelating molecules and phlebotomy. An early diagnostic can be decisive to limit the progression of musculoskeletal complications like osteoarthritis and osteoporosis because of iron toxicity. In iron-related osteoarthritis, aggravation can be associated to a few events that can contribute to joints articular cartilage exposure to high iron concentrations, which can promote articular degeneration with very little chance of tissue regeneration. In contrast, bone metabolism is much more dynamic than cartilage, but progressive iron accumulation and ageing can be decisive factors for bone health. The iron overload associated with hereditary diseases like hemochromatosis, hemophilias, thalassemias and other hereditary anaemias increase the negative impact of iron toxicity in joints and bone, as well as in life quality, even when iron levels can be controlled. The molecular mechanisms by which iron can compromise cartilage and bone have been illusive and only in the last 20 years studies have started to shed some light into the molecular mechanisms associated with iron toxicity. Ferroptosis and the regulation of intracellular iron levels is instrumental in the balance between detoxification and induced cell death. In addition, these complications are accompanied with multiple susceptibility factors that can aggravate iron toxicity and should be identified. Therefore, understanding tissues microenvironment and cell communication is fundamental to contextualize iron toxicity.
- Contribution to the molecular characterization of osteoarthritis and osteoporosis phenotypes associated with hereditary hemochromatosisPublication . F. Simao, Marcio; Cancela, Leonor; Pinto, Idílio Jorge Matias PereiraIn this study, we aimed to investigate the molecular mechanisms responsible for development of osteoarthritis (OA) and osteoporosis (OP) phenotypes in hereditary hemochromatosis using the Hfe-KO mouse model. Results from the analysis of Hfe-KO mice articular cartilage showed that Hfe loss of function alone is not enough to promote an OA-like phenotype and there were no evidences for high iron deposition in articulations of Hfe-KO mice. However, in vitro characterization of Hfe-KO primary chondrocytes exposed to 50μM of iron citrate produced an OA-like phenotype, with lower extracellular matrix production and increase metalloproteases expression. In addition, we identified the presence of iron metabolism dysregulation in Hfe-KO cells which showed an increase in intracellular iron levels relatively to wild type (WT). These results suggested that OA phenotype results from the conjugation of Hfe-KO with direct iron overload exposure and / or other synergistic variables. In contrast, Hfe-KO mice (12- months-old) showed a bone loss phenotype with significant decrease in trabecular and subchondral bone. At 6 months of age no differences were observed in WT and Hfe-KO mice bone kept under standard diet but when subject to iron rich diets they showed opposite bone phenotypes, with increased bone mineralization for WT, likely resulting from a decrease in osteoclast activity, and bone loss for Hfe-KO mice associated with iron toxicity in bone tissue. In conclusion, our results indicate that iron rich diet promotes bone loss phenotype in Hfe-KO mouse model while it is not sufficient to induce OA in the absence of other insults.
- Intracellular iron uptake is favored in Hfe-KO mouse primary chondrocytes mimicking an osteoarthritis-related phenotypePublication . F. Simao, Marcio; J. Gavaia, Paulo; Camacho, Antonio; Porto, Graca; Jorge Pinto, I.; Ea, Hang-Korng; Cancela, M. LeonorHFE-hemochromatosis is a disease characterized by a systemic iron overload phenotype mainly associated with mutations in the HFE protein (HFE) gene. Osteoarthritis (OA) has been reported as one of the most prevalent complications in HFE-hemochromatosis patients, but the mechanisms associated with its onset and progression remain incompletely understood. In this study, we have characterized the response to high iron concentrations of a primary culture of articular chondrocytes isolated from newborn Hfe-KO mice and compared the results with that of a similar experiment developed in cells from C57BL/6 wild-type (wt) mice. Our data provide evidence that both wt- and Hfe-KO-derived chondrocytes, when exposed to 50 mu M iron, develop characteristics of an OA-related phenotype, such as an increased expression of metalloproteases, a decreased extracellular matrix production, and a lower expression level of aggrecan. In addition, Hfe-KO cells also showed an increased expression of iron metabolism markers and MMP3, indicating an increased susceptibility to intracellular iron accumulation and higher levels of chondrocyte catabolism. Accordingly, upon treatment with 50 mu M iron, these chondrocytes were found to preferentially differentiate toward hypertrophy with increased expression of collagen I and transferrin and downregulation of SRY (sex-determining region Y)-box containing gene 9 (Sox9). In conclusion, high iron exposure can compromise chondrocyte metabolism, which, when simultaneously affected by an Hfe loss of function, appears to be more susceptible to the establishment of an OA-related phenotype.
- Identificação e caracterização de duas isoformas da Ferritina da amêijoa R. decussatus e avaliação de algumas das suas propriedades biológicasPublication . F. Simao, Marcio; Cancela, Leonor; Leite, Ricardo B.Este trabalho teve como objectivos principais a identificação e caracterização, pela primeira vez, da sequência completa de dois transcritos que codificam para isoformas da Ferritina na amêijoa R. decussatus. Uma vez identificadas as sequências das ORFs, as proteínas correspondentes foram expressas num sistema in vitro o que permitiu analisar algumas das suas propriedades biológicas como a capacidade de oxidação. Os dois transcritos diferentes identificados foram denominados de Ferritina alfa e beta, tendo respectivamente 915nt e 808nt e codificando para proteínas com 171 e 170 aminoácidos. A análise das sequências de ADNc dos transcritos revelou a conservação de um elemento de regulação do ferro (IRE) unicamente na subunidade beta, por outro lado na alfa a sequência que poderia estar associada a este domínio forma uma estrutura secundária diferente provavelmente sem capacidade de contribuir para a regulação do ferro. A análise da expressão dos transcritos alfa e beta foi realizada em 4 tecidos diferentes da amêijoa R. decussatus submetida a duas condições experimentais diferentes, um grupo controlo e um grupo sujeito a um tratamento com um suplemento de ferro (1mM FeCl2). Analisando em cada tecido (Brânquias, Pé, Sifões e Hepatopâcreas) a expressão dos transcritos da Ferritina alfa e beta em resposta ao tratamento com ferro foi possível observar que ocorre um aumento significativo de expressão da subunidade beta nessas condições nos tecidos do Pé (2,97, p<0,001), dos Sifões (2,61, p=0,003) e do Hepatopâncreas (2,31, p=0,011) enquanto para a subunidade alfa ocorre uma diminuição significativa da expressão nas Brânquias (0,40, p=0,010) e no Pé (0,45, p=0,015). A análise das sequências de aminoácidos codificadas pelos transcritos das Ferritinas alfa e beta revelaram uma identidade entre si de 78%, apresentando cada subunidade 4 hélices alfa principais (A, B, C e D) e uma quinta hélice (E) mais pequena na extremidade C-terminal, sendo as hélices B e C separadas por uma longa cadeia de aminoácidos sem estrutura secundária definida (coil). Foi também confirmada a conservação dos centros ferroxidase nas duas proteínas, assim como os domínios de nucleação do ferro e aminoácidos chave envolvidos na formação dos canais de transporte do ferro. iii A análise filogenética das sequências das Ferritinas alfa e beta da amêijoa R. decussatus indica que estas se enquadram filogeneticamente nos moluscos e no subgrupo das amêijoas, confirmando também que são duas proteínas independentes que evoluíram paralelamente. Após a expressão das Ferritinas alfa e beta in vitro foi desenvolvido um ensaio para avaliar o potencial de oxidação de cada subunidade (recombinante não digerida vs recombinante digerida pela protéase TEV). Os resultados demonstraram que as Ferritinas recombinantes digeridas (alfa e beta) apresentam um potencial de oxidação significativamente superior relativamente às proteínas recombinantes não digeridas, não apresentando no entanto quaisquer diferenças entre as subunidades alfa e beta. Resumindo, este trabalho permitiu caracterizar pela primeira vez as sequências completas de dois transcritos diferentes de Ferritinas na amêijoa R. decussatus, que codificam para isoformas distintas desta família de proteínas. A estrutura e os principais domínios são extremamente conservados e a análise das suas propriedades biológicas mostrou que não existem diferenças significativas no potencial de oxidação entre as isoformas alfa e beta. A análise das relações filogenéticas destas proteínas com as de outros organismos, moluscos e vertebrados, permitiu obter informação relevante sobre as relações filogenéticas entre as Ferritinas dos vertebrados e dos moluscos.
- Effect of C282Y genotype on self-reported musculoskeletal complications in hereditary hemochromatosisPublication . Brito Camacho, António; Funck-Brentano, Thomas; Simão, Márcio; Cancela, Leonor; Ottaviani, Sebastien; Cohen-Solal, Martine; Richette, PascalObjective Arthropathy that mimics osteoarthritis (OA) and osteoporosis (OP) is considered a complication of hereditary hemochromatosis (HH). We have limited data comparing OA and OP prevalence among HH patients with different hemochromatosis type 1 (HFE) genotypes. We investigated the prevalence of OA and OP in patients with HH by C282Y homozygosity and compound heterozygosity (C282Y/H63D) genotype. Methods A total of 306 patients with HH completed a questionnaire. Clinical and demographic characteristics and presence of OA, OP and related complications were compared by genotype, adjusting for age, sex, body mass index (BMI), current smoking and menopausal status. Results In total, 266 of the 306 patients (87%) were homozygous for C282Y, and 40 (13%) were compound heterozygous. The 2 groups did not differ by median age [60 (interquartile range [IQR] 53 to 68) vs. 61 (55 to 67) years, P=0.8], sex (female: 48.8% vs. 37.5%, P=0.18) or current smoking habits (12.4% vs. 10%, P=0.3). As compared with compound heterozygous patients, C282Y homozygous patients had higher median serum ferritin concentration at diagnosis [1090 (IQR 610 to 2210) vs. 603 (362 to 950) mu g/L, P<0.001], higher median transferrin saturation [80% (IQR 66 to 91%) vs. 63% (55 to 72%), P<0.001]) and lower median BMI [24.8 (22.1 to 26.9) vs. 26.2 (23.5 to 30.3) kg/m2, P<0.003]. The overall prevalence of self-reported OA was significantly higher with C282Y homozygosity than compound heterozygosity (53.4% vs. 32.5%; adjusted odds ratio [aOR] 2.4 [95% confidence interval 1.2-5.0]), as was self-reported OP (25.6% vs. 7.5%; aOR 3.5 [1.1-12.1]). Conclusion Patients with C282Y homozygosity may be at increased risk of musculoskeletal complications of HH.