Study of Matrix Gla Protein (MGP) deficiency in the zebrafish and its interaction with elastin: contribution to the study of Keutel syndrome

• Autor(a) principal: Nascimento, Pedro Manuel Fortes
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.1/19631
• topic: Calcificação ectópica; Sindrome keutel; Peixe-zebra; Proteína gla de matriz (mgp); Domínio/Área Científica::Ciências Sociais::Outras Ciências Sociais

The Keutel Syndrome (KS) is a rare autosomal recessive disorder that manifests in humans due to loss-of-function mutations in the MATRIX GLA PROTEIN (MGP) gene, which is correlated with ectopic calcification within cartilaginous and vascular tissues. The mechanisms by which MGP inhibits calcium deposition are still poorly understood, thus contributing to a major limitation in the comprehension of the disease pathophysiology and therefore the development of effective therapies. The Mgp-deficient mice model has been very used to favor the understanding of the disease. However, the symptomatology in Mgp-deficient mice is far more severe from what is observed in KS patients, which generates interest in the development of new animal models that can mimic the disease. The objectives of this work were to contribute to the establishment for the first time of a zebrafish model to study KS pathophysiology and to investigate whether MGP is a crucial inhibitor of vascular and cartilage calcification in the zebrafish model. For that, we identified and characterized an mgp zebrafish through RT-PCR and sequencing, and found an 18-nucleotide deletion at exon 4. Next, we performed a morphometric analysis at the larval and adult stages of development, where we found that the homozygous mgp mutant larvae are smaller than their wild-type siblings. To investigate the impact of the mutation in the process of ectopic mineralization, the zebrafish mutants were treated with warfarin, a potent inhibitor of the vitamin K cycle that induces ectopic calcification, and we found that the homozygous mutants die more. To determine the pathological accumulation of calcium, we performed Alizarin red S and von Kossa stainings, and we found that the mgp homozygous mutants have an extensive accumulation of calcium in the abdominal intervertebral space, caudal fin ray area, heart and parasphenoidal carotid artery. To confirm that these effects were due to the loss of expression of Mgp, we performed an immunohistochemistry analysis with an anti-BGP lab produced antibody and found a reduction in the expression of Mgp in the mgp mutants. In conclusion, we characterized a zebrafish mutant for mgp and found that they have a reduced size and extensive accumulation of calcium in bone and soft tissues. The mgp zebrafish mutant mimics the conditions observed in human KS patients suggesting that it might be a good model to study the pathophysiology of the KS.