Updating the role of matrix metalloproteinases in mineralized tissue and related diseases

Bone development and healing processes involve a complex cascade of biological events requiring well-orchestrated synergism with bone cells, growth factors, and other trophic signaling molecules and cellular structures. Beyond health processes, MMPs play several key roles in the installation of heart and blood vessel related diseases and cancer, ranging from accelerating metastatic cells to ectopic vascular mineralization by smooth muscle cells in complementary manner. The tissue inhibitors of MMPs (TIMPs) have an important role in controlling proteolysis. Paired with the post-transcriptional efficiency of specific miRNAs, they modulate MMP performance. If druggable, these molecules are suggested to be a platform for development of “smart” medications and further clinical trials. Thus, considering the pleiotropic effect of MMPs on mammals, the purpose of this review is to update the role of those multifaceted proteases in mineralized tissues in health, such as bone, and pathophysiological disorders, such as ectopic vascular calcification and cancer

Evaluation of cell viability and membrane transporters in kidney epithelial cells lineage treated of fluoride

O balanço do fluoreto (F) dentro do corpo é modulado por sua ingestão, absorção e remoção, e os rins, responsáveis pela sua excreção do organismo, são particularmente vulneráveis à toxicidade do F. Os efeitos nefrotóxicos do F envolvem mudanças estruturais marcantes nos rins, além disso, estudos proteomicos têm mostrado grandes alterações no perfil de proteínas envolvidas em pontos chave na transdução de sinal. Estes efeitos podem influenciar negativamente no transporte iônico nos rins. A vista deste fato, o transporte iônico no canal de sódio epitelial (ENaC) é limitante para a taxa de reabsorção de sódio nos rins, sendo essencial para a manutenção do equilíbrio eletrolítico e homeostase do corpo. Assim, este trabalho objetivou investigar os efeitos do F, utilizando concentrações semelhantes às que podem ser encontradas no néfron durante a fluorose dentária, na viabilidade celular e expressão de transportadores de membrana (ENaC) em linhagem celular renal M-1. Para os ensaios de viabilidade das células da linhagem M-1 foi empregado os testes colorimétricos Cristal Violeta e MTT, utilizando as concentrações de tratamento com fluoreto de sódio (NaF) a 10, 40, 100, 200 e 400 M, durante períodos experimentais de 24, 48, 72 e 96 h. As mesmas concentrações e os mesmos tempos foram utilizados no tratamento com cloreto de sódio (NaCl), utilizado como controle na possível interferência de Na+ na modulação das células. Para a investigação da influência do F: 1) nos canais ENaC foi utilizada a técnica de imunofluorescência e 2) para a análise de expressão gênica das subunidades que formam o ENaC, foi utilizada a técnica RT-PCR. Em nossos resultados pudemos observar que as maiores concentrações tanto de NaF quanto de NaCl provocaram a diminuição da viabilidade celular para ambos os ensaios de viabilidade, no entanto, foi possível observar algumas diferenças na resposta do tratamento com NaF em comparação com NaCl, por meio do ensaio Cristal Violeta. Não foi observado diferenças nas imagens de imunofluorescêcia, mas outros aspectos morfológicos foram vistos nessas imagens, como o aparecimento de domes celular, sugerindo que até mesmo a maior concentração de F não foi capaz de inibir a proliferação celular. Nosso resultado mais significativo foi em relação à expressão das subunidades dos canais de ENaC, onde a concentração de 400 M foi capaz de diminuir bruscamente a expressão das três subunidades do ENaC, enquanto as concentrações de 100 e 200 M mostraram apresentar expressão igual e em alguns casos até maior que o grupo controle. Pudemos concluir que doses de F na ordem de micromolares podem modular a expressão das subunidades formadoras do ENaC, positivamente quando em baixas concentrações e negativamente quando em concentrações elevadas.The balance of fluoride (F) within the body is modulated by its ingestion, absorption and removal, and the kidneys, responsible for their excretion of the organism, are particularly vulnerable to the toxicity of F. The nephrotoxic effects of F involve marked structural changes in the kidneys , in addition, proteomic studies have shown large changes in the profile of proteins involved in key points in signal transduction. These effects may negatively influence ion transport in the kidneys. In view of this fact, the ionic transport in the epithelial sodium channel (ENaC) is limiting to the rate of sodium reabsorption in the kidneys, being essential for the maintenance of the electrolyte balance and homeostasis of the body. Thus, the objective of this work was to investigate the effects of F, using concentrations similar to those found in the nephron during dental fluorosis, cell viability and expression of membrane transporters (ENaC) in renal cell line M-1. For the M-1 cell line viability assays, the Crystal Violet and MTT colorimetric assays were used, using the 10, 40, 100, 200 and 400 M sodium fluoride (NaF) treatment concentrations during experimental periods of 24, 48, 72 and 96 h. The same concentrations and the same times were used in the treatment with sodium chloride (NaCl), used as control in the possible interference of Na + in the modulation of the cells. For the investigation of the influence of F: 1) in the ENaC channels, the immunofluorescence technique was used and 2) for the analysis of gene expression of the subunits that form the ENaC, the RT-PCR technique was used. In our results it was observed that the higher concentrations of NaF and NaCl caused a decrease in cell viability for both viability assays, however, it was possible to observe some differences in NaF treatment response in comparison to NaCl, through test Crystal Violet. No differences were observed in immunofluorescence images, but other morphological aspects were seen in these images, such as the appearance of cellular \"domes\", suggesting that even the highest F concentration was not able to inhibit cell proliferation. Our most significant result was the expression of the subunits of the ENaC channels, where the concentration of 400 M was able to decrease expression of the three subunits of the ENaC, whereas the concentrations of 100 and 200 M showed equal expression and in some cases even higher than the control group. We can conclude that doses in the order of micromolar the F can modulate the expression of the ENaC forming subunits, positively when in low concentrations and negatively when in high concentrations

CSChighE-cadherinlow immunohistochemistry panel predicts poor prognosis in oral squamous cell carcinoma

Abstract Identifying marker combinations for robust prognostic validation in primary tumour compartments remains challenging. We aimed to assess the prognostic significance of CSC markers (ALDH1, CD44, p75NTR, BMI-1) and E-cadherin biomarkers in OSCC. We analysed 94 primary OSCC and 67 metastatic lymph node samples, including central and invasive tumour fronts (ITF), along with clinicopathological data. We observed an increase in ALDH1+/CD44+/BMI-1- tumour cells in metastatic lesions compared to primary tumours. Multivariate analysis highlighted that elevated p75NTR levels (at ITF) and reduced E-cadherin expression (at the tumour centre) independently predicted metastasis, whilst ALDH1high exhibited independent predictive lower survival at the ITF, surpassing the efficacy of traditional tumour staging. Then, specifically at the ITF, profiles characterized by CSChighE-cadherinlow (ALDH1highp75NTRhighE-cadherinlow) and CSCintermediateE-cadherinlow (ALDH1 or p75NTRhighE-cadherinlow) were significantly associated with worsened overall survival and increased likelihood of metastasis in OSCC patients. In summary, our study revealed diverse tumour cell profiles in OSCC tissues, with varying CSC and E-cadherin marker patterns across primary tumours and metastatic sites. Given the pivotal role of reduced survival rates as an indicator of unfavourable prognosis, the immunohistochemistry profile identified as CSChighE-cadherinlow at the ITF of primary tumours, emerges as a preferred prognostic marker closely linked to adverse outcomes in OSCC