Sinalização parácrina mediada por proteínas e microvesículas entre células ósseas e seu microambiente
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Data
2021-04-08
Autores
Fernandes, Célio Junior da Costa
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Compreender os mecanismos envolvidos na manutenção e homeostase óssea requer elucidar o comportamento de células em seu microambiente. O objetivo deste estudo foi investigar como células ósseas (MC3t3-E1, HOEL) ou mesenquimais são moduladas através da sinalização parácrina desencadeada por fibroblastos (3t3-NIH), células endoteliais de veias (HUVEC) e de aorta (HCAEC), além de células de músculo liso (AoSMC/VSMC). Para avaliar essa capacidade de modulação de células ósseas, fibroblastos, células endoteliais e de musculatura lisa foram mantidas em cultura por 72 horas com o intuito de condicionar o meio, o qual foi coletado e utilizado para tratar MC3T3 ou HOEL ou células mesenquimais, com o intuito de fazer uso de ferramentas de biologia celular e molecular para investigar alterações de expressão gênica e fenotípica. Nossos resultados mostram que 3T3-NIH é capaz de modular a expressão de marcadores de diferenciação osteoblasto-osteócito (DMP1. SOST, RankL e OPG). Além disso, vimos que AoSMC em duas condições distintas - Estatico e Shear-Stress - são capazes de promover uma transição osteoblasto-osteócito através de vesículas extracelulares (EVs), ativando a expressão de biomarcadores moleculares deste processo (miR23-a, DMP1, SOST, FGF23 e GP38) e, neste contexto, nossos dados também mostram que o meio condicionado por HUVEC modula marcadores de diferenciação de osteoblastos (Runx2, Osterix e ALP) e genes que são traduzidos em proteínas e que atuam na homeostase dos minerais da matriz óssea (NPP1 e TNAP). Considerando que as células AoSMC e HUVEC são capazes de modular a osteocitogênese e a osteoblastogênese, respectivamente, o meio condicionado por essas células foi coletado e as vesículas foram extraídas coletadas para melhor caracterização em termos de tamanho, concentração, biomarcadores de membrana, carga elétrica de membrana e conteúdo proteico, o que revelou que o perfil AoSMC não apresentou diferença em termos de condições experimentais (estática e Shear-Stress), também vimos que as células mesenquimais que recebem o tratamento com meio de cultura condicionado por AoSMC aumentou os marcadores de osteocitogênese, enquanto os EVs isolados do meio condicionado por HUVEC modularam positivamente esses mesmos marcadores (PHEX, MEPE, MMP-14, GP-38, DMP1). Esses dados permitem concluir que o tecido endotelial e os fibroblastos contribuem para a homeostase do osso e sua remodelação dinâmica é modulada pela transição osteoblastos-osteócitos por meio de fatores tróficos e vesículas extracelulares.
Understanding the mechanisms involved in bone maintenance and homeostasis requires elucidating the behavior of bone tissue in relation to communication with other adjacent tissues. The aim of this study was to investigate how bone cells (MC3t3-E1, HOEL) or mesenchymal cells are modulated through paracrine signaling triggered by fibroblasts (3t3-NIH), vein endothelial cells (HUVEC) and aortic endothelial cells (HCAEC), in addition to smooth muscle cells (AoSMC/VSMC). The cells of the tissues adjacent to the bone remained in culture for 72 hours to condition the medium, this same medium was collected and used to treat MC3t3 or HOEL or mesenchymal cells. Our results show that 3t3-NIH is capable of modulating the expression of osteoblast-osteocyte differentiation markers (DMP1. SOST, RankL and OPG). In addition, we saw that AoSMC in two different conditions: Static and Shear-Stress, are capable of promoting an osteoblast-osteocyte transition through extracellular vesicles (EVs), activating the expression of molecular biomarkers of this process (miR23-a, DMP1, SOST, FGF23 and GP38), in this context our data also show that the medium conditioned by HUVEC modulates osteoblast differentiation markers (Runx2, Osterix and ALP) and genes that are translated into proteins and that act in the homeostasis of bone matrix minerals (NPP1 and TNAP). Considering that AoSMC and HUVEC cells are capable of modulating osteocytogenesis and osteoblastogenesis, respectively, the medium conditioned by these cells was collected and the vesicles were extracted for treatment: 1. medium rich in vesicles and other trophic factors, and 2. treatment only with vesicles. EVs were characterized in terms of size, concentration, membrane biomarkers, membrane electrical charge and protein content, which revealed that the AoSMC profile showed no difference in terms of experimental conditions (static and Shear-Stress), we also saw that the mesenchymal cells receiving treatment with AoSMC-conditioned culture medium increased osteocytogenesis markers, while EVs isolated from HUVEC-conditioned medium positively modulated these same markers (PHEX, MEPE, MMP-14, GP-38, DMP1). These data allow us to conclude that the endothelial tissue and fibroblasts contribute to bone balance by modulating the osteoblast-osteocyte transition through trophic factors and extracellular vesicles.
Understanding the mechanisms involved in bone maintenance and homeostasis requires elucidating the behavior of bone tissue in relation to communication with other adjacent tissues. The aim of this study was to investigate how bone cells (MC3t3-E1, HOEL) or mesenchymal cells are modulated through paracrine signaling triggered by fibroblasts (3t3-NIH), vein endothelial cells (HUVEC) and aortic endothelial cells (HCAEC), in addition to smooth muscle cells (AoSMC/VSMC). The cells of the tissues adjacent to the bone remained in culture for 72 hours to condition the medium, this same medium was collected and used to treat MC3t3 or HOEL or mesenchymal cells. Our results show that 3t3-NIH is capable of modulating the expression of osteoblast-osteocyte differentiation markers (DMP1. SOST, RankL and OPG). In addition, we saw that AoSMC in two different conditions: Static and Shear-Stress, are capable of promoting an osteoblast-osteocyte transition through extracellular vesicles (EVs), activating the expression of molecular biomarkers of this process (miR23-a, DMP1, SOST, FGF23 and GP38), in this context our data also show that the medium conditioned by HUVEC modulates osteoblast differentiation markers (Runx2, Osterix and ALP) and genes that are translated into proteins and that act in the homeostasis of bone matrix minerals (NPP1 and TNAP). Considering that AoSMC and HUVEC cells are capable of modulating osteocytogenesis and osteoblastogenesis, respectively, the medium conditioned by these cells was collected and the vesicles were extracted for treatment: 1. medium rich in vesicles and other trophic factors, and 2. treatment only with vesicles. EVs were characterized in terms of size, concentration, membrane biomarkers, membrane electrical charge and protein content, which revealed that the AoSMC profile showed no difference in terms of experimental conditions (static and Shear-Stress), we also saw that the mesenchymal cells receiving treatment with AoSMC-conditioned culture medium increased osteocytogenesis markers, while EVs isolated from HUVEC-conditioned medium positively modulated these same markers (PHEX, MEPE, MMP-14, GP-38, DMP1). These data allow us to conclude that the endothelial tissue and fibroblasts contribute to bone balance by modulating the osteoblast-osteocyte transition through trophic factors and extracellular vesicles.
Descrição
Palavras-chave
Endotelio, Cross-talk, Vesículas Extracelulares, Osteócito, Endothelial tissue, Cross-talk, Extracellular vesicles, Osteocytes