Estudo dos efeitos dos bifosfonatos na angiogénese/osteogénese do tecido ósseo: avaliação in vitro em co-culturas de osteoblastos e células endoteliais
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2013-11-07
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A relação entre os processos de osteogénese e angiogénese, regulada pelas interacções existentes entre as células osteoblásticas e as células endoteliais, respectivamente, é crucial para a manutenção da integridade do tecido ósseo. A formação óssea é sempre precedida pela invasão vascular, uma vez que a osteogénese ocorre na proximidade dos vasos sanguíneos recentemente formados. Desta forma, as propriedades anti-angiogénicas dos bifosfonatos (BPs) podem ter efeitos directos na actividade das células osteoblásticas, contribuindo para a diminuição da formação óssea. No entanto, o exacto mecanismo pelo qual os bifosfonatos afectam as interacções existentes entre estes dois processos no tecido ósseo é desconhecido.
No presente estudo, analisamos os efeitos do alendronato e do zoledronato no comportamento de células osteoblásticas e de células endoteliais humanas, em sistema de monocultura e co-cultura.
As células da linha osteoblástica humana MG63 e as células endoteliais humanas, provenientes da microvascularização dérmica (HDMECs), foram cultivadas durante 14 dias, em meio de cultura específico, na ausência (controlo) e na presença do alendronato e do zoledronato, nas concentrações de 10-12, 10-10, 10-8 e 10-6 M. Avaliou-se a viabilidade/proliferação celular pelo ensaio colorimétrico MTT, o impacto dos compostos nas características fenotípicas celulares foi avaliado pela visualização celular, utilizando uma coloração imunofluorescente específica, seguida de análise por microscopia laser confocal (CLSM). Recorrendo à técnica de RT-PCR fez-se a caracterização das culturas quanto à expressão génica dos marcadores osteoblásticos ALP, BMP-2 e OPG, e dos marcadores endoteliais CD31 e CAD-VE.
Os resultados obtidos demonstram que as células MG63 em monocultura apresentaram uma proliferação gradual e expressaram os marcadores associados ao fenótipo osteoblástico. As monoculturas de células endoteliais apresentaram um padrão de crescimento típico, com expressão da molécula de adesão CD31. As células MG63 e HDMECs em co-cultura apresentaram uma proliferação crescente e expressaram os marcadores associados ao fenótipo osteoblástico e endotelial. O alendronato e o zoledronato, em todas as concentrações testadas, não interferiram com a proliferação das células MG63, estimulando para as concentrações testadas (10-12 e 10-6 M), a expressão dos marcadores osteoblásticos. O mesmo padrão foi observado para a proliferação das células endoteliais, no entanto, estes compostos causaram uma inibição da expressão dos seus marcadores. Em sistema de co-cultura, o alendronato e o zoledronato, causaram efeitos similares aos obtidos para as células osteoblásticas e para as células endoteliais, cultivadas individualmente.
Assim, através da realização deste estudo, pode concluir-se que a inibição da expressão dos marcadores endoteliais estabelecidos, na presença de concentrações altas e baixas de alendronato e de zoledronato, pode constituir um mecanismo contributivo para a inibição da angiogénese, com eventuais repercussões no processo de formação óssea. Além disso, a exposição destes compostos em sistema de co-cultura de células osteoblásticas e células endoteliais, pode ser importante para clarificar os efeitos dos bifosfonatos na relação entre os processos de osteogénese e angiogénese, in vivo.
The interplay between osteogenesis and angiogeneses, regulated by the interactions between osteoblastic and endothelial cells, respectively, is extremely important to maintain skeletal integrity. Bone formation is always preceded by vascular invasion and osteogenesis occurs in the vicinity of the new blood vessels. Thus, the anti-angiogenic properties of bisphosphonates may have direct effects on osteoblastic cells activity, decreasing bone formation events. However, the exact mechanism as bisphosphonates affect the interplay between these two processes in bone tissue is unknown. This study describes the effects of alendronate and zoledronate on human osteoblastic and endothelial cells behavior, in monoculture and co-culture system. MG63 osteoblastic-like cells and human dermal microvascular endothelial cells (HDMECs), were cultured for 14 days, in specific culture medium, in the absence (control) and in the presence of alendronate and zoledronate, 10-12, 10-10, 10-8 e 10-6 M. Cell viability/proliferation was evaluated by the MTT colorimetric assay, the impact on phenotypic characteristics was assessed by cell visualization, using a specific immunofluorescent staining followed by confocal laser scanning microscopy (CLSM) and the gene expression characterization of osteoblast and endothelial-associated markers, ALP, BMP-2, OPG, CD31 and CAD-VE, was made by RT-PCR. The results show that monocultures of MG63 cells proliferated gradually and expressed osteoblast-associated markers. Monocultures of endothelial cells presented a typical pattern of cell growth and expressed the adhesion molecule CD31. Co-cultures of MG63 and HDMECs showed an increased proliferation and expressed osteoblast and endothelial-associated markers. Alendronate and zoledronate, at all tested concentrations did not interfere with MG63 cells proliferation, however, at concentrations of 10-12 and 10-6 M stimulated the osteoblastic markers expression. The same pattern was observed for endothelial cells proliferation, however, these compounds caused inhibition of the endothelial markers expression. In co-culture system, alendronate and zoledronate caused similar effects to those obtained for osteoblastic and endothelial cells in individual cultures. Thus, it can be concluded that the inhibition of the endothelial markers expression, in the presence of low and high concentrations of alendronate and zoledronate, might be a contributing mechanism to an impairment of the angiogenesis, with an eventual repercussion in the bone formation process. Furthermore, these compounds exposure in co-culture system of osteoblastic and endothelial cells may be important to clarify the effects of bisphosphonates in the interplay between osteogenesis and angiogenesis in vivo.
The interplay between osteogenesis and angiogeneses, regulated by the interactions between osteoblastic and endothelial cells, respectively, is extremely important to maintain skeletal integrity. Bone formation is always preceded by vascular invasion and osteogenesis occurs in the vicinity of the new blood vessels. Thus, the anti-angiogenic properties of bisphosphonates may have direct effects on osteoblastic cells activity, decreasing bone formation events. However, the exact mechanism as bisphosphonates affect the interplay between these two processes in bone tissue is unknown. This study describes the effects of alendronate and zoledronate on human osteoblastic and endothelial cells behavior, in monoculture and co-culture system. MG63 osteoblastic-like cells and human dermal microvascular endothelial cells (HDMECs), were cultured for 14 days, in specific culture medium, in the absence (control) and in the presence of alendronate and zoledronate, 10-12, 10-10, 10-8 e 10-6 M. Cell viability/proliferation was evaluated by the MTT colorimetric assay, the impact on phenotypic characteristics was assessed by cell visualization, using a specific immunofluorescent staining followed by confocal laser scanning microscopy (CLSM) and the gene expression characterization of osteoblast and endothelial-associated markers, ALP, BMP-2, OPG, CD31 and CAD-VE, was made by RT-PCR. The results show that monocultures of MG63 cells proliferated gradually and expressed osteoblast-associated markers. Monocultures of endothelial cells presented a typical pattern of cell growth and expressed the adhesion molecule CD31. Co-cultures of MG63 and HDMECs showed an increased proliferation and expressed osteoblast and endothelial-associated markers. Alendronate and zoledronate, at all tested concentrations did not interfere with MG63 cells proliferation, however, at concentrations of 10-12 and 10-6 M stimulated the osteoblastic markers expression. The same pattern was observed for endothelial cells proliferation, however, these compounds caused inhibition of the endothelial markers expression. In co-culture system, alendronate and zoledronate caused similar effects to those obtained for osteoblastic and endothelial cells in individual cultures. Thus, it can be concluded that the inhibition of the endothelial markers expression, in the presence of low and high concentrations of alendronate and zoledronate, might be a contributing mechanism to an impairment of the angiogenesis, with an eventual repercussion in the bone formation process. Furthermore, these compounds exposure in co-culture system of osteoblastic and endothelial cells may be important to clarify the effects of bisphosphonates in the interplay between osteogenesis and angiogenesis in vivo.
Descrição
Dissertação de Mestrado em Biologia Clínica Laboratorial.
Palavras-chave
Bifosfonatos , Osteoblastos , Células endoteliais , Co-culturas