Ação de heparinas na atividade antiangiogênica in vitro e in vivo: potencialização do efeito de anti-VEGF pela heparina 6-O-dessulfatada
Arquivos
Data
2024-11-07
Autores
Fetter, Bruna Zancanelli 
Orientadores
Dreyfuss, Juliana Luporini
Tipo
Tese de doutorado
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ISSN da Revista
Título de Volume
Resumo
A angiogênese, processo de formação de novos vasos sanguíneos a partir de uma vasculatura preexistente, desempenha um papel crucial em várias doenças oculares, como a Degeneração Macular Relacionada à Idade (DMRI). Seu processo é altamente controlado por uma interação complexa de fatores pró-angiogênicos e antiangiogênicos. A molécula reguladora pró-angiogênica mais estudada nesse processo é o fator de crescimento endotelial vascular (VEGF), que desempenha uma função importante na sobrevivência, proliferação e migração de células endoteliais. O Bevacizumabe (Bev), uma versão humanizada do anticorpo monoclonal anti-VEGF, foi desenvolvido para atuar na redução da angiogênese em várias doenças, como o câncer e neovascularização ocular. A heparina, um glicosaminoglicano sulfatado, também apresenta capacidade de se ligar e modular a atividade do VEGF, tendo demonstrado através de suas formas quimicamente modificadas um comportamento de fator antiangiogênico. Nesse contexto, o objetivo do trabalho foi avaliar o potencial antiangiogênico da heparina quimicamente modificada 6-O-dessulfatada (Hep6-O) e a possível potencialização da ação antiangiogênica da combinação de Hep6-O + Bev para o desenvolvimento de novas terapias antiangiogênicas. Para isso, Hep6-O foi modificada a partir da heparina suína não fracionada (HNF); em seguida, foi avaliada a atividade anticoagulante de HNF e Hep6-O. Para os ensaios in vitro, utilizaram-se as Células Endoteliais da Aorta de Coelho (RAEC) para investigar a viabilidade, proliferação, migração celular, formação de estruturas do tipo capilar e síntese de glicosaminoglicanos sulfatados (GAGs) após o tratamento com Hep6-O, Bev e Hep6-O + Bev. Os ensaios in vivo utilizaram o modelo de neovascularização de coroide induzida por laser (CNV) em ratos Zucker magros tratados com injeção intravítrea de Hep6-O, Bev e Hep6-O + Bev, analisados em microscópio de imunofluorescência (CEUA 9900220818). Além disso, estudos de Ressonância Plasmônica de Superfície (SPR) e Dicroísmo Circular (CD) foram conduzidos para avaliar a interação molecular e as alterações na estrutura secundária de fatores de crescimento (FGF-2, PDGF-BB e VEGF-165) frente a HNF, Hep6-O, Bev e suas combinações. Os resultados indicam que Hep6-O apresentou baixa atividade anticoagulante quando comparada à HNF. Os estudos in vitro mostraram que RAECs tratadas com Hep6-O, Bev e Hep6-O + Bev não apresentaram citotoxicidade em nenhuma concentração testada. Por outro lado, Hep6-O apresentou efeito antiangiogênico, sendo este efeito potencializado por Hep6-O + Bev quando comparado com Hep6-O ou Bev isolados, levando a uma redução significativa na proliferação, formação de estruturas tipo tubo capilar e migração das células endoteliais em todas as concentrações testadas quando comparadas ao controle. Em relação a síntese de GAGS, Hep6-O + Bev estimulou a biossíntese de GAGs sulfatados, principalmente o heparam sulfato secretado para o meio de cultura, sendo este aumento maior quando comparado aos tratamentos isolados com Hep6-O ou Bev. Os estudos in vivo mostraram uma redução significativa na área de neovascularização de coróide após 14 dias de injeção intravítrea de Hep6-O e Bev quando comparadas ao controle (salina), sendo este efeito potencializado por Hep6-O + Bev. Os dados de SPR mostraram que dependendo do fator de crescimento, há ligação entre as heparinas e o domínio de ligação aos fatores de crescimento aqui avaliados, enquanto os espectros de CD indicaram mudanças conformacionais nas proteínas e modulação de suas atividades. Com isso conclui-se que Hep6-O apresenta características de fator antiangiogênico, potencializando a ação do Bev, sugerindo uma possível sinergia entre as duas moléculas na inibição da angiogênese. Hep6-O não apenas mantém suas propriedades antiangiogênicas, mas também apresenta baixa atividade anticoagulante, o que pode reduzir os riscos associados a terapias prolongadas. Os ensaios de interação molecular mostraram que Hep6-O modula a ligação do VEGF e do FGF aos seus receptores, interferindo na ativação desses fatores e inibindo a angiogênese de forma mais eficaz quando em combinação com Bev. Estes achados sugerem que a coadministração de Hep6-O + Bev pode representar uma estratégia promissora para o tratamento de doenças neovasculares, como a DMRI. Estudos clínicos futuros são necessários para validar os efeitos terapêuticos dessa combinação e explorar o seu potencial completo.
Angiogenesis, the process of new blood vessels from pre-existing vasculature, plays a crucial role in several ocular diseases, such as Age-Related Macular Degeneration (AMD). This process is tightly regulated by a complex interaction between pro-angiogenic and anti-angiogenic factors. The most studied pro-angiogenic regulatory molecule is vascular endothelial growth factor (VEGF), which is critically involved in endothelial cell survival, proliferation, and migration. Bevacizumab (Bev), a humanized monoclonal anti-VEGF antibody, was developed to reduce angiogenesis in various diseases, including cancer and ocular neovascularization. Heparin, a sulfated glycosaminoglycan, also has the capacity to bind and modulate VEGF activity, demonstrating anti-angiogenic properties through its chemically modified forms. In this context, the aim of this study was to evaluate the anti-angiogenic potential of chemically modified 6-O-desulfated heparin (Hep6-O) and the possible enhancement of the anti-angiogenic effect of the Hep6-O + Bev combination for the development of novel anti-angiogenic therapies. To achieve this, Hep6-O was derived from unfractionated porcine heparin (UFH); subsequently, the anticoagulant activity of UFH and Hep6-O was assessed. For in vitro assays, Rabbit Aortic Endothelial Cells (RAEC) were used to investigate cell viability, proliferation, migration, tube formation, and sulfated glycosaminoglycan (GAG) synthesis after treatment with Hep6-O, Bev, and Hep6-O + Bev. In vivo assays employed a laser-induced choroidal neovascularization (CNV) model in lean Zucker rats treated with intravitreal injections of Hep6-O, Bev, and Hep6-O + Bev, analyzed through immunofluorescence microscopy (CEUA 9900220818). Additionally, Surface Plasmon Resonance (SPR) and Circular Dichroism (CD) studies were conducted to assess molecular interactions and secondary structure changes in growth factors (FGF-2, PDGF-BB, and VEGF-165) in response to UFH, Hep6-O, Bev, and their combinations. The results indicate that Hep6-O exhibited lower anticoagulant activity compared to UFH. In vitro studies showed that RAECs treated with Hep6-O, Bev, and Hep6-O + Bev did not exhibit cytotoxicity at any tested concentration. On the other hand, Hep6-O showed anti-angiogenic effects, which were enhanced by Hep6-O + Bev when compared to Hep6-O or Bev alone, leading to a significant reduction in endothelial cell proliferation, tube formation, and migration across all tested concentrations compared to the control. Regarding GAG synthesis, Hep6-O + Bev stimulated the biosynthesis of sulfated GAGs, primarily heparan sulfate secreted into the culture medium, with this increase being more pronounced compared to treatments with Hep6-O or Bev alone. The in vivo studies demonstrated a significant reduction in the area of choroidal neovascularization after 14 days of intravitreal injection of Hep6-O and Bev compared to the control (saline), with this effect being enhanced by Hep6-O + Bev. SPR data showed that, depending on the growth factor, there was binding between the heparins and the growth factor binding domains evaluated here, while CD spectra indicated conformational changes in the proteins and modulation of their activities. These findings suggest that Hep6-O exhibits anti-angiogenic properties, enhancing the action of Bev, indicating a possible synergy between the two molecules in inhibiting angiogenesis. Hep6-O not only retains its anti-angiogenic properties but also demonstrates low anticoagulant activity, which may reduce the risks associated with prolonged therapies. Molecular interaction assays revealed that Hep6-O modulates the binding of VEGF and FGF to their receptors, interfering with the activation of these factors and inhibiting angiogenesis more effectively when combined with Bev. These findings suggest that the co-administration of Hep6-O + Bev may represent a promising strategy for treating neovascular diseases such as AMD. Future clinical studies are necessary to validate the therapeutic effects of this combination and explore its full potential.
Angiogenesis, the process of new blood vessels from pre-existing vasculature, plays a crucial role in several ocular diseases, such as Age-Related Macular Degeneration (AMD). This process is tightly regulated by a complex interaction between pro-angiogenic and anti-angiogenic factors. The most studied pro-angiogenic regulatory molecule is vascular endothelial growth factor (VEGF), which is critically involved in endothelial cell survival, proliferation, and migration. Bevacizumab (Bev), a humanized monoclonal anti-VEGF antibody, was developed to reduce angiogenesis in various diseases, including cancer and ocular neovascularization. Heparin, a sulfated glycosaminoglycan, also has the capacity to bind and modulate VEGF activity, demonstrating anti-angiogenic properties through its chemically modified forms. In this context, the aim of this study was to evaluate the anti-angiogenic potential of chemically modified 6-O-desulfated heparin (Hep6-O) and the possible enhancement of the anti-angiogenic effect of the Hep6-O + Bev combination for the development of novel anti-angiogenic therapies. To achieve this, Hep6-O was derived from unfractionated porcine heparin (UFH); subsequently, the anticoagulant activity of UFH and Hep6-O was assessed. For in vitro assays, Rabbit Aortic Endothelial Cells (RAEC) were used to investigate cell viability, proliferation, migration, tube formation, and sulfated glycosaminoglycan (GAG) synthesis after treatment with Hep6-O, Bev, and Hep6-O + Bev. In vivo assays employed a laser-induced choroidal neovascularization (CNV) model in lean Zucker rats treated with intravitreal injections of Hep6-O, Bev, and Hep6-O + Bev, analyzed through immunofluorescence microscopy (CEUA 9900220818). Additionally, Surface Plasmon Resonance (SPR) and Circular Dichroism (CD) studies were conducted to assess molecular interactions and secondary structure changes in growth factors (FGF-2, PDGF-BB, and VEGF-165) in response to UFH, Hep6-O, Bev, and their combinations. The results indicate that Hep6-O exhibited lower anticoagulant activity compared to UFH. In vitro studies showed that RAECs treated with Hep6-O, Bev, and Hep6-O + Bev did not exhibit cytotoxicity at any tested concentration. On the other hand, Hep6-O showed anti-angiogenic effects, which were enhanced by Hep6-O + Bev when compared to Hep6-O or Bev alone, leading to a significant reduction in endothelial cell proliferation, tube formation, and migration across all tested concentrations compared to the control. Regarding GAG synthesis, Hep6-O + Bev stimulated the biosynthesis of sulfated GAGs, primarily heparan sulfate secreted into the culture medium, with this increase being more pronounced compared to treatments with Hep6-O or Bev alone. The in vivo studies demonstrated a significant reduction in the area of choroidal neovascularization after 14 days of intravitreal injection of Hep6-O and Bev compared to the control (saline), with this effect being enhanced by Hep6-O + Bev. SPR data showed that, depending on the growth factor, there was binding between the heparins and the growth factor binding domains evaluated here, while CD spectra indicated conformational changes in the proteins and modulation of their activities. These findings suggest that Hep6-O exhibits anti-angiogenic properties, enhancing the action of Bev, indicating a possible synergy between the two molecules in inhibiting angiogenesis. Hep6-O not only retains its anti-angiogenic properties but also demonstrates low anticoagulant activity, which may reduce the risks associated with prolonged therapies. Molecular interaction assays revealed that Hep6-O modulates the binding of VEGF and FGF to their receptors, interfering with the activation of these factors and inhibiting angiogenesis more effectively when combined with Bev. These findings suggest that the co-administration of Hep6-O + Bev may represent a promising strategy for treating neovascular diseases such as AMD. Future clinical studies are necessary to validate the therapeutic effects of this combination and explore its full potential.
Descrição
Citação
FETTER, Bruna Zancanelli. Ação de heparinas na atividade antiangiogênica in vitro e in vivo: potencialização do efeito de anti-VEGF pela Heparina 6-O-dessulfatada. 2024. 203 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Univesidade Federal de São Paulo (UNIFESP). São Paulo, 2024.