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PlGF silencing combined with PEDF overexpression: Modeling RPE secretion as potential therapy for retinal neovascularization

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Abstract

Ocular neovascularization is a defining feature of several blinding diseases. We have previously described the effectiveness of long-term pigment epithelium-derived factor (PEDF) expression in the retina of diabetic mice in ameliorating some diabetic retinopathy hallmarks. In this study, we aimed to investigate if the antiangiogenic potential of PEDF overexpression was enhanced in combination with placental growth factor (PlGF) silencing. Human RPE cells were transfected with a self-replicating episomal vector (pEPito) for PEDF overexpression and/or a siRNA targeting PlGF gene. Conditioned media from PEDF overexpression, from PlGF inhibition and from their combination thereof were used to culture human umbilical vein endothelial cells, and their proliferation rate, migration capacity, apoptosis and ability to form tube-like structures were analyzed in vitro. We here demonstrate that pEPito-driven PEDF overexpression in combination with PlGF silencing in RPE cells does not affect their viability and results in an enhanced antiangiogenic activity in vitro. We observed a significant decrease in the migration and proliferation of endothelial cells, and an increase in apoptosis induction as well as a significant inhibitory effect on tube formation. Our findings demonstrate that simultaneous PEDF overexpression and PlGF silencing strongly impairs angiogenesis compared with the single approaches, providing a rationale for combining these therapies as a new treatment for retinal neovascularization.

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Acknowledgements

The authors acknowledge the financial support of Fundação para a Ciência e Tecnologia (Grant No. SFRH/BD/114051/2016 individual fellowship to Rute Araújo), Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT), Grant No. 02/SAICT/2017/028121 (to Gabriela A. Silva), and the Marie Curie Reintegration Grant (Grant No. PIRG-GA-2009-249314 to Gabriela A. Silva) under the FP7 program. iNOVA4Health—UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia / Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is also acknowledged.

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Authors and Affiliations

  1. CEDOC – Chronic Diseases Research Center, NOVA Medical School, Universidade Nova de Lisboa, Campo Mártires da Pátria 130, 1169-056, Lisboa, Portugal

    Rute S. Araújo & Gabriela A. Silva

  2. Bioengineering- Cell Therapies and Regenerative Medicine PhD Program, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal

    Rute S. Araújo

  3. NOVA Medical School, Universidade Nova de Lisboa, Campo Mártires da Pátria 130, 1169-056, Lisboa, Portugal

    Gabriela A. Silva

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Correspondence to Gabriela A. Silva.

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Araújo, R.S., Silva, G.A. PlGF silencing combined with PEDF overexpression: Modeling RPE secretion as potential therapy for retinal neovascularization. Mol Biol Rep 47, 4413–4425 (2020). https://doi.org/10.1007/s11033-020-05496-2

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