Angiogenesis in ocular neovascular diseases : from multitarget inhibition to gene therapy tools

Ocular neovascular diseases, such as neovascular age-related macular degeneration (nAMD) or proliferative retinopathies (PRs) are characterized by the growth of abnormal blood vessels in the eye, leading to vision impairment. Hypoxia-mediated responses trigger angiogenesis, the growth of new blood vessels from existing ones, a pivotal feature for the development of these diseases. Hypoxia-inducible factors (HIFs) and its target genes regulate various aspects of the angiogenic cascade. The vascular endothelial growth factor (VEGF) is one of the main initiators of the angiogenic process, and the urokinase plasminogen activator receptor (uPAR), is responsible for the extracellular matrix (ECM) remodeling, allowing endothelial cell migration. Due to angiogenesis complexity, current treatments for ocular neovascular diseases present various limitations, creating a need for novel therapies. In this thesis, I investigated novel approaches of treatments for ocular neovascular diseases by pursuing a multitarget inhibition strategy along with evaluating specific tools for the targeted delivery of gene therapy constructs in the eye.

In Paper I we investigated the inhibition of hypoxia-induced angiogenesis by antagonizing the uPA/uPAR system with UPARANT using a novel ex vivo human iris organotypic cultures model. We discovered an unidentified endothelial-specific antagonistic effect of UPARANT on the uPA/uPAR system by disrupting the interaction between uPAR and the low-density lipoprotein receptor-related protein-1 (LRP-1). This led to the inhibition of _-catenin-mediated angiogenesis through the blockage of the uPA/uPAR system, rather than through VEGF inhibition. These findings expanded our understanding of the effects of uPA/uPAR system antagonism in the context of ocular angiogenesis.

In Paper II we evaluated the efficacy of echinomycin, an inhibitor of HIF-1_ DNAbinding activity, in reducing hypoxia-driven angiogenesis for the first time in the eye. We examined the effects of echinomycin in vitro on retinal cells, and in vivo using a murine model of laser-induced choroidal neovascularization (CNV). The inhibition of HIF-1_ led to a decreased response of retinal cells to hypoxia, and a reduction of vascular area in CNV-induced mice. These data suggested that targeting HIF-1_ is pivotal for future implications in the treatment of patients with ocular neovascular diseases.

In Paper III we aimed to establish a protocol for the redifferentiation of ARPE-19 cells into a more mature retinal pigment epithelium (RPE) phenotype, to improve the evaluation of RPE-specific gene therapy strategies. The combination of laminin (LN) 521 coating with nicotinamide (NAM) supplementation promoted the reorganization and expression of mature RPE signature proteins and genes, leading ARPE-19 closer to their in vivo phenotype. Redifferentiated ARPE-19 transduced adeno associated viral vectors (AAVs) at low multiplicity of infection, mimicking in vivo AAV tropism, essential to test new gene therapies for RPE-centered diseases.

List of scientific papers

I. Pesce NA*, Plastino F*, Reyes-Goya C, Bernd J, Pavone V, Kvanta A, Dal Monte M, Locri F#, AndrŽ H#. Mitigation of human iris angiogenesis through uPAR/LRP-1 interaction antagonism in an organotypic ex vivo model. FASEB J. 2024 Mar 15;38(5):e23533. *Co-first authors. #Co-last authors.
https://doi.org/10.1096/fj.202301892RR

II. Plastino F*, Santana-Garrido ç*, Pesce NA, Aronsson M, Lardner E, Mate A, Kvanta A, V‡zquez CM, AndrŽ H. Echinomycin mitigates ocular angiogenesis by transcriptional inhibition of the hypoxia-inducible factor-1. Exp Eye Res. 2021 May;206:108518. *Co-first authors.
https://doi.org/10.1016/j.exer.2021.108518

III. Bernd J, Plastino F, Karayannis J J, Kvanta A, Filippo Locri#, Helder AndrŽ#. Accelerated maturation of ARPE-19 cells for the translational assessment of gene therapy. #Co-last authors. [Submitted]