Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Stephen Redenti

Committee Members

Hyungsik Lim

Derrick Brazill

Greg Phillips

Jean Hérbert

Subject Categories

Bioinformatics | Biology | Cell Biology | Life Sciences | Pharmacology | Systems Neuroscience

Keywords

Visual Neuroscience, Cell Migration and Invasion, Retinal Degeneration, Systems Pharmacology, Bioinformatics

Abstract

Disease or trauma induced loss of rod and cone photoreceptors is a major cause of vision loss worldwide. To replace lost photoreceptors and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal injection of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). A major obstacle in the field is that transplanted photoreceptor replacement cells exhibit extremely limited migration into host retina following injection into the subretinal space. In this work, we show that PPCs and host retinal interaction bioinformatics modeling can identify key pathways involved in photoreceptor replacement cell migration and invasion behaviors. In addition, we demonstrate that using systems pharmacology, small molecules and drugs can be targeted to upregulate receptors and signaling pathways enhancing replacement photoreceptor cell invasion and migration behaviors. Previously, we developed a comprehensive database of retinal ligand and PPC receptor pairs involved in migration. Ligand-receptor pairs significantly expressed and predicted to facilitate chemotactic migration included stromal derived factor-1/ C-X-C chemokine receptor type 4 (SDF-1/CXCR4), vascular endothelial growth factor/ Kinase insert domain receptor (VEGF/KDR) and Osteopontin/Integrin Alpha V (Osteopontin/ITGAV). In this work, we modeled the ligand-receptor activated cytoplasmic and nuclear signaling of SDF-1/CXCR4, VEGF/KDR, and Osteopontin/ITGAV to identify downstream mechanisms facilitating PPC and RPC migration. We then visualized these three target receptors on the surface of both PPCs and RPCs. In addition to analyzing migration associated signaling, transplantable PPC and RPC invasion mechanisms were investigated. We modeled receptor activated cytoplasmic and nuclear signaling for actin related protein 2/3 (ACTR2/3), cortactin (CCTN), cell division cycle 42 (CDC42), ezrin (EZR), matrix metallopeptidase 9 (MMP9), urokinase plasminogen activator surface receptor (PLAUR) and ras-related protein (RAB5A), to investigate mechanisms that may facilitate PPC and RPC invasion into retinal tissue. Next, we modulated signaling pathways using the systems pharmacologic database, Genebudger, to identify FDA approved drugs to enhance expression of PPC and RPC invasion and migration associated receptors. Viability was evaluated in RPCs treated with drugs and ligand-receptor driven PPC and RPC chemotactic migration was quantified using microfluidic chambers with steady-state ligand gradients of VEGF, SDF-1 and Osteopontin. Invasion was analyzed using Boyden chambers modified with extracellular matrix for cell invasion assays. The data supports a novel method of combined bioinformatics modeling and systems pharmacology that promotes transplantable photoreceptor replacement cell migration and invasion to improve cell-replacement and vision restoration.

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