Anti-VEGF-mediated barrier closure

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This project’s long-term goal is to elucidate how anti-VEGF (i.e. acute neutralization of VEGF (vascular endothelial growth factor)) stops leakage of blood vessels that are chronically exposed to excess VEGF. Anti- VEGF’s ability to curb blood vessel permeability has improved and/or sustained the quality of life of patients afflicted with pervasive, incurable diseases such as proliferative diabetic retinopathy (PDR), diabetic macular edema (DME) and neovascular age-related macular degeneration (nAMD). Such clinical observations demonstrate anti-VEGF’s efficacy, and beg the question of how anti-VEGF calms pathological blood vessels. We posit that chronically elevated VEGF alters gene expression in the endothelium of blood vessels in a way that relaxes the endothelial barrier, and that anti-VEGF reverses such changes. Aim 1. Test the hypothesis that anti-VEGF acts transcriptionally to close the barrier. Our preliminary data reveal that prolonged exposure to VEGF changes the expression of many genes, including those that encode known regulators of the endothelial cell barrier. These discoveries support our work hypothesis that anti-VEGF acts transcriptionally to close the barrier. This hypothesis will be tested as follows. The initial, RNAseq-based phase will compare the gene expression profile in cells that have or have not been treated with anti-VEGF and thereby identify anti-VEGF differentially expressed genes (DEGs). Candidates DEGs will be prioritized based on their known function, and then their contribution to VEGF-/anti- VEGF-mediated control of barrier function will be determined. We will identify genes that anti-VEGF depends on to close the endothelial cell barrier that has been breached with VEGF. Aim 2. Investigate the mechanism of action of anti-VEGF on patient-derived retinal endothelial cells. Aim 1 will be done with primary human retinal endothelial cells (HRECs) from a healthy adult donor. Aim 2 will be a repeat of aim 1, except using PRECs, retinal endothelial cells isolated from pathological blood vessels that develop in patients with PDR. This is the target cell type of anti-VEGF therapy. Determining the mechanism of action of anti-VEGF in these cells will provide clinically relevant information. Aim 3. Determine how anti-VEGF calms pathological blood vessels in patients. In aim 3 we will learn how anti-VEGF acts in patients by comparing the gene expression profile in freshly isolated endothelium from pathological blood vessels of treatment naïve and anti-VEGF-treated patients. In addition, we will compare the results from all 3 aims to determine which of the anti-VEGF-mediated effects that occur in patients are faithfully modeled by anti-VEGF treatment of cultured PRECs (aim 2) or HRECs (aim 1). This project will unveil the molecular mediators of anti-VEGF’s therapeutic benefit. Such information will remove current roadblocks to developing biomarkers and alternatives to anti-VEGF, which are needed to address the needs of patients afflicted with a variety of blinding conditions such as DME, PDR and nAMD.