Research Projects

Glaucoma is a leading cause of permanent blindness in India, with an estimated 11 million individuals affected [1]. Among its variants, neovascular glaucoma (NVG) is a particularly aggressive form, characterized by pathological neoangiogenesis in the anterior segment of the eye [2]. NVG remains poorly understood and largely untreatable. Retinal degeneration in NVG is thought to occur secondary to intraocular pressure (IOP) elevation, which in turn is driven by vascular remodeling in the anterior chamber of the eye. However, current anti-angiogenic therapies have shown limited efficacy in NVG. Thus, there is a need to understand the mechanisms of disease progression and identify new therapeutic strategies. To address this, we propose to use zebrafish as an in vivo model for studying NVG pathogenesis. Zebrafish share ~70% genetic similarity with humans and possess conserved ocular architecture, making them ideal for investigating eye development and disease [3,4]. We have identified casper zebrafish (a mutant lacking pigment due to mitfa and mpv17 mutations [5]), that displays progressive anterior segment hypervascularization mimicking features of human NVG. Using this model, we aim to test the hypothesis that anterior segment neoangiogenesis is initiated by local microenvironmental cues that trigger endothelial remodeling, leading ultimately to retinal dysfunction and vision loss. Preliminary single-cell RNA sequencing (scRNA-seq) of adult casper zebrafish eyes has revealed alterations in endothelial and stromal cell populations. Notably, components of the extracellular matrix, such as emilin2a, are upregulated in fibroblasts of mutant eyes. Since emilin2a is known to modulate angiogenesis [6], these findings provide a promising entry point to explore molecular mediators of disease. Multiple gene dysregulated in endothelium has also been identified. However, a more comprehensive profiling is necessary to uncover additional ligand-receptor interactions and signaling networks involved during the onset of neoangiogenesis. The project will begin with a spatiotemporal characterization of anterior segment angiogenesis and its correlation with changes in retinal layer structure and visual function. We will define the precise stages of onset and progression of vascular pathology and identify the time points most relevant for downstream analysis. Next, we will perform scRNA-seq on anterior segment tissues at early and late stages of vasculopathy in both wild-type and casper fish. Using computational tools such as Seurat and NicheNet, we will map cell-type-specific gene expression changes and infer intercellular signaling events [7,8]. Ligands overexpressed in stromal cells and their corresponding endothelial receptors will be identified as prime candidates. Candidates will then be tested in vivo through gain- and loss-of-function experiments: endothelial-specific overexpression and CRISPR-Cas9-mediated gene knockouts. These studies will establish the causal role of specific cues in driving anterior segment hypervascularization. To assess the conservation and translational relevance of these findings, we will test the identified pathways in human endothelial cell cultures. Functional assays, including tube formation, migration, and proliferation, will be used to examine angiogenic responses to the addition of ligands or receptor agonists identified from the zebrafish studies. These in vitro studies will serve as proof-of-concept for the therapeutic targeting of conserved angiogenic mechanisms identified in the zebrafish model. If successful, this project will offer significant insights into the cellular and molecular basis of anterior segment vascular pathology in NVG. It will define how microenvironmental signals sculpt the endothelial landscape, and reveal novel mediators of NVG. Ultimately, this work will expand our understanding of ocular vascular biology and support the development of therapeutic strategies for NVG.