ORCID
https://orcid.org/0000-0002-7151-0011
Date of Award
12-18-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss among the elderly, affecting nearly 200 million people worldwide. AMD impacts the macula, which is the small, central area of the retina responsible for high-acuity vision. The exudative (wet) form of late-stage AMD is the most severe, accounting for ninety percent of the legally blind patients with AMD. Wet AMD is characterized by the abnormal growth of blood vessels and fluid leakage beneath the retina known as choroidal neovascularization (CNV), directly resulting in vision loss. While anti-VEGFa targeted therapies have been the gold standard for ameliorating wet AMD for the past two decades, patients can become unresponsive to these therapies over time. Therefore, further investigation into the molecular mechanisms of CNV is urgently needed to develop novel therapies. Given that advanced age is the primary risk factor for wet AMD, we focused on the highly conserved microRNA miR-34a, which becomes upregulated with age in several tissues in both mice and humans and exacerbates pathology in various age-related disease models. Moreover, a recent study found miR-34a was the most upregulated microRNA in both the whole retina and central macula of patients with AMD. Additionally, miR-34a is known to inhibit several genes that are involved in angiogenesis, epithelial-to-mesenchymal transition, immune response, and cellular senescence, all processes associated with wet AMD. Thus, we hypothesized that miR-34a may play a significant role in the progression of CNV as seen in wet AMD. In this study, we used a well-established in vivo laser-injury mouse model of wet AMD to investigate mechanisms of pathological ocular neovascularization. We found that miR-34a was upregulated in the vascular endothelial cells of murine CNV lesions after laser injury. Using both global miR-34a knockout mice (miR-34a-/-) and vascular endothelial-specific knockout mice, we demonstrated that miR-34a promotes CNV in both contexts. Interestingly, upregulating miR-34a in vitro and knocking it out in vivo did not impact the expression of VEGFa or its receptor KDR. To identify direct targets of miR-34a in vascular endothelial cells, we used an integrative transcriptomic approach combining our bulk RNA-sequencing data of transfected human vascular endothelial cells with a publicly available single-cell RNA-sequencing dataset of murine CNV lesions. This analysis identified the potently anti-angiogenic Krüppel-like factor 2 (KLF2) as a possible target of miR-34a, which we validated via dual-luciferase assays. Additionally, miR-34a-/- mice exhibited significantly reduced KLF2 expression in CNV lesions. Moreover, we discovered that suppressing KLF2 resulted in the upregulation of the proangiogenic C-X-C chemokine receptor type 4 (CXCR4) and its ligand stromal derived factor 1 (SDF-1, or CXCL12) in vascular endothelial cells. To further explore the importance of KLF2 expression in CNV, we knocked down KLF2 in vivo, which resulted in enlarged CNV lesions. After aging miR-34a-/- mice for twenty months, we found that miR-34a also promoted CNV in advanced age. By staining excised neovascular tufts from patients with wet AMD, we confirmed the presence of miR-34a in human CNV. Finally, after analyzing two different publicly available single-cell RNA-sequencing datasets from human donors, we observed a decrease in KLF2 expression across several types of vascular endothelial cells in the eye with age, while CXCL12 expression increased in the choroidal arteries of patients with wet AMD. Taken together, our investigation revealed that miR-34a may drive CNV pathology in wet AMD, potentially through mechanisms independent of VEGFa. This research highlights the role of miR-34a in influencing the KLF2/CXCR4/CXCL12 pathway in ocular neovascularization. These findings could lead to novel therapies that complement existing anti-VEGFa treatments.
Language
English (en)
Chair and Committee
Rajendra Apte
Committee Members
Amber Stratman; David Ornitz; Philip Williams; Steven Teitelbaum; Thorold Theunissen
Recommended Citation
Colasanti, Jason, "The Role of MiR-34a in Pathological Neovascularization as Seen in Age-Related Macular Degeneration" (2024). Arts & Sciences Electronic Theses and Dissertations. 3351.
https://openscholarship.wustl.edu/art_sci_etds/3351