Investigating the role of perivascular mesenchymal stem cells in macular fibrosis secondary to neovascular age-related macular degeneration

Age-related macular degeneration (AMD) is a disease that affects the macula, the central part of the retina at the back of the eye, causing progressive loss of central vision in the elderly. Globally, 200 million people are affected by AMD and this number is expected to increase to 300 million by 2040. In the developed world, AMD is the most common cause of blindness in the elderly. AMD has two advanced forms: wet (also known as neovascular AMD) and dry (also known as geographic atrophy); the former accounts for ~80% of AMD-related visual impairment.
Wet AMD occurs when diseased blood vessels grow into the macula, causing fluid leakage and bleeding that impairs vision. It is currently treated with injections of VEGF inhibitor (e.g. Lucentis or Avastin) into the eye, which reduces the growth of blood vessels. Although such treatment can stabilise or even improve visual function, 50% of treated eyes eventually develop fibrosis (scarring) in the macula. Unlike scarring of the skin, which heals the wound, scarring in the macula, instead reduces the efficacy of VEGF inhibitors. The fibrovascular membranes that emerge from diseased blood vessels eventually destroy the entire macula. Currently, there are no medications to prevent or treat this condition due to a poor understanding of the disease mechanism.
New blood vessels in wet AMD develop into fibrovascular scar when cells, called myofibroblasts, infiltrate and accumulate in the macula. Myofibroblasts produce excessive amounts of scar-forming extracellular matrix such as collagens and fibronectins. Myofibroblasts are absent from the healthy retina, including the macula, and we do not yet know where they come from and how they are activated in wet AMD. This knowledge is crucial for developing therapeutic interventions to wet AMD and macular fibrosis.
Recently, perivascular mesenchymal stem cells (pMSC), a unique type of cell residing around blood vessels were identified as a major source of myofibroblasts in injury-induced scars in multiple organs, including the lung, kidney, heart, and skin. These pMSCs are known to safeguard blood vessels and maintain their integrity. During injury, they detach from blood vessel walls and travel to the site of damage, where they participate in tissue repair and regeneration. When the injury persists or when injury-mediated inflammation does not resolve promptly, these pMSCs expand and become myofibroblasts leading to organ fibrosis.
We have found that within the eye, the retina and choroid also contain a network of pMSCs. In an experimental model of wet AMD-mediated retinal fibrosis, over 60% of myofibroblast originated from pMSCs, and genetic deletion of pMSCs reduced retinal fibrosis. Our aim is to understand why pMSCs become myofibroblasts in wet AMD and how we might prevent or reverse this process. We will use advanced genomic techniques to uncover the gene expression profile of individual pMSCs within the diseased tissue at different stages of retinal fibrosis. This will inform us of which pMSC subtypes give rise to myofibroblasts and the pathways that control this process. We will verify these pathways in pMSC cultures derived from the retina, choroid, and retinal scar tissue. We will then target these pathways with pharmacological approaches to prevent or reverse the differentiation of pMSCs into myofibroblasts using in vitro and in vivo models of retinal fibrosis.
Our results will inform further research and enhance our understanding of retinal repair and fibrosis in wet AMD and other sight-threatening diseases caused by abnormal tissue repair (e.g. proliferative diabetic retinopathy [PDR] and proliferative vitreoretinopathy [PVR]). Ultimately, our research will aim in developing effective treatments for retinal fibrosis.

Intravitreal injection of VEGF inhibitors is the standard of care for nAMD patients. However, ~50% of treated eyes develop macular fibrosis, which causes anti-VEGF resistance and eventually irreversible blindness. Myofibroblasts drive new blood vessels into fibrovascular lesion in nAMD by producing excessive amounts of extracellular matrix proteins but their source is unknown. Recent studies suggest that Gli1+ perivascular mesenchymal stem cells (Gli1+ pMSCs) can differentiate into myofibroblasts and contribute critically to tissue fibrosis in the lung, liver, and heart. We found that the retina and choroid also contain a network of Gli1+ pMSC. In retinal injury and inflammation, they detach from blood vessels, migrate to the site of damage, and participate in retinal scar formation. This proposal aims to understand the mechanisms by which Gli1+ pMSCs differentiation into myofibroblasts in nAMD. We have found that myofibroblasts in macular fibrosis express Gli1. We will first characterise the phenotype of myofibroblasts obtained from a cohort of donated nAMD eyes. We will then induce retinal fibrosis in Gli1-CreERt2:tdTomato mice and isolate Gli1-Tomato cells from the retina and choroid at different stages of the disease for single-cell RNA sequencing. Bioinformatic analysis will be conducted to understand the trajectories of retinal and choroidal Gli1+ pMSCs as they transition into myofibroblasts, and key pathways involved in this process. These pathways will be confirmed in retinal and choroidal pMSC cultures from Gli1-CreERt2:tdTomato mice, and bioinformatic analysis will also uncover high impact hub genes. We will use the acquired knowledge to design strategies to prevent or reverse myofibroblast differentiation. These strategies will be tested in pMSC cultures from mouse retina and choroid, bone marrow-derived human MSCs, and an in vivo model of retinal fibrosis in Gli1-CreERt2:tdTomato and CreERt2:iDTR mice.