Investigation of the role of Glil+ cells in retinal angiogenesis and fibrosis.
Background: In ocular diseases such as Age-related Macular Degeneration (AMD) and diabetic retinopathy, dysregulated inflammation in the retina can lead to the formation of a fibrovascular membrane, which can cause severe structural damage to the retina thus leading to severe visual defects and blindness. The process of pathological neovascularisation into the formation of a fibrovascular membrane is not completely understood. Recent research has implicated the Hedgehog pathway transcription factor, Gli1, in the development of fibrosis in numerous organs. This study aims to characterise Gli1 expression in ocular tissues and to elucidate the role of Gli1 in the development of fibrovascular membranes in the retina utilising transgenic mice, and specific small molecule inhibitor of Gli1, GANT61. Methods: Gli1CreERT2 mice were crossed with Ai9(RCL-tdT) (Tomato) mice to produce Gli1-Cre x Tomato crossbreeds. 6–7-week-old Heterozygous Gli1-Cre x Tomato mice received 100 mg/kg Tamoxifen daily (I.P.) for 5 days. Mice were monitored for 80 days to determine the duration Gli1-Tomato expression. Eyes, and other organs, were collected for immunofluorescent staining. For the one laser choroidal neovascularisation (CNV) model, Gli1-Cre x Tomato mice received 3-4 laser burns per eye to induce choroidal neovascularisation and were monitored over 21 days to assess the expression of Gli1 in the injured retina using fundus imaging and immunofluorescent staining. For the two-stage CNV model, Gli1-Cre x Tomato mice received 3-4 laser burns per eye to induce choroidal neovascularisation followed by a second laser directed towards CNV lesions 7 days later. Mice were then monitored over a period of 30 days. Two-stage laser mice received either GANT61 or diphtheria toxin (for Gli1-cre x Rosa mice) to determine the effects of Gli1 inhibition/ablation on fibrovascular lesion development. Cell survival assays of retinal pigment epithelial and microvascular endothelial cells in the presence of TGFβ2 together with/without Gli1 inhibitor GANT61 were utilised to interrogate the role of Gli1 in mesenchymal transition. Results: Expression of Gli1-Tomato was detected as far 90 days in the eye, predominantly in the choroid, sclera, cornea, and ciliary body, although the RPE layer appeared to be Gli1-tomato negative. Immunofluorescent staining displayed co-expression of Gli1-tomato with CD31+, NG2+, and Collagen 4+ retinal microvasculature. Fundus imaging of CNV lesions in one laser model displayed expression of Gli1-tomato in and around the lesion. Immunofluorescent imaging of cryosections and flatmounts showed co-expression of Gli1-tomato with endothelial markers in choroidal neovascular lesions, which persisted up to 21 days after endothelial components diminish. Two-stage laser model CNV lesions displayed co-expression of Gli1-tomato with collagen-1+ lesions up to 30 days post-laser. Knockdown or inhibition of Gli1 did not appear to significantly affect fibrovascular lesion development, however a trend towards decreases in lesion size and collagen-1+ intensity was observed. GANT61 mediated inhibition of Gli1 in vitro demonstrated that epithelial cells undergoing epithelial-to-mesenchymal transition (EMT) were more sensitised than in homeostatic conditions, in contrast endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT) were less sensitised than in homeostatic conditions. Conclusions: Gli1+ cells are expressed throughout the eye, most notably within the uveal tract and retinal microvasculature. In the context of injury, Gli1+ cells are closely associated with endothelial and perivascular components of the CNV lesion. In contrast to published data, results suggest that Gli1+ cells do not drive the development of fibrovascular lesions in a two-stage laser CNV model. Epithelial cells induced to undergo EMT became particularly sensitised to Hh signalling inhibition, suggesting a potential therapeutic avenue for ocular fibrosis. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 722717. Thesis embargoed until 31 July 2026.
Hedgehog Signalling, Gli1, Fibrosis, CNV, retina, retinal microvascular, neovascularisation, laser-induced neovascularisation, two-staged laser model
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