Indian hedgehog (Ihh) secreted by adult choroid endothelial cells` regulates choroidal homeostasis and immune response

Abstract

Purpose : Patients with atrophic AMD display photoreceptor loss secondary to RPE dysfunction and choroidal damage. However, the exact etiology of AMD remains unknown due to lack of information on RPE/choroid cell diversity and intercellular crosstalk mechanisms. Here we report the selective high expression of Indian Hedgehog (Ihh) in adult choroid endothelial cells (ECs) and its function as an angio- and immunomodulatory choroid signaling molecule likely relevant to AMD.

Methods : Cells from tissue of wildtype RPE/choroid were single-cell sorted and scRNAseq was performed. Bulk RNAseq was used to compare the transcriptomes of purified RPE/choroid from wildtype and mutant mice after laser-induced CNV. To study crosstalk between EC-derived Ihh and stromal, Gli1⁺ perivascular cells, adult tamoxifen-induced knockout mice were generated by crossing Ihh^flox/flox with Cdh5-Ert2Cre⁺ transgenic mice, and cDNA of eGFP was inserted into exon 1 of mouse Gli1 gene to generate Gli1^+/eGFP knock-in animals. Photoreceptor function was measured by ERG and OKT, and morphology of RPE/choroid and photoreceptors was evaluated by immunohistochemistry. In-vitro assays such as flow-cytometry and RT-qPCR were used to examine crosstalk between RPE and choroid.

Results : We report the first single cell RNAseq analysis of adult mouse RPE/choroid tissue identifying 13 main cell types including 3 subtypes of ECs. By combining these results with a transcriptomic analysis of tissue-specific ECs from other organs, we found a marked enrichment of Ihh expression in choroid ECs, particularly in the choriocapillaries located underneath the RPE. Using reporter mice, we identified the target of choroidal Ihh signaling as a large population of stromal, Gli1⁺ perivascular mesenchymal stem cell (MSC)-like cells. EC-specific Ihh KO and Gli1^eGFP/eGFP mice displayed loss of mast cells, altered inflammatory and vasculatory response, visual impairment and retinal degeneration indicating a novel Ihh/Gli1-dependent signaling circuit in adult choroid regulating choroidal homeostasis and retinal function.

Conclusions : Our study uncovers the cellular and molecular landscape of the adult mouse RPE/choroid and stromal Gli1⁺ MSC-like cells as key player in response to EC-derived Ihh implicated in choroidal neovascularization and immune response, thus providing possible new therapeutic targets for atrophic AMD and choroidal disorders.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.