Single-cell RNA transcriptome reveals the underlying mechanisms by which losartan alleviates nitrogen mustard induced corneal injury

Current therapies used for managing limbal stem cell deficiency (LSCD) fail to fully reverse the damage to limbal epithelial stem cells (LESCs) and are often accompanied by side effects following long-term usage. Thus, more effective and safer therapeutic options are required. Here, in contrast to acute injury in other chemical burn models (e.g., alkali burn), we utilized a nitrogen mustard-induced corneal injury model featuring a chronic inflammation and a prolonged chronic/delayed phase of damage, which leads to LSCD hallmarks (corneal haze, chronic inflammation, neovascularization, conjunctivalization). Previously, we demonstrated that Angiotensin-converting enzyme 2 (ACE2) knockout mouse corneas developed haze and neovascularization due to induction of exaggerated inflammation. Such corneal inflammatory response induced by loss of ACE2 can be reversed by treatment of losartan, a widely used anti-hypertension drug with approved long-term safety. Here, we showed that systemic treatment with losartan markedly decreased corneal haze and inflammation during the acute and delayed phases of NM corneal injury. More importantly, in the delayed phase, losartan treatment also reduced the NM-induced neo-vascularized areas in the cornea by 56%. Goblet cells that were detected within the corneal epithelium during the delayed phase, were significantly reduced (43%) following losartan treatment, suggesting that systemic treatment with losartan significantly resolves classical hallmarks of LSCD. Single cell RNA sequencing (scRNA-seq) provided evidence that NM injury induced a dramatic increase of monocytes/macrophages in the cornea and that such increase was attenuated by losartan treatment. Flow cytometry analysis confirmed that losartan attenuated corneal inflammation via reducing the monocytes/macrophages in the cornea. Our findings strongly support a new therapeutic use for losartan in blunting LSCD, which is a consequence associated with numerous corneal inflammatory conditions (e.g., dry eye, bacterial keratitis, diabetic keratopathy). Overall design: Methods: We conducted scRNA-seq on ocular anterior segmental tissue from 1 uninjured wild-type, 1 nitrogen mustard (NM) injured + saline treated, and 1 nitrogen mustard (NM) injured + losartan treated mice, using a 10X Gemomics pipeline. Cell populations were distinguished by UMAP embedding. Seurat analysis was conducted to compare gene expression profiles between these groups of mice.