Endothelial-perivascular mural partitioning and multicellular signaling in injury-associated corneal...

Cornea, a transparent avascular tissue, develops inflammation, neovascularization, and vision-limiting scarring following injury. This study sought to characterize vascular-focused single-cell RNA-seq (scRNA-seq) profiling between naïve and traumatic rabbit corneas collected 2 weeks after alkali trauma. Seurat-based vascular clustering, ligand‒receptor (LR) niche signaling, interpretable downstream directional pathway prioritization, and LR-focused pathway annotation were used. Among the fourteen identified clusters, the neovessel cluster was subclustered. Vascular heterogeneity and curated vascular gene sets were identified/quantified across vascular subclusters using percentage-expressing cells, average normalized expression, and module scores. The expression-supported LR scoring framework was implemented after integrating the mean normalized expression and the fraction of expressing cells for analyzing the cellular communication of major surrounding corneal cell populations, including keratocyte, myofibroblast, immune, remodeling, sensory-nerve and Schwann cells with receptors in vascular cells. Subclustering of the neovessel cluster identified two major vascular states, an endothelial-enriched population and a perivascular mural-enriched population. Injury-driven expansion of the neovascular niche was indicated by an increase in both vascular cell populations in traumatic cornea than naïve. Across curated gene sets, endothelial cells expressed gene signatures consistent with angiogenic activation and endothelial barrier junction features, whereas perivascular mural cells were more enriched for contractile stabilization, remodeling and fibrosis coupling. Both endothelial and perivascular mural ligand/receptor had a broad incoming LR connectivity to keratocyte, myofibroblast, remodeling, immune, sensory-nerve and Schwann cell populations. Neovessel-targeted sc-RNAseq profiling revealed that VEGF, PDGF, ANGPT/TEK, chemokines related axes, vascular gene programs, and signaling interactions in traumatic cornea as prominent contributors.