ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation

**Background:** The cornea is normally avascular, and the limbus (the vascularized junction between cornea and conjunctiva) is thought to act as a barrier against corneal neovascularization. Limbal epithelial stem cells (LESCs) maintain corneal transparency, but their exact role in regulating (lymph)angiogenesis and inflammation is unclear. ABCB5 is a specific LESC marker. This study investigated the role of ABCB5+ LESCs in corneal neovascularization and inflammation using ABCB5 knockout (ABCB5KO) mice and human limbal epithelial cells.

**Methods:** In vivo, corneal whole-mounts from ABCB5KO and WT mice at 4, 8, and 26 weeks of age were stained for CD31 (blood vessels) and LYVE-1 (lymphatic vessels) to assess baseline vascular morphology. In a separate experiment, 8-week-old mice underwent suture-induced inflammatory corneal neovascularization; corneas were harvested after 14 days and stained for CD31, LYVE-1, and CD45 (leukocytes). In vitro, human limbal epithelial cells were magnetically sorted into ABCB5-positive (ABCB5+) and ABCB5-negative (ABCB5-) fractions. Conditioned media from these fractions were applied to human blood endothelial cells (BECs) and lymphatic endothelial cells (LECs). Metabolic activity (Alamar Blue), migration (scratch wound closure), and tube formation (Matrigel assay) were assessed. Proteomic analysis (LC-MS/MS) compared ABCB5+ vs. ABCB5- cells, focusing on inflammation and angiogenesis-related proteins.

**Key Results:** In developing mice (4 weeks), ABCB5KO animals showed significantly more blood vessel sprouts per mm² (KO 8.0±1.34 vs. WT 2.34±1.23, p<0.05) and more lymphatic vessel endpoints (KO 2.47±0.58 vs. WT 1.98±0.5, p<0.05) and branching points (KO 0.7±0.24 vs. WT 0.44±0.16, p<0.05) compared to WT. These differences were absent in adult mice (8 and 26 weeks). In the suture-induced inflammation model, WT mice had significantly more lymphatic vessel sprouts than ABCB5KO mice (WT 2.22±0.21 vs. KO 1.57±0.61, p<0.05). The fold change in CD45+ cell area (inflamed/naïve) was significantly higher in ABCB5KO than WT (KO 1.931±0.045 vs. WT 1.77±0.055, p<0.05). In vitro, conditioned media from ABCB5+ cells significantly increased BEC metabolic activity (ABCB5+ 32±0.37 vs. ABCB5- 27.7±0.62, p<0.05) and accelerated scratch wound closure for both LECs (at 16h: ABCB5+ 95.95±3.02% vs. ABCB5- 57.16±30.44%, p<0.05) and BECs (at 4h: ABCB5+ 66.5±17.3% vs. ABCB5- 35.2±18.4%; at 16h: ABCB5+ 95.5±4.68% vs. ABCB5- 53.8±24.21%, both p<0.05). Tube formation was not significantly different. Proteomics identified 606 significantly differentially expressed proteins between ABCB5+ and ABCB5- cells. In ABCB5+ cells, proangiogenic factors (e.g., heme oxygenase 1, prostaglandin G/H synthase 2, ephrin type-A receptor 1) were upregulated, while the antiangiogenic thrombospondin-1 was downregulated. Anti-inflammatory proteins (e.g., interleukin-1 receptor antagonist, integrin beta-6, antithrombin III) were upregulated, along with some pro-inflammatory factors (IFI16, EPHX2).

**Clinical Implications:** This study reveals a novel dual role of ABCB5+ LESCs: they inhibit developmental (lymph)angiogenesis but promote inflammatory (lymph)angiogenesis in adulthood, while also exerting anti-inflammatory effects. These findings are clinically relevant for LESC therapy, as ABCB5+ cells are used in ongoing clinical trials for limbal stem cell deficiency (NCT03549299). The anti-inflammatory profile of ABCB5+ cells may prevent excessive immune cell recruitment and offset their mild proangiogenic activity, potentially leading to successful corneal regeneration without neovascularization or inflammation.