Prox1 Protein in Corneal Limbal Lymphatic Vessels Maintains Limbal Stem Cell Stemness and Regulates Corneal Injury Repair.

Purpose: The purpose of this study was to elucidate the role of the corneal lymphatic Prox1 gene in modulating limbal stem cell stemness and facilitating corneal injury repair.

Methods: The limbal Prox1 gene was knocked down by adeno-associated virus (AAV). The alkali burn model was induced in the naive group, the AAV-sham group, and the AAV-shProx1 group. Anterior segment photography, fluorescein sodium staining, and hematoxylin and eosin (H&E) staining were conducted immediately on days 1, 3, 7, 14, and 21 post-injury. Immunofluorescent (IF) staining was used to assess Ki67, ΔNp63, and K14. Additionally, seq-mRNA technology facilitated a comparative transcriptomic analysis between the AAV-sham and the AAV-shProx1 groups 7 days post-injury. Key regulated genes were verified by protein level. Furthermore, a co-culture model of lymphatic endothelial cells (LECs) and limbal stem cells (LSCs) was used to investigate the proliferation capacity and stemness expression of LSCs.

Results: Fluorescein sodium staining revealed that the epithelial defect area was significantly larger in the AAV-shProx1 group than in the AAV-sham group on days 1 and 3 post-injury (P < 0.05). Ki67, ΔNp63, and K14 expressions were consistently lower in the AAV-shProx1 group than in the AAV-sham group at distinct time points. Additionally, seq-mRNA results demonstrated that genes (Prox1 and Lyve1) were downregulated while inflammatory factors (Ccl2, Ccl7, IL16, IL1R, and TNFsf11) were upregulated in the AAV-shProx1 group compared with the AAV-sham group. When Prox1 was silenced in LECs, the proliferation and stemness of LSCs were markedly downregulated.

Conclusions: The Prox1 and Lyve1 proteins in lymphatic vessels served as pivotal regulated proteins in corneal injury repair. The draining role of lymphatic vessels during corneal injury was indispensable.