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.