Abstract
Purpose :
For decades, it has been well accepted that corneal epithelial stem cells and their immediate progeny, the early transit amplifying (eTA) cells, reside in the limbal epithelial basal layer. Activation of quiescent stem/eTA cells is required for proper re-epithelialization during wound healing. The molecular profile of activated stem/eTA cells remains unclear because of difficulties in obtaining discrete cell populations for analyses. Single cell RNA sequencing (scRNA-seq) technology can profile the transcriptome at a single cell level, providing information on how stem/eTA cell activation is regulated. Using this technology, we report that ACE2, a key component in the renin-angiotensin system (RAS), functions as a negative regulator of stem/eTA activation.
Methods :
Mouse corneal epithelium was exposed to 1M NaOH for 30s or mechanically removed with a diamond burr. Corneas were processed for scRNA-seq and data was analyzed using R with a Seurat package. Limbal epithelial cell proliferation was assessed using BrdU incorporation. RT-qPCR, western blotting and immunostaining were conducted to determine the change of gene expression.
Results :
ACE2 was predominantly expressed in the stem cell-enriched limbal basal epithelium. scRNA-seq combined with GO analysis suggested that ACE2 was involved in limbal stem/eTA cell proliferation. Interestingly, immunostaining and RT-qPCR indicated that ACE2 expression was reduced following corneal injuries. Reduction in ACE2 promoted proliferation in human limbal epithelial cell culture as well as in mouse limbal epithelium after corneal epithelial debridement. Significantly, the negative effect of ACE2 on proliferation was not reversed following treatment with the angiotensin II receptor blocker losartan, indicating that the function of ACE2 in limbal epithelium is independent of RAS. scRNA-seq also revealed that reduction of ACE2 caused activation of the TGFA/EGFR pathway, which reduced expression of Lcn2. Lcn2 is a negative regulator of proliferation in a variety of cells. Inhibition of EGFR or overexpression of Lcn2 reversed the increased proliferation in limbal epithelial cells lacking ACE2.
Conclusions :
Our findings strongly support the idea that in response to corneal injury, ACE2 is downregulated, which results in the activation of stem/eTA cell proliferation via a novel TGFA/EGFR/Lcn2 signaling pathway in an angiotensin-independent way.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.