Fig 1: The limbal epithelial cell proliferation induced by knockdown of ACE2 can be rescued by overexpression of LCN2(A–E) hTCEpi cells transduced with lentiviral-LCN2 (LV-LCN2) or lentiviral—empty vector (EV) were transfected with siControl, siACE2, or siLCN2. Immunofluorescent microscopy visualized the BrdU+ cells 72 h after transfection.(F) The percentage of BrdU+ cells was quantified by ImageJ. N = 3 of independent experiments. One-way ANOVA was conducted, ∗p < 0.05.(G) RT-qPCR results showed a significant reduction of LCN2 gene expression after siLCN2 transfection compared with siControl. Bar graph showed the fold changes. N = 3 of independent experiments. ∗p < 0.05. (H) hTCEpi cells were transduced with lentiviral-LCN2 (LV-LCN2) or lentiviral—empty vector (EV). LV-LCN2 transduction resulted in a significant increase of LCN2 while had no significant effect on the expression level of TGFA. Bar graph showed the fold changes. N = 3 of independent experiments. ∗p < 0.05.(I) RT-qPCR showed that LCN2 expression level in hTCEpi cells was increased when treated with AG1478 (1μM) and decreased when treated with TGFA (50 ng/ml), compared with untreated cells (nt). Graph showed the fold changes. N = 4 of independent experiments. ∗p < 0.05.
Fig 2: ACE2 expression is reduced in response to corneal injury(A) WT mouse central corneas were subjected to debridement wounding. Three hours post injury, immunofluorescent staining showed that ACE2 expression was reduced in limbal epithelium (N = 7) compared to uninjured controls (N = 5). ACE2 relative fluorescence intensity was quantified using ImageJ. ∗p < 0.05. PC: peripheral cornea.(B) A diagram summarizing how ACE2 negatively regulates limbal epithelial proliferation via inhibiting TGFA/EGFR signaling, which consequently modulates LCN2 expression.
Fig 3: ACE2 negatively modulates TGFA/EGFR signaling(A) Network analysis of scRNA-seq data predicts the connection between ACE2 and its downstream gene using Genemania. This indicates that ACE2 connects with GSN and LCN2 through EGFR.(B) Western blotting showed that knockdown of ACE2 increased p-EGFR, an active form of EGFR, as well as p-Akt, a downstream effector of EGFR signaling. Densitometry was performed using ImageStudio Lite. Bar graph showed the fold changes. N = 3 of independent experiments. ∗p < 0.05.(C) Cell-cell communication network and relative contribution of each ligand-receptor pair within the EGF signaling were analyzed by CellChat package. Tgfa interaction was only observed in Ace2 KO mice.(D) Violin plots showed that in cluster 8 of our scRNA-seq data, Tgfa, a ligand of EGFR, was increased in Ace2 KO mice compared to WT.(E) RT-qPCR showed that knockdown of ACE2 in hTCEpi cells increased TGFA expression. Bar graph showed the fold changes. N = 5 of independent experiments. ∗p < 0.05.
from Cell Signaling Technology for Human TGF-alpha Recombinant Protein