Fig 2: Increased proliferation, and dedifferentiation of epithelial cells, and fibroblast activation in the corneas of HE4-OE mice. A. Proliferation of corneal epithelial cells were determined by immunohistochemistry using specific antibodies against KI67, and cell nuclei were visualized by DAPI staining. An increased number of KI67-positive epithelial cells (stained in red fluorescence, indicated by white arrow) were detected in the corneas of 6-month-old HE4-OE mice. Scale bars = 50 µm, N = 3, two male mice and one female mouse. B. The epithelial cells in the cornea of wild-type mice were Pax6-positive (green fluorescence, indicated by white arrow). But the epithelial cells in the cornea of 6-month-old HE4-OE mice were Pax6-negative. Scale bars = 50 µm, N = 3, two male mice and one female mouse. C. The Keratin12-positive (red fluorescence, indicated by the white arrow) and Keratin12-negative epithelial cells in the cornea of 6-month-old wild-type and HE4-OE mice, respectively. Scale bars = 50 µm, N = 3, two male mice and one female mouse. D. In comparison with 6-month-old wild-type mice, multiple FSP1-positive fibroblasts (red fluorescence, indicated by the white arrow) were detected near the base of epithelial layer of HE4-OE mice. Scale bars = 50 µm, N = 3, two male mice and one female mouse.

Fig 3: The keratopathy of HE4-OE mice. A. Corneal opacity was observed in HE4-OE mice. The left is a representative photograph of a 6-month-old wild-type mouse, and the right is a representative photograph of a 6-month-old HE4-OE mouse. Note the opaque and untransparent look of the cornea, and hair loss around the eye and at the back, of the HE4-OE mouse. B. C. and D. are stereomicroscopic photographs of the corneas of a wild-type mouse, a 3-month-old HE4-OE mouse before development of opacity, and a 6-month-old HE4-OE mouse with the development of opacity, respectively. Correspondingly, E. F. and G. are microscopic pictures of HE4 antibody-stained corneal sections for the wild-type mouse, the HE4-OE mouse before development of opacity, and the HE4-OE mouse with the development of opacity, respectively. Note the diminished HE4 expression in the cornea of wild-type mice, but high HE4 expression in the corneal epithelium before development of opacity (indicated by arrow). The development of opacity in HE4-OE was accompanied by an increased HE4 expression in the corneal epithelium and stroma (indicated by arrow). Scale bars = 50 µm, N = 3, two female mice and one male mouse.

Fig 4: The keratopathy of HE4-OE mice. A. The histological structures of the corneas of 6-month-old wild-type and HE4-OE mice were examined after H&E staining. Note the increased thickness of the corneal epithelium (indicated by the black bars), increased cellular components, and the presence of multiple vessel-like structures (indicated by the blue arrow) in the cornea of a HE4-OE mouse. Scale bars = 50 µm, N = 3, two female mice and one male mouse. B. Immunohistochemistry with anti-HE4 antibody. Increased HE4 expression was observed in the corneal epithelium of HE4-OE mouse. Scale bars = 50 µm, N = 3, two female mice and one male mouse.

Fig 5: Dramatic upregulation of HE4 in the corneal epithelium following alkali burn. Alkali burn was performed in 12-week-old mice, and the control mice were treated with PBS. The corneal tissues were isolated at 12 h post-treatment for H&E staining and immunostaining. A. H&E staining showed that the corneal epithelium was disrupted and epithelial cells were dispersed. The stromal fibers were disorganized and disrupted. Scale bars = 50 µm, N = 3, two female mice and one male mouse. B. Results of immunohistochemistry with HE4 antibody, showing a dramatic increase of HE4 expression after alkali burn. Scale bars = 50 µm, sample size N = 3, two female mice and one male mouse, for each time point. Corneal tissues were harvested at 3 h, 9 h and 24 h after alkali burn. Real-time PCR was performed to determine mRNA (C, D and E), and ELISA was conducted to assess the protein levels of HE4, IL-6 and TNF-α (F, G and H). Alkali burn led to increased HE4 mRNA and protein levels. The HE4 upregulation was accompanied by the increase of pro-inflammatory factors IL-6 and TNF-α mRNA as well as protein levels. N = 3, two female mice and one male mouse, for each time point. The data were expressed as mean ± SD. *p < 0.05, **p < 0.01.