Volume 25, Issue 8, Pages 1881-1888 (August 2017) Hepatocyte Growth Factor Suppresses Inflammation and Promotes Epithelium Repair in Corneal Injury  Masahiro Omoto, Kunal Suri, Afsaneh Amouzegar, Mingshun Li, Kishore R. Katikireddy, Sharad K. Mittal, Sunil K. Chauhan  Molecular Therapy  Volume 25, Issue 8, Pages 1881-1888 (August 2017) DOI: 10.1016/j.ymthe.2017.04.020 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 HGF Promotes Corneal Epithelial Cell Proliferation Human corneal epithelial cells (HCECs) were cultured in keratinocyte serum-free medium (K-SFM, left panel), supplemented K-SFM (middle panel), or supplemented K-SFM with 100 ng/mL hepatocyte growth factor (HGF, right panel) for 48 hr. (A) Representative bright field microscopic images showing confluent HCECs in supplemented keratinocyte serum-free medium (K-SFM, middle) compared to basal medium without supplements (left) and increased cell numbers when cultured with HGF (scale bar, 25 μm). (B) HCEC proliferation was measured with bromodeoxyuridine (BrdU) incorporation assay after culture in K-SFM or supplemented K-SFM with or without HGF. (C) HCECs were cultured in K-SFM, supplemented K-SFM in the presence or absence of 100 ng/mL IL-1β, or supplemented K-SFM with the addition of IL-1β and HGF. Cell proliferation was measured using the BrdU incorporation assay. Data are represented as mean ± SEM. Experiments were repeated three times, and data in each group are from triplicate wells. Molecular Therapy 2017 25, 1881-1888DOI: (10.1016/j.ymthe.2017.04.020) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 HGF Inhibits Activation of Inflammatory Cells and Their Expression of Pro-inflammatory Cytokines CD11b+ cells isolated from murine spleens were grown in either RPMI medium alone or medium with IL-1β with or without HGF. (A) Flow cytometry analysis showed increased protein expression (mean fluorescent intensity, MFI) of activation marker MHC class II upon treatment with IL-1β, which was substantially downregulated with HGF. (B and C) After culture of CD11b+ cells with IL-1β in the presence or absence of HGF, mRNA expression of the pro-inflammatory cytokines TNF-α (B) and IL-1β (C) were assessed using real-time PCR. Data are represented as mean ± SEM. Experiments were repeated three times on samples pooled from four or five mice, and data in each group are from triplicate wells. Molecular Therapy 2017 25, 1881-1888DOI: (10.1016/j.ymthe.2017.04.020) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 HGF Promotes Epithelial Cell Proliferation in Corneal Injury Mechanical injury was induced in murine corneas by scraping the epithelium, and either topical recombinant hepatocyte growth factor (HGF) or mouse serum albumin (MSA) was applied twice daily. Normal corneas without injury or injured corneas receiving MSA served as controls. Corneas were harvested at day 3 after injury. (A) Representative immunofluorescent images of sections of mouse cornea stained for Ki-67 (green) showing proliferating cells (scale bar, 100 μm). Quantification of Ki-67+ cells in immunohistochemistry micrographs shows higher numbers of Ki-67+ cells in HGF-treated corneas compared to MSA-treated or non-injured corneas in both the peripheral and the central cornea. (B and C) mRNA expression of (B) p63 and (C) HGF-R in corneas was assessed using real-time PCR. Data are represented as mean ± SEM. Representative data from two independent experiments are shown, and each group consists of six mice. Epi, epithelium; Str., stroma; Endo., endothelium. Molecular Therapy 2017 25, 1881-1888DOI: (10.1016/j.ymthe.2017.04.020) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 Topical HGF Suppresses Inflammation in Corneal Injury Mechanical injury was induced in murine corneas by scraping the epithelium, and either topical recombinant hepatocyte growth factor (HGF) or mouse serum albumin (MSA) was applied twice daily. Normal corneas without injury or injured corneas receiving MSA served as controls. Corneas were harvested at day 3 after injury. (A) Representative immunofluorescence images of corneal cross sections showing higher expression of CD45 (green) in MSA-treated controls, compared to HGF-treated eyes (scale bar, 50 μm). (B and C) mRNA expression of the pro-inflammatory cytokines IL-1β (B) and TNF-α (C) was measured using real-time PCR. Data are represented as mean ± SEM. Representative data from two independent experiments are shown, and each group consists of six mice. Epi, epithelium; Str., stroma; Endo., endothelium. Molecular Therapy 2017 25, 1881-1888DOI: (10.1016/j.ymthe.2017.04.020) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 Topical HGF Promotes Epithelial Wound Healing in Corneal Injury Mechanical injury was induced in murine corneas by scraping the epithelium, and either topical recombinant hepatocyte growth factor (HGF) or mouse serum albumin (MSA) was applied twice daily for 7 days. Normal corneas without injury or injured corneas receiving MSA served as controls. (A) Biomicroscopic images of fluorescein staining in injured corneas 1, 3, 5, and 7 days after corneal epithelial injury. Green indicates an epithelial defect. A smaller area of fluorescein staining represents faster repair or closure of the epithelial wound. (B) The mean area of fluorescein staining measured with ImageJ software was significantly reduced in HGF-treated corneas at 3, 5, and 7 days. Data are represented as mean ± SEM. Representative data from two independent experiments are shown, and each group consists of six mice. Molecular Therapy 2017 25, 1881-1888DOI: (10.1016/j.ymthe.2017.04.020) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions