title: Osmoprotective effects of supplemental epidermal growth factor in an ex vivo multilayered human conjunctival model under hyperosmotic stress.
abstract: BACKGROUND
  To analyze the effects of supplemental epidermal growth factor (EGF) and the roles of inflammatory cytokines (interleukin [IL]-6) in an ex vivo dry-eye model under hyperosmotic stress using a multilayered culture of human conjunctival epithelial cells (HCECs).
  
  
  METHODS
  Multilayered cultures of HCECs were exposed to hyperosmotic stress (400 mOsm/L) for 24 h in addition to 0.5 ng/mL EGF (low-EGF group) or 25 ng/mL EGF (high-EGF group). Apoptosis was analyzed using the TUNEL assay. Cell proliferation was measured using the [3H]-thymidine incorporation assay. The expression of IL-6, EGF, EGF receptor (EGFR), and phosphorylated extracellular signal-regulated kinase (p-ERK) was measured by western blot analysis. The secretion of IL-6 was measured using ELISA. Western blot analysis was also performed using antibodies against cleaved caspase-3.
  
  
  RESULTS
  The percentage of apoptotic cells was lower in the high-EGF group (6.7%) than in the low-EGF group (10.3%). The high-EGF group demonstrated increased proliferation (323.7 counts/min in the low-EGF group vs 649.1 counts/min in the high-EGF group). EGF induced higher phosphor-EGFR expression and upregulated p-ERK in HCECs. In addition, EGF significantly decreased the secretion of IL-6 and cleaved caspase-3 in HCECs.
  
  
  CONCLUSIONS
  The level of IL-6 was increased in the ex vivo HCEC dry-eye model that was under hyperosmotic stress. Supplemental EGF reduces the level of IL-6, decreases apoptosis, and increases proliferation. These findings indicate that EGF has potential as a therapeutic agent for the treatment of dry eyes.
authors.affiliation: Department of Ophthalmology, Chungbuk National University College of Medicine, Cheongju, South Korea.
authors.forename: Jae-hyung
authors.initials: JH
authors.lastname: Kim
authors.forename: Soon-Suk
authors.initials: SS
authors.lastname: Kang
authors.forename: Eun Soon
authors.initials: ES
authors.lastname: Kim
authors.forename: Jae Yong
authors.initials: JY
authors.lastname: Kim
authors.forename: Myoung Joon
authors.initials: MJ
authors.lastname: Kim
authors.forename: Hungwon
authors.initials: H
authors.lastname: Tchah
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003228:Conjunctiva / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015352:Dry Eye Syndromes / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009997:Osmotic Pressure; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014019:Tissue Donors
chemical_list: C508600:IL6 protein, human; D015850:Interleukin-6; D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D053148:Caspase 3