Osmoprotective effects of supplemental epidermal growth factor in an ex vivo multilayered human conjunctival model under hyperosmotic stress

• Published in: Graefe's archive for clinical and experimental ophthalmology 2013-08, Vol.251 (8), p.1945-1953
• Main Authors: Kim, Jae-hyung, Kang, Soon-Suk, Kim, Eun Soon, Kim, Jae Yong, Kim, Myoung Joon, Tchah, Hungwon
• Format: Article
• Language: English
• Subjects: Apoptosis; Basic Science; Blotting, Western; Caspase 3 - metabolism; Cell Count; Cell Proliferation - drug effects; Cells, Cultured; Conjunctiva - drug effects; Conjunctiva - metabolism; Conjunctiva - pathology; Dry Eye Syndromes - genetics; Dry Eye Syndromes - pathology; Dry Eye Syndromes - prevention & control; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor - pharmacology; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Epithelial Cells - pathology; Extracellular Signal-Regulated MAP Kinases - genetics; Extracellular Signal-Regulated MAP Kinases - metabolism; Humans; In Situ Nick-End Labeling; Interleukin-6 - genetics; Interleukin-6 - metabolism; Medicine; Medicine & Public Health; Models, Biological; Ophthalmology; Osmotic Pressure; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptor, Epidermal Growth Factor - genetics; Receptor, Epidermal Growth Factor - metabolism; RNA, Messenger - metabolism; Tissue Donors
• ISSN: 0721-832X; 1435-702X
• DOI: 10.1007/s00417-013-2369-5

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.