Skip to main content

Advertisement

SpringerLink
Log in

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

  • Basic Science
  • Published:
Graefe's Archive for Clinical and Experimental Ophthalmology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Brewitt H, Sistani F (2001) Dry eye disease: the scale of the problem. Surv Ophthalmol 45(Suppl 2):S199–202

    Article  PubMed  Google Scholar 

  2. Foulks GN (2008) Pharmacological management of dry eye in the elderly patient. Drugs Aging 25:105–118

    Article  PubMed  CAS  Google Scholar 

  3. Behrens A, Doyle JJ, Stern L, Chuck RS, McDonnell PJ, Azar DT, Dua HS, Hom M, Karpecki PM, Laibson PR, Lemp MA, Meisler DM, Del Castillo JM, O’Brien TP, Pflugfelder SC, Rolando M, Schein OD, Seitz B, Tseng SC, van Setten G, Wilson SE, Yiu SC (2006) Dysfunctional tear syndrome: a Delphi approach to treatment recommendations. Cornea 25:900–907

    Article  PubMed  Google Scholar 

  4. WorkShop DE (2007) The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop. Ocul Surf 5:75–92

    Article  Google Scholar 

  5. Lemp MA (2008) Management of dry eye disease. Am J Manag Care 14:S88–101

    PubMed  Google Scholar 

  6. Pflugfelder SC (2004) Antiinflammatory therapy for dry eye. Am J Ophthalmol 137:337–342

    Article  PubMed  Google Scholar 

  7. WorkShop DE (2007) Management and therapy of dry eye disease: report of the Management and Therapy Subcommittee of the International Dry Eye WorkShop. Ocul Surf 5:163–178

    Article  Google Scholar 

  8. Latkany R (2008) Dry eyes: etiology and management. Curr Opin Ophthalmol 19:287–291

    Article  PubMed  Google Scholar 

  9. Narayanan S, Manning J, Proske R, McDermott AM (2006) Effect of hyperosmolality on beta-defensin gene expression by human corneal epithelial cells. Cornea 25:1063–1068

    Article  PubMed  Google Scholar 

  10. Bildin VN, Yang H, Crook RB, Fischbarg J, Reinach PS (2000) Adaptation by corneal epithelial cells to chronic hypertonic stress depends on upregulation of Na:K:2Cl cotransporter gene and protein expression and ion transport activity. J Membr Biol 177:41–50

    Article  PubMed  CAS  Google Scholar 

  11. Gilbard JP, Carter JB, Sang DN, Refojo MF, Hanninen LA, Kenyon KR (1984) Morphologic effect of hyperosmolarity on rabbit corneal epithelium. Ophthalmology 91:1205–1212

    PubMed  CAS  Google Scholar 

  12. Li DQ, Chen Z, Song XJ, Luo L, Pflugfelder SC (2004) Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells. Invest Ophthalmol Vis Sci 45:4302–4311

    Article  PubMed  Google Scholar 

  13. Li DQ, Luo L, Chen Z, Kim HS, Song XJ, Pflugfelder SC (2006) JNK and ERK MAP kinases mediate induction of IL-1beta, TNF-alpha and IL-8 following hyperosmolar stress in human limbal epithelial cells. Exp Eye Res 82:588–596

    Article  PubMed  CAS  Google Scholar 

  14. Pan Z, Wang Z, Yang H, Zhang F, Reinach PS (2011) TRPV1 activation is required for hypertonicity-stimulated inflammatory cytokine release in human corneal epithelial cells. Invest Ophthalmol Vis Sci 52:485–493

    Article  PubMed  CAS  Google Scholar 

  15. Enriquez-de-Salamanca A, Calder V, Gao J, Galatowicz G, García-Vázquez C, Fernández I, Stern ME, Diebold Y, Calonge M (2008) Cytokine responses by conjunctival epithelial cells: an in vitro model of ocular inflammation. Cytokine 44:160–167

    Article  PubMed  CAS  Google Scholar 

  16. Pflugfelder SC, Jones D, Ji Z, Afonso A, Monroy D (1999) Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjogren’s syndrome keratoconjunctivitis sicca. Curr Eye Res 19:201–211

    Article  PubMed  CAS  Google Scholar 

  17. Shatos MA, Gu J, Hodges RR, Lashkari K, Dartt DA (2008) ERK/p44p42 mitogen-activated protein kinase mediates EGF-stimulated proliferation of conjunctival goblet cells in culture. Invest Ophthalmol Vis Sci 49:3351–3359

    Article  PubMed  Google Scholar 

  18. Shatos MA, Rios JD, Horikawa Y, Hodges RR, Chang EL, Bernardino CR, Rubin PA, Dartt DA (2003) Isolation and characterization of cultured human conjunctival goblet cells. Invest Ophthalmol Vis Sci 44:2477–2486

    Article  PubMed  Google Scholar 

  19. Chung SH, Lee JH, Yoon JH, Lee HK, Seo KY (2007) Multi-layered culture of primary human conjunctival epithelial cells producing MUC5AC. Exp Eye Res 85:226–233

    Article  PubMed  CAS  Google Scholar 

  20. Lee J, Jun EJ, Sunwoo JH, Kim ES, Kim JH, Kim JY, Kim MJ, Kim YK, Lee H, Tchah H (2011) An ex vivo model of coxsackievirus infection using multilayered human conjunctival epithelial cells. Graefes Arch Clin Exp Ophthalmol 249:1327–1332

    Article  PubMed  Google Scholar 

  21. van Setten GB, Tervo T, Viinikka L, Perheentupa J, Tarkkanen A (1991) Epidermal growth factor in human tear fluid: a minireview. Int Ophthalmol 15:359–362

    Article  PubMed  Google Scholar 

  22. Enriquez-de-Salamanca A, Castellanos E, Stern ME, Fernández I, Carreño E, García-Vázquez C, Herreras JM, Calonge M (2010) Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease. Mol Vis 16:862–873

    PubMed  CAS  Google Scholar 

  23. Rao K, Farley WJ, Pflugfelder SC (2010) Association between high tear epidermal growth factor levels and corneal subepithelial fibrosis in dry eye conditions. Invest Ophthalmol Vis Sci 51:844–849

    Article  PubMed  Google Scholar 

  24. Luo L, Li DQ, Corrales RM, Pflugfelder SC (2005) Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface. Eye Contact Lens 31:186–193

    Article  PubMed  Google Scholar 

  25. Corrales RM, Luo L, Chang EY, Pflugfelder SC (2008) Effects of osmoprotectants on hyperosmolar stress in cultured human corneal epithelial cells. Cornea 27:574–579

    Article  PubMed  Google Scholar 

  26. Li J, Zhu HY, Beuerman RW (2009) Stimulation of specific cytokines in human conjunctival epithelial cells by defensins HNP1, HBD2, and HBD3. Invest Ophthalmol Vis Sci 50:644–653

    Article  PubMed  Google Scholar 

  27. Adhikary G, Sun Y, Pearlman E (2008) C-Jun NH2 terminal kinase (JNK) is an essential mediator of Toll-like receptor 2-induced corneal inflammation. J Leukoc Biol 83:991–997

    Article  PubMed  CAS  Google Scholar 

  28. Yeh S, Song XJ, Farley W, Li DQ, Stern ME, Pflugfelder SC (2003) Apoptosis of ocular surface cells in experimentally induced dry eye. Invest Ophthalmol Vis Sci 44:124–129

    Article  PubMed  Google Scholar 

  29. Turner K, Pflugfelder SC, Ji Z, Feuer WJ, Stern M, Reis BL (2000) Interleukin-6 levels in the conjunctival epithelium of patients with dry eye disease treated with cyclosporine ophthalmic emulsion. Cornea 19:492–496

    Article  PubMed  CAS  Google Scholar 

  30. Luo L, Li DQ, Doshi A, Farley W, Corrales RM, Pflugfelder SC (2004) Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface. Invest Ophthalmol Vis Sci 45:4293–4301

    Article  PubMed  Google Scholar 

  31. Massingale ML, Li X, Vallabhajosyula M, Chen D, Wei Y, Asbell PA (2009) Analysis of inflammatory cytokines in the tears of dry eye patients. Cornea 28:1023–1027

    Article  PubMed  Google Scholar 

  32. Singh AB, Harris RC (2005) Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell Signal 17:1183–1193

    Article  PubMed  CAS  Google Scholar 

  33. Grant MB, Khaw PT, Schultz GS, Adams JL, Shimizu RW (1992) Effects of epidermal growth factor, fibroblast growth factor, and transforming growth factor-beta on corneal cell chemotaxis. Invest Ophthalmol Vis Sci 33:3292–3301

    PubMed  CAS  Google Scholar 

  34. Fredj-Reygrobellet D, Plouet J, Delayre T, Baudouin C, Bourret F, Lapalus P (1987) Effects of aFGF and bFGF on wound healing in rabbit corneas. Curr Eye Res 6:1205–1209

    Article  PubMed  CAS  Google Scholar 

  35. van Setten GB, Schultz GS, Macauley S (1994) Growth factors in human tear fluid and in lacrimal glands. Adv Exp Med Biol 350:315–319

    Article  PubMed  CAS  Google Scholar 

  36. Barton K, Nava A, Monroy DC, Pflugfelder SC (1998) Cytokines and tear function in ocular surface disease. Adv Exp Med Biol 438:461–469

    Article  PubMed  CAS  Google Scholar 

  37. Nezu E, Ohashi Y, Kinoshita S, Manabe R (1992) Recombinant human epidermal growth factor and corneal neovascularization. Jpn J Ophthalmol 36:401–406

    PubMed  CAS  Google Scholar 

  38. Lu L, Wang L, Shell B (2003) UV-induced signaling pathways associated with corneal epithelial cell apoptosis. Invest Ophthalmol Vis Sci 44:5102–5109

    Article  PubMed  Google Scholar 

  39. Krishnamoorthy M, Heimburg-Molinaro J, Bargo AM, Nash RJ (2009) Heparin binding epidermal growth factor-like growth factor and PD169316 prevent apoptosis in mouse embryonic stem cells. J Biochem 145:177–184

    Article  PubMed  CAS  Google Scholar 

  40. Li DQ, Tseng SC (1996) Differential regulation of cytokine and receptor transcript expression in human corneal and limbal fibroblasts by epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor B, and interleukin-1 beta. Invest Ophthalmol Vis Sci 37:2068–2080

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was partially supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A101538) and partially by the National Research Foundation of Korea Grant funded by the Korean Government (2012R1A1A2003278).

Author information

Authors and Affiliations

  1. Department of Ophthalmology, Chungbuk National University College of Medicine, Cheongju, South Korea

    Jae-hyung Kim

  2. Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, 388-1 Pungnap-2dong, Songpa-gu, Seoul, 138-736, South Korea

    Soon-Suk Kang, Eun Soon Kim, Jae Yong Kim, Myoung Joon Kim & Hungwon Tchah

Authors
  1. Jae-hyung Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Soon-Suk Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eun Soon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jae Yong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Myoung Joon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hungwon Tchah
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hungwon Tchah.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, Jh., Kang, SS., Kim, E.S. et al. Osmoprotective effects of supplemental epidermal growth factor in an ex vivo multilayered human conjunctival model under hyperosmotic stress. Graefes Arch Clin Exp Ophthalmol 251, 1945–1953 (2013). https://doi.org/10.1007/s00417-013-2369-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00417-013-2369-5

Keywords

Search

Navigation