The in vitro effect of platelet-derived growth factor isoforms on the proliferation of bovine corneal stromal fibroblasts depends on cell density

  • Peter Oliver Denk
  • Marcus Knorr
Laboratory Investigation

Abstract

• Purpose: Recently, it has been shown that corneal stromal fibroblasts express the mRNA for PDGF-β-type receptors, while corneal epithelial cells express the mRNA for the PDGF B-chain, suggesting a role of PDGF isoforms in the regulation of corneal homeostasis and wound healing via an unidirectional epithelial to stromal paracrine interaction. The purpose of this study was to characterize the proliferative response of cultured bovine corneal stromal fibroblasts to PDGF isoforms. • Methods: Bovine corneal stromal fibroblasts were seeded at a cell density of 60 cells/mm2 (low density) and 120 cells/mm2 (high density) and were cultured under serum-free conditions. Except for corresponding controls, PDGF AA, BB and AB (obtained by separate expression of cloned genes inE. coli) were added in concentrations ranging from 3.125 to 100 ng/ml. Cell numbers were determined after an incubation period of 6 days using a cell counter. • Results: Stromal fibroblasts, when cultured at a high density, revealed constant cell numbers during the whole incubation period. Under these culture conditions, stimulation with PDGF AA, BB and AB led to a significant dose-dependent increase in cell proliferation. When cultured at a low cell density, stromal fibroblasts revealed a significant reduction of cell numbers after 6 days of incubation. This reduction was prevented by PDGF AA and AB isoforms in a dose-dependent manner. In contrast, PDGF BB was not effective. • Conclusion: The results of the “high-density” assays suggest that PDGF isoforms act as mitogens for stromal fibroblasts during wound healing, when density of fibroblasts is high. The results of the “low-density” assays support the idea that PDGF AA and AB can prevent cell loss during corneal homeostasis when density of keratocytes is low.

Keywords

Cell Density Wound Healing Proliferative Response Constant Cell Stromal Fibroblast 

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References

  1. 1.
    Antoniades HN, Galanopoulos T, Neville-Golden J, Kiritsy CP, Lynch SE (1991) Injury induces in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts. Proc Natl Acad Sci USA 88:565–569Google Scholar
  2. 2.
    Assouline M, Chew SJ, Thompson HW, Beuerman R (1992) Effect on growth factors on collagen lattice contraction by human keratocytes. Invest Ophthalmol Vis Sci 33:1742–1755Google Scholar
  3. 3.
    Barnes D, Sato G (1980) Methods for growths of cultured cells in serum-free medium. Anal Biochem 102: 255–70Google Scholar
  4. 4.
    Bennett NT, Schultz GS (1991) Growth factors and wound healing: biochemical properties of growth factors and their receptors. Am J Surg 165: 728–736Google Scholar
  5. 5.
    Bergman RH, Spigelman AV (1994) The role of fibroblast inhibitors on corneal healing following photorefractive keratectomy with 193 nanometer excimer laser in rabbits. Ophthalmic Surg 25: 170–174Google Scholar
  6. 6.
    Bonner JC, Badgett A, Lindroos PM, Osornio-Vargas AR (1995) Transforming growth factor β1 downregulates the platelet-derived growth factor a-receptor subtype on human lung fibroblasts in vitro. Am J Respir Cell Mol Biol 13:496–505Google Scholar
  7. 7.
    Grinspan JB, Franceschini B (1995) Platelet-derived growth factor is a survival factor for PSA-NCAM+ oligodendrocyte pre-progenitor cells. J Neurosci Res 41:540–551Google Scholar
  8. 8.
    Hanna KD, Pouliquen Y, Waring GO, Savoldelli M, Cotter J, Morton K, Menasche M (1989) Corneal stromal wound healing in rabbits after 193 nm excimer laser surface ablation. Arch Ophthalmol 107: 895–901Google Scholar
  9. 9.
    Herbst H, Schuppan D, Milani S (1995) Fibrogenese und Fibrolyse in der Leber. Verh Dtsch Ges Pathol 79:15–27Google Scholar
  10. 10.
    Hersh PS, Schein OD, Steinert R, Summit Photorefractive Keratectomy Phase III Study Group (1996) Characteristics influencing outcomes of excimer laser photorefractive keratectomy. Ophthalmology 103:1962–1969Google Scholar
  11. 11.
    Hoppe J, Weich HA, Eichner W (1989) Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed inE. coli. Biochemistry 28:2956–2960Google Scholar
  12. 12.
    Hoppe J, Weich HA, Eichner W, Tatje D (1990) Preparation of biologically active platelet-derived growth factor isoforms AA and AB. Eur J Biochem 187:207–214Google Scholar
  13. 13.
    Hoppenreijs VPT, Pels E, Vrensen GFJM, Felten PC, Treffers WF (1993) Platelet-derived growth factor: receptor expression in corneas and effects on corneal cells. Invest Ophthalmol Vis Sci 34:637–649Google Scholar
  14. 14.
    Hoppenreijs VPT, Pels E, Vrensen GFJM, Treffers WF (1994) Effects of platelet-derived growth factor on endothelial wound healing of human corneas. Invest Ophthalmol Vis Sci 35:150–161Google Scholar
  15. 15.
    Katakami C, Sahori A, Kazusa R, Yamamoto M (1991) Keratocyte activity in wound healing after epikeratophakia in rabbits. Invest Ophthalmol Vis Sci 32: 1837–1845Google Scholar
  16. 16.
    Li DQ, Tseng SCG (1995) Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of human ocular surface. J Cell Physiol 163:61–79Google Scholar
  17. 17.
    Li DQ, Tseng SCG (1996) Differential regulation of cytokine and receptor transcript expression in human corneal and limbal fibroblasts by epidermal growth factor, transforming growth factor-α, platelet-derived growth factor B, and interleukin-1β. Invest Ophthalmol Vis Sci 37:2068–2080Google Scholar
  18. 18.
    Martinet Y, Bitterman PB, Mornex JF, Grotendorst GR, Martin GR, Crystal RG (1986) Activated human monocytes express the c-sis photo-oncogene and release a mediator showing PDGF-like activity. Nature 319:158–160Google Scholar
  19. 19.
    Murali S, Hardten DR, DeMartelaere S, et al (1994) Effect of topically administered platelet-derived growth factor on corneal wound strength. Curr Eye Res 13: 857–862Google Scholar
  20. 20.
    Peng Q, Solomon KD, Kent DG, Ahmad OF, Apple DJ (1996) Comparison of keratocyte activation in laser in situ keratomileusis and photorefracive keratectomy. Invest Ophthalmol Vis Sci 37:S63Google Scholar
  21. 21.
    Pierce GF, Mustoe TA, Altrock BW, Deue1 TF, Thomason A (1991) Role of platelet-derived growth factor in wound healing. J Cell Biochem 45:319–326Google Scholar
  22. 22.
    Pop M, Aras M (1996) Photorefractive keratectomy retreatments for regression. One year follow-up. Ophthalmology 103:1979–1984Google Scholar
  23. 23.
    Remmers EF, Sano H, Wilder RL (1991) Platelet-derived growth factors and heparin-binding (fibroblast) growth factors in the synovial tissue pathology of rheumatoid arthritis. Semin Arthritis Rheum 21: 191–199Google Scholar
  24. 24.
    Sporn MB, Roberts AB (1993) A major advance in the use of growth factors to enhance wound healing. J Clin Invest 92:2565–2566Google Scholar
  25. 25.
    Szerenyi KD, Xun Wei W, Gabrielian K, McDonnell PJ (1994) Keratocyte loss and repopulation of anterior corneal stroma after de-epithelialization. Arch Ophthalmol 112: 973–976Google Scholar
  26. 26.
    Tripathi RC, Borisuth NSC, Tripathi BJ (1991) Growth factors in the aqueous humor and their therapeutic implications in glaucoma and anterior segment disorders of the human eye. Drug Dev Res 22:1–23Google Scholar
  27. 27.
    Wilson SE, He Y, Lloyd SA (1992) EGF, EGF Receptor, basic FGF, TGF beta-1, and IL-1 alpha mRNA in human corneal epithelial cells and stromal fibroblasts. Invest Ophthalmol Vis Sci 33:1756–1765Google Scholar
  28. 28.
    Wilson SE, He YG, Weng J, Li Q, McDowall AW, Vital M, Chwang EL (1996a) Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 62:325–327Google Scholar
  29. 29.
    Wilson SE, Li Q, Weng J, Barry Lane PA, Jester JV, Liang Q, Wordinger RJ (1996b) The Fas-Fas ligand system and other modulators of apoptosis in the cornea. Invest Ophthalmol Vis Sci 37:1582–1592Google Scholar
  30. 30.
    Zhang JZ, Maruyama K, Ono I, Kaneko F (1995) Production and secretion of platelet-derived growth factor AB by cultured human keratinocytes: regulatory effects of phorbol 12-myristate 13-acetate, etretinate, 1,25-dihydroxyvitamin D3, and several cytokines. J Dermatol 22: 305–309Google Scholar

Copyright information

© Springer-Verlag 1997

Authors and Affiliations

  • Peter Oliver Denk
    • 1
  • Marcus Knorr
    • 1
  1. 1.University Eye Hospital TübingenTübingenGermany

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