Role of Urokinase Type Plasminogen Activator (u-PA) in Corneal Epithelial Migration

Summary

The role of plasminogen activator (PA) in the migration of comeal reepithelialization was studied. Rabbit corneal blocks were cultured, and both the extent of epithelial migration over the exposed corneal stroma and the activity of PA released into the culture media were measured. A significant, direct correlation between epithelial migration and PA activity in the medium was observed, even when the migration was stimulated by fibronectin or EGF, or was inhibited by cytochalasin B or cycloheximide. Zymography confirmed that the PA released into the culture medium was of the urokinase type (u-PA). Immunohistochemical studies showed that u-PA and plasmin(ogen) were present at the leading edge of the migrating epithelium. Studies of corneal cell cultures indicated that epithelial cells rather than endothelial cells or fibroblasts were the source of the u-PA. The addition of anti-human u-PA IgG or protease inhibitors retarded the migration of the comeal epithelium in a dose-dependent manner, indicating that u-PA activity is essential for the migration of the corneal epithelium. These findings suggest that the migration of corneal epithelial cells requires not only cell attachment to the extracellular matrix through the fibronectin but also degradation of the fibronectin by the release of cellular u-PA.

• References

• 1 Fujikawa LS, Foster CS, Harrist TJ, Lanigan JM, Colvin RB. Fibronectin in healing rabbit corneal wounds. Lab Invest 1981; 45: 120-129
• 2 Nishida T, Nakagawa S, Watanabe K, Yamada KM, Otori T, Berman MB. A peptide from fibronectin cell-binding domain inhibits attachment of epithelial cells. Invest Ophthalmol Vis Sci 1988; 29: 1820-1825
• 3 Berman M, Manseau E, Law M, Aiken D. Ulceration is correlated with degradation of fibrin and fibronectin at the corneal surface. Invest Ophthalmol Vis Sci 1983; 24: 1358-1366
• 4 Trinkaus-Randall V, Newton AW, Franzblau C. The synthesis and role of integrin in corneal epithelial cells in culture. Invest Ophthalmol Vis Sci 1990; 31: 440-447
• 5 Grushkin-Lerner LS, Trinkaus-Randall V. Localization of integrin and syndecan in vivo in a corneal epithelial abrasion and keratectomy. Curr Eye Res 1991; 10: 75-85
• 6 Paallysaho T, Williams DS. Epithelial cell-substrate adhesion in the cornea of actin, talin, integrin and fibronectin. Exp Eye Res 1991; 52: 261-267
• 7 Tervo K, Tervo T, van Setten G-B, Virtanen I. Integrins in human corneal epithelium. Cornea 1991; 10: 461-465
• 8 Murakami J, Nishida T, Otori T. Coordinated appearance of β₁ integrins and fibronectin during corneal wound healing. J Lab Clin Med 1992; 120: 86-93
• 9 Ossowski L, Quigley JP, Reich E. Plasminogen, a necessary factor for cell migration in vitro. In: Proteases and Biological Control. Reich E, Rifkin DB, Shaw E. (eds) Cold Spring Harbor Laboratory Press; New York: 1975: 901-913
• 10 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
• 11 Mignatti P, Robbins E, Rifkin DB. Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade. Cell 1986; 47: 487-498
• 12 Saksela O, Rifkin DB. Cell associated plasminogen activation: regulation and physiological functions. Annu Rev Cell Biol 1988; 4: 93-126
• 13 Morioka S, Jensen PJ, Lazarus GSD. Human epidermal plasminogen activator: characterization, localization, and modulation. Exp Cell Res 1985; 161: 364-372
• 14 Morioka S, Lazarus GS, Baird JL, Jensen PJ. Migrating keratinocytes express urokinase-type plasminogen activator. J Invest Dermatol 1987; 88: 418-423
• 15 Tervo T, Salonen E-M, Vaheri A, Immonen I, van Setten G-B, Himmberg JJ, Tarkkanen A. Elevation of tear fluid plasmin in corneal disease. Acta Ophthalmol 1988; 66: 393-399
• 16 Salonen E-M, Tervo T, Törma E, Tarkkanen A, Vaheri A. Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy. Acta Ophthalmol 1987; 65: 3-12
• 17 Hayashi K, Sueishi K. Fibrinolytic activity and species of plasminogen activator in human tears. Exp Eye Res 1988; 46: 131-137
• 18 Barlati S, Marchina E, Quaranta CA, Vigasio F, Semeraro F. Analysis of fibronectin, plasminogen activators and plasminogen in tear fluid as markers of corneal damage and repair. Exp Eye Res 1990; 51: 1-9
• 19 Nishida T, Nakagawa S, Awata T, Ohashi Y, Watanabe K, Manabe R. Fibronectin promotes epithelial migration of cultured rabbit cornea in situ. J Cell Biol 1983; 97: 1653-1657
• 20 Friberger P, Knös M, Gustavsson S, Aurell L, Cleason G. Methods for determination of plasmin, antiplasmin and plasminogen by means of substrate S-2251. Haemostasis 1978; 7: 138-145
• 21 Matsuo O, Sakai T, Bando H, Okada K, Nakajima S, Takagi O, Izaki S. Plasminogen activator in bronchoalveolar fluid. Haemostasis 1986; 16: 43-50
• 22 Awata T, Nishida T, Nakagawa S, Manabe R. Differential regulation of fibronectin synthesis in three different types of corneal cells. Jpn J Ophthalmol 1989; 33: 132-143
• 23 Nishida T, Ueda A, Fukuda M, Mishima H, Yasumoto K, Otori T. Interactions of extracellular collagen and corneal fibroblasts: Morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix. In Vitro Cell Dev Biol 1988; 24: 1009-1014
• 24 Nishida T, Otori T. Effects of intraocular irrigating solutions on the spreading of rabbit corneal endothelial cells on extracellular matrices. Jpn J Ophthalmol 1991; 35: 61-67
• 25 Berman M, Kenyon K, Hayashi K, L’Hernault N. The pathogenesis of epithelial defects and stromal ulceration. In: The Cornea: Transactions of the World Congress on the Cornea III. Cavanagh HD. (ed) Raven Press; New York: 1988: 35-43
• 26 Zieske JD, Bukusoglu G. Effect of protease inhibitors on corneal epithelial migration. Invest Ophthalmol Vis Sci 1991; 32: 2073-2078
• 27 Pandolfi M, Lantz E. Partial purification and characterization of keratokinase, the fibrinolytic activator of the cornea. Exp Eye Res 1979; 29: 563-571
• 28 Salvesen G, Nagase H. Inhibition of proteolytic enzymes. In: Proteolytic Enzymes, A Practical Approach. Beynon RJ, Bond JS. (eds) IRL Press; Oxford: 1989: 83-104
• 29 Nishida T, Ohashi Y, Awata T, Manabe R. Fibronectin: a new therapy for corneal trophic ulcer. Arch Ophthalmol 1983; 101: 1046-1048
• 30 Nishida T, Nakagawa S, Ohashi Y, Awata T, Manabe R. Fibronectin in corneal wound healing: appearance in cultured rabbit cornea. Jpn J Ophthalmol 1982; 26: 410-415
• 31 Gold LI, Schwimmer R, Quigley JP. Human plasma fibronectin as a substrate for human urokinase. Biochem J 1989; 262: 529-534
• 32 Jensen PJ, John M, Baird J. Urokinase and tissue type plasminogen activators in human keratinocyte culture. Exp Cell Res 1990; 187: 162-169
• 33 Mignatti P, Tsuboi R, Robbins E, Rifkin DB. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases. J Cell Biol 1989; 108: 671-682
• 34 Sato Y, Rifkin DB. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-β1-like molecule by plasmin during co-culture. J Cell Biol 1989; 109: 309-315