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Plasminogen activator and its inhibitor in the experimental corneal wound
Exp. Eye Res. (1989) 48. 445-449
P l a s m i n o g e n Activator and its Inhibitor in the E x p e r i m e n t a l Corneal W o u n d TIMO T E R V O * t , K A A R I ~ ' A TERVO~, GYSBERT-BOTHO VAN SETTESIt, [SMO VIRTANE~N~ AND AHT1 TARKKANEN t
Departments of Ophthalmologyr and Anatomy$ University of Helsinki, Finland (Received 23 August 1988) Anterior keratectomy was performed to 10 eyes of 8 adult rabbits. The animals were killed 3-24 hr after wound closure. The corneas were excised and subjected to histochemical demonstration of urokinase-type plasminogen activator (u-PA), tissue-type PA (t-PA) and plasminogen activator inhibitor ((PAL 1) using commercial antibodies. A strong immunoreaction for u-PA, a weaker reaction for PAL1 and a very weak t-PA-like immunoreaction appeared in the anterior stroma of the wounded area. None of the antisera used showed any immunostaining in the cornea outside the wound. The role of the plasminogen activator-plasmin system in the healing of the corneal wound is discussed. Key words:cornea, keratectomy, plasminogen activator, plasminogen activator-inhibitor.
1. I n t r o d u c t i o n P l a s m i n o g e n a c t i v a t o r s (PAs) refer to the serine p r o t e i n a s e s t h a t c o n v e r t p l a s m i n o g e n to active p l a s m i n (Christman, Silverstein a n d Acs, 1977 ; D a n o et al., 1985). There are two d i s t i n c t t y p e s of PA, tissue or v a s c u l a r t y p e of P A ( t : P A ) a n d urokinase (u-PA) (Dano et al., 1985). F i b r i n o l y t i c a c t i v i t y in t h e cornea was first d e t e c t e d h i s t o c h e m i c a l l y b y Pandolfi a n d A s t r u p (1967) a n d l a t e r p a r t i a l l y purified a n d c h a r a c t e r i z e d as similar to u - P A (Pandolfi a n d L a n t z , 1979). I n culture fluid from corneas the p r o t e o l y t i c a c t i v i t y was shown to be lower t h a n in c u l t u r e fluid o b t a i n e d from c o n j u n c t i v a l tissue ( L a n t z a n d Pandolfi, 1986). W a n g , B e r m a n a n d L a w (1983) d e m o n s t r a t e d t h a t corneal u l c e r a t i o n is c o r r e l a t e d w i t h the a p p e a r a n c e of active p l a s m i n o g e n a c t i v a t o r s . R e c e n t l y Chan (1986) showed using an in v i t r o m o d e l t h a t w o u n d i n g of t h e c u l t u r e d corneal e p i t h e l i u m increases p r o d u c t i o n of P A significantly. W e h a v e d e m o n s t r a t e d p r o t e o l y t i c a c t i v i t y due to p l a s m i n in t h e t e a r fluid of p a t i e n t s with corneal epithelial lesions. Moreover, use of a t o p i c a l serine p r o t e a s e inhibitor, a p r o t i n i n , was followed b o t h b y decrease of p l a s m i n a c t i v i t y in the t e a r s a n d resulted in r a p i d corneal healing (Tervo, Salonen a n d Vaheri, 1986; Salonen, Tervo, TSrmii, T a r k k a n e n a n d Vaheri, 1987). R e c e n t studies confirmed the occurrence of p l a s m i n in the t e a r fluid of p a t i e n t s with corneal epithelial disorders (Tervo et al., 1988a) a n d d u r i n g c o n t a c t lens wear (Tervo et al., 1988b).
2. M a t e r i a l s a n d M e t h o d s In order to define the site of plasminogen activation in vivo as a response to corneal wounds an experimental 350/~m deep anterior keratectomy was performed to 10 rabbit eyes using a 6 mm trephine. Post-operatively, gentamycin ointment was administered once a day on both eyes. The re-epithelialization was followed by daily staining with fluorescein. The * To whom correspondance should be addressed at Department of Ophthalmology, University of Helsinki, SF-00290 Helsinki, Finland. 00l 4-4835/89/030445 + 05 $03.00/0
9 1989 Acadenlic Press Limited
446
T. T E R V O
E T AL.
PA AND PAI IN CORNEAL WOUND
447
animals were killed 3-24 hr after wound closure. The corneas were immediately fixed at +4~ in 96% ethanol for 1-2 hr, rehydrated and immersed overnight in 0"l M phosphatebuffered saline (PBS), pH 7-4, containing 25% w/v sucrose and then cut on a cryostat. The indirect immunofluorescence method was used for demonstration of t-PA, u-PA and P A L l (inhibitor of plasminogen activator, type 1). The primary antibodies used were monoclonal mouse hybridoma antibodies against human t-PA, u-PA and P A L I (American Diagnostiea, Greenwich, CT) diluted 1 : 50. Fluoreseein isothioeyanate-eonjugated sheep anti-mouse IgG (Cappel Laboratories, Malvern, PA) served as secondary antibody. In control sections either the primary or secondary antibodies were omitted. Some of the sections were also incubated with an antiserum preabsorbed either with human u-PA or t-PA (immunoblocking).
3. R e s u l t s a n d D i s c u s s i o n
Re-epithelialization after Keratectomy L a m e l l a r , 350 # m deep a n t e r i o r k e r a t e c t o m y resulted in a s t a n d a r d i z e d corneal wound. I n eight eyes epithelial healing t i m e was 109-144 hr. In two eyes the epithelial defect persisted d u r i n g the time of o b s e r v a t i o n (7 days).
Immunohistochemistry I n the n o n - w o u n d e d areas of the corneas e x a m i n e d no t - P A , u - P A or P A I - l i k e i m m u n o r e a c t i v i t i e s were observed. H o w e v e r , a v e r y weak epithelial i m m u n o r e a c t i o n m i g h t have r e m a i n e d u n d e t e c t e d due to t h e b a c k g r o u n d fluorescence. A clear staining for t - P A of the e p i t h e l i u m as r e p o r t e d earlier b y T r i p a t h i , Geanon and T r i p a t h i (1987) could n o t be observed. I n the corneal w o u n d s e x a m i n e d 3 24 hr after healing a strong i m m u n o r e a c t i v i t y for u - P A (Fig. 1) a n d a weak r e a c t i o n for t - P A (Fig. 2) was found in the a n t e r i o r s t r o m a . T h e w o u n d edge (arrows) showed t h e m o s t intense immunofluorescence (Fig. 1). A t the same site, also a m o d e r a t e P A I - l i k e i m m u n o r e a c t i o n was o b s e r v e d (Fig. 3). Tissue sections which were i n c u b a t e d w i t h o u t either t h e p r i m a r y or s e c o n d a r y a n t i b o d y were negative. The role of the p l a s m i n o g e n a c t i v a t o r - p l a s m i n s y s t e m in corneal p a t h o p h y s i o l o g y has become a s u b j e c t of g r e a t interest. P l a s m i n o g e n a c t i v a t o r is p r o b a b l y i n v o l v e d in collagen d e g r a d a t i o n in u l c e r a t e d corneas (Berman, L e a f y a n d Gage, 1980). A c t i v a t e d p l a s m i n can d e g r a d e m a n y m a t r i x proteins, such as fibronectin a n d laminin, a n d can f u r t h e r m o r e a c t i v a t e o t h e r e n z y m e s such as procollagenases a n d l a t e n t m a c r o p h a g e elastase, t h u s p o t e n t i a l l y leading to c o m p l e t e m a t r i x d e g r a d a t i o n (Dano et al., 1985). Consequently, the p l a s m i n g e n e r a t e d m a y i m p a i r e p i t h e l i a l w o u n d healing b y cleaving t h e b a s a l l a m i n a m a t r i x p r o t e i n s (Berman, Manseau, L a w a n d Aiken, 1983). This m i g h t e x p l a i n t h e p r o m i s i n g t h e r a p e u t i c results achieved in the t r e a t m e n t of corneal ulcers with fibronectin alone (Nishida, Ohashi, A w a t a a n d Manabe, 1983; H a r n i s h a n d Sinha, 1985), a n d / o r with a topical t r e a t m e n t w i t h p r o t e i n a s e i n h i b i t o r a p r o t i n i n (Tervo et al., 1986; Salonen et al., 1987).
Fro. 1. u-PA like immunoreaction in the healed keratectomy wound. Note the shang reaction at the wound edge (arrow). x 320. Fro. 2. t-PA-like immunoreaction at the corneal wound. The epithelium shows more intense background reaction than the sections incubated with antibodies to u-PA but the sub-epithelial reaction is less intense (compared to Fig. 1). x 320. FIG. 3. Immunoreaction for PAI-I in the corneal wound. A moderate immunoreaction can be observed in the anterior strom~ at the wound area. x 320.
448
T. TERVO ET AL.
The present results suggest that corneal wounding leads to an expression of u-PA gene in the epithelium especially in the wound margins. Matrix components as fibronectin and laminin are capable of binding PA (Salonen, Zitting and Vaheri, 1984; Salonen et al., 1985). These are exposed at the bottom of the wound, where u-PA was demonstrable by means of immunohistochemistry. Our results also point out that the wound edge is the most critical area in corneal wound healing. Here PAs have been suggested to initiate plasmin-dependent 'limited proteotysis' (Berman, Kenyon, Hayashi and L'Hernault, 1988). I t not only contains u-PA, but also other extracellular matrix proteins such as fibronectin (Fujikawa, Foster, Gibson and Colvin, 1984), laminin (Fujikawa et al., 1984) and tenascin (unpublished results by the authors). ACKNOWLEDGMENTS The authors are very grateful to the Paulo Foundation, Helsinki, the University of Helsinki, the Finnish Medical Research Council, the Sigrid Juselius Foundation and the Finnish Cancer Research Fund for financial support. REFERENCES Berman, M., Leary, R. and Gage, J. (1980). Evidence for a role of the plasminogen activatorplasmin system in corneal ulceration. Invest. Ophthalmol. Vis. Sci. 19, 1204-21. Berman, M., Manseau, E., Law, M. and Aiken, D. (1983). Ulceration is correlated with degradation of fibrin and fibronectin at the corneal surface. Invest. Ophthalmol. Vis. Sci. 24, 1358 66. Berman, M., Kenyon, K., Hayashi, K. and L'Hernault, N. (1988). The pathogenesis of epithelial defects and stromal ulceration. In The Cornea: Transactions of the World Congress on the Cornea III. (Ed. Cavannagh, H. D.) Pp. 35-43. Raven Press: New York. Chan, K. Y. {1986). Release of plasminogen activator by cultured corneal epithelial cells during differentiation and wound closure. Exp. Eye Res. 42, 417-31. Christman, J. K., Silverstein, S. C. and Acs, G. {1977). Plasminogen activators. Res. Monogr. Cell Tiss. Physiol. 2, 91-149. Dano, K., Andreasen, P.A., Grondahl-Hansen, J., Kristensen, P., Nielsen, L.S. and Skriver, L. (1985). Plasminogen activators and cancer. Adv. Cancer Res. 44, 139-266. Fujikawa, L. S., Foster, C. S., Gibson, J. K. and Colvin, R. B. (1984). Basement membrane components in healing rabbit corneal epithelial wounds: immunofiuorescence and ultrastructural studies. J. Cell Biol. 98, 128-38. Harnisch, J.-P. and Sinha, P.K. (1985). Fibronectin: Eine BehandlungsmSglichkeit therapieresistenter Hornhautulcera. Klin. Mbl. Augenheilk. 187, 53-6. Lantz, E. and Pandolfi, M. (1986). Fibrinolysis in cornea and conjunctiva: evidence of two types of activators. Albrecht yon Graefe's A~h. Clin. Exp. Ophthalmol. 224, 393-6. Nishida, T., Ohashi, Y., Awata, T. and Manabe, R. (1983). Fibronectin : a new therapy for corneal trophic ulcer. Arch. Ophthalmol. 101, 1046 8. Pandolfi, M. and Astrup, T. (1967). A histochemical study of the fibrinolytic activity. Cornea, conjunctiva and lacrimal gland. Arch. Ophthalmol. 77, 258--64. Pandolfi, M. and Lantz, E. (1979). Partial purifcation and characterization of keratokinase, the fibrinolytic activator of the cornea. Exp. Eye Res. 29, 563-71. Salonen, E.-M., Zitting, A. and Vaheri, A. (1984). Laminin interacts with plasminogen and its tissue type activator. FEBS Letters 172, 29-32. Salonen, E.-M., Saksela, O., Vartio, T., Vaheri, A., Nielsen, L. and Zeuthen, J. (1985). Plasminogen and its tissue-type activator bind to immobilized fibronectin. J. Biol. Chem. 260, 12302-7. Salonen, E.-M., Tervo, T., TSrm~:, E., Tarkkanen, A. and Vaheri, A. (1987). Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy. Acta Ophthalmol. 65, 3-12. Tervo, T., Salonen, E.-M. and Vaheri, A. (1986). A new therapy to promote corneal healing. Invest. Ophthalmol. Vis. Sci. (Suppl.) 27, 53.
PA AND PAI IN CORNEAL WOUND
449
Tervo, T., Salonen, E.-M., Vaheri, A., Immonen, I., van Setten, G.-B., Himberg, J. J. and Tarkkanen, A. (1988a). Elevation of tear fluid plasmin in corneal disease. Acta Ophthalmol. (in press). Tervo, T., van Setten, G.-B., Andersson, R., Salonen, E.-M., Vaheri, A., Immonen, I. and Tarkkanen, A. (1988b). Contact lens wear is associated with the appearence of plasmin in the tear fluid--preliminary results. Graef's Arch. Clin. Exp. Ophthalmol. (in press). Tripathi, B. J., Geanon, J. D. and Triphathi, R. C. (1987). Distribution of tissue plasminogen activator in human and monkey eyes. Ophthalmology 94, 1434-8. Wang, H.-M., Berman, M. and Law, M. (1983). Latent and active forms of plasminogen activator in corneal ulceration. Invest. Ophthalmol. Vis. Sci. (Suppl.) 24, 44.
P l a s m i n o g e n Activator and its Inhibitor in the E x p e r i m e n t a l Corneal W o u n d TIMO T E R V O * t , K A A R I ~ ' A TERVO~, GYSBERT-BOTHO VAN SETTESIt, [SMO VIRTANE~N~ AND AHT1 TARKKANEN t
Departments of Ophthalmologyr and Anatomy$ University of Helsinki, Finland (Received 23 August 1988) Anterior keratectomy was performed to 10 eyes of 8 adult rabbits. The animals were killed 3-24 hr after wound closure. The corneas were excised and subjected to histochemical demonstration of urokinase-type plasminogen activator (u-PA), tissue-type PA (t-PA) and plasminogen activator inhibitor ((PAL 1) using commercial antibodies. A strong immunoreaction for u-PA, a weaker reaction for PAL1 and a very weak t-PA-like immunoreaction appeared in the anterior stroma of the wounded area. None of the antisera used showed any immunostaining in the cornea outside the wound. The role of the plasminogen activator-plasmin system in the healing of the corneal wound is discussed. Key words:cornea, keratectomy, plasminogen activator, plasminogen activator-inhibitor.
1. I n t r o d u c t i o n P l a s m i n o g e n a c t i v a t o r s (PAs) refer to the serine p r o t e i n a s e s t h a t c o n v e r t p l a s m i n o g e n to active p l a s m i n (Christman, Silverstein a n d Acs, 1977 ; D a n o et al., 1985). There are two d i s t i n c t t y p e s of PA, tissue or v a s c u l a r t y p e of P A ( t : P A ) a n d urokinase (u-PA) (Dano et al., 1985). F i b r i n o l y t i c a c t i v i t y in t h e cornea was first d e t e c t e d h i s t o c h e m i c a l l y b y Pandolfi a n d A s t r u p (1967) a n d l a t e r p a r t i a l l y purified a n d c h a r a c t e r i z e d as similar to u - P A (Pandolfi a n d L a n t z , 1979). I n culture fluid from corneas the p r o t e o l y t i c a c t i v i t y was shown to be lower t h a n in c u l t u r e fluid o b t a i n e d from c o n j u n c t i v a l tissue ( L a n t z a n d Pandolfi, 1986). W a n g , B e r m a n a n d L a w (1983) d e m o n s t r a t e d t h a t corneal u l c e r a t i o n is c o r r e l a t e d w i t h the a p p e a r a n c e of active p l a s m i n o g e n a c t i v a t o r s . R e c e n t l y Chan (1986) showed using an in v i t r o m o d e l t h a t w o u n d i n g of t h e c u l t u r e d corneal e p i t h e l i u m increases p r o d u c t i o n of P A significantly. W e h a v e d e m o n s t r a t e d p r o t e o l y t i c a c t i v i t y due to p l a s m i n in t h e t e a r fluid of p a t i e n t s with corneal epithelial lesions. Moreover, use of a t o p i c a l serine p r o t e a s e inhibitor, a p r o t i n i n , was followed b o t h b y decrease of p l a s m i n a c t i v i t y in the t e a r s a n d resulted in r a p i d corneal healing (Tervo, Salonen a n d Vaheri, 1986; Salonen, Tervo, TSrmii, T a r k k a n e n a n d Vaheri, 1987). R e c e n t studies confirmed the occurrence of p l a s m i n in the t e a r fluid of p a t i e n t s with corneal epithelial disorders (Tervo et al., 1988a) a n d d u r i n g c o n t a c t lens wear (Tervo et al., 1988b).
2. M a t e r i a l s a n d M e t h o d s In order to define the site of plasminogen activation in vivo as a response to corneal wounds an experimental 350/~m deep anterior keratectomy was performed to 10 rabbit eyes using a 6 mm trephine. Post-operatively, gentamycin ointment was administered once a day on both eyes. The re-epithelialization was followed by daily staining with fluorescein. The * To whom correspondance should be addressed at Department of Ophthalmology, University of Helsinki, SF-00290 Helsinki, Finland. 00l 4-4835/89/030445 + 05 $03.00/0
9 1989 Acadenlic Press Limited
446
T. T E R V O
E T AL.
PA AND PAI IN CORNEAL WOUND
447
animals were killed 3-24 hr after wound closure. The corneas were immediately fixed at +4~ in 96% ethanol for 1-2 hr, rehydrated and immersed overnight in 0"l M phosphatebuffered saline (PBS), pH 7-4, containing 25% w/v sucrose and then cut on a cryostat. The indirect immunofluorescence method was used for demonstration of t-PA, u-PA and P A L l (inhibitor of plasminogen activator, type 1). The primary antibodies used were monoclonal mouse hybridoma antibodies against human t-PA, u-PA and P A L I (American Diagnostiea, Greenwich, CT) diluted 1 : 50. Fluoreseein isothioeyanate-eonjugated sheep anti-mouse IgG (Cappel Laboratories, Malvern, PA) served as secondary antibody. In control sections either the primary or secondary antibodies were omitted. Some of the sections were also incubated with an antiserum preabsorbed either with human u-PA or t-PA (immunoblocking).
3. R e s u l t s a n d D i s c u s s i o n
Re-epithelialization after Keratectomy L a m e l l a r , 350 # m deep a n t e r i o r k e r a t e c t o m y resulted in a s t a n d a r d i z e d corneal wound. I n eight eyes epithelial healing t i m e was 109-144 hr. In two eyes the epithelial defect persisted d u r i n g the time of o b s e r v a t i o n (7 days).
Immunohistochemistry I n the n o n - w o u n d e d areas of the corneas e x a m i n e d no t - P A , u - P A or P A I - l i k e i m m u n o r e a c t i v i t i e s were observed. H o w e v e r , a v e r y weak epithelial i m m u n o r e a c t i o n m i g h t have r e m a i n e d u n d e t e c t e d due to t h e b a c k g r o u n d fluorescence. A clear staining for t - P A of the e p i t h e l i u m as r e p o r t e d earlier b y T r i p a t h i , Geanon and T r i p a t h i (1987) could n o t be observed. I n the corneal w o u n d s e x a m i n e d 3 24 hr after healing a strong i m m u n o r e a c t i v i t y for u - P A (Fig. 1) a n d a weak r e a c t i o n for t - P A (Fig. 2) was found in the a n t e r i o r s t r o m a . T h e w o u n d edge (arrows) showed t h e m o s t intense immunofluorescence (Fig. 1). A t the same site, also a m o d e r a t e P A I - l i k e i m m u n o r e a c t i o n was o b s e r v e d (Fig. 3). Tissue sections which were i n c u b a t e d w i t h o u t either t h e p r i m a r y or s e c o n d a r y a n t i b o d y were negative. The role of the p l a s m i n o g e n a c t i v a t o r - p l a s m i n s y s t e m in corneal p a t h o p h y s i o l o g y has become a s u b j e c t of g r e a t interest. P l a s m i n o g e n a c t i v a t o r is p r o b a b l y i n v o l v e d in collagen d e g r a d a t i o n in u l c e r a t e d corneas (Berman, L e a f y a n d Gage, 1980). A c t i v a t e d p l a s m i n can d e g r a d e m a n y m a t r i x proteins, such as fibronectin a n d laminin, a n d can f u r t h e r m o r e a c t i v a t e o t h e r e n z y m e s such as procollagenases a n d l a t e n t m a c r o p h a g e elastase, t h u s p o t e n t i a l l y leading to c o m p l e t e m a t r i x d e g r a d a t i o n (Dano et al., 1985). Consequently, the p l a s m i n g e n e r a t e d m a y i m p a i r e p i t h e l i a l w o u n d healing b y cleaving t h e b a s a l l a m i n a m a t r i x p r o t e i n s (Berman, Manseau, L a w a n d Aiken, 1983). This m i g h t e x p l a i n t h e p r o m i s i n g t h e r a p e u t i c results achieved in the t r e a t m e n t of corneal ulcers with fibronectin alone (Nishida, Ohashi, A w a t a a n d Manabe, 1983; H a r n i s h a n d Sinha, 1985), a n d / o r with a topical t r e a t m e n t w i t h p r o t e i n a s e i n h i b i t o r a p r o t i n i n (Tervo et al., 1986; Salonen et al., 1987).
Fro. 1. u-PA like immunoreaction in the healed keratectomy wound. Note the shang reaction at the wound edge (arrow). x 320. Fro. 2. t-PA-like immunoreaction at the corneal wound. The epithelium shows more intense background reaction than the sections incubated with antibodies to u-PA but the sub-epithelial reaction is less intense (compared to Fig. 1). x 320. FIG. 3. Immunoreaction for PAI-I in the corneal wound. A moderate immunoreaction can be observed in the anterior strom~ at the wound area. x 320.
448
T. TERVO ET AL.
The present results suggest that corneal wounding leads to an expression of u-PA gene in the epithelium especially in the wound margins. Matrix components as fibronectin and laminin are capable of binding PA (Salonen, Zitting and Vaheri, 1984; Salonen et al., 1985). These are exposed at the bottom of the wound, where u-PA was demonstrable by means of immunohistochemistry. Our results also point out that the wound edge is the most critical area in corneal wound healing. Here PAs have been suggested to initiate plasmin-dependent 'limited proteotysis' (Berman, Kenyon, Hayashi and L'Hernault, 1988). I t not only contains u-PA, but also other extracellular matrix proteins such as fibronectin (Fujikawa, Foster, Gibson and Colvin, 1984), laminin (Fujikawa et al., 1984) and tenascin (unpublished results by the authors). ACKNOWLEDGMENTS The authors are very grateful to the Paulo Foundation, Helsinki, the University of Helsinki, the Finnish Medical Research Council, the Sigrid Juselius Foundation and the Finnish Cancer Research Fund for financial support. REFERENCES Berman, M., Leary, R. and Gage, J. (1980). Evidence for a role of the plasminogen activatorplasmin system in corneal ulceration. Invest. Ophthalmol. Vis. Sci. 19, 1204-21. Berman, M., Manseau, E., Law, M. and Aiken, D. (1983). Ulceration is correlated with degradation of fibrin and fibronectin at the corneal surface. Invest. Ophthalmol. Vis. Sci. 24, 1358 66. Berman, M., Kenyon, K., Hayashi, K. and L'Hernault, N. (1988). The pathogenesis of epithelial defects and stromal ulceration. In The Cornea: Transactions of the World Congress on the Cornea III. (Ed. Cavannagh, H. D.) Pp. 35-43. Raven Press: New York. Chan, K. Y. {1986). Release of plasminogen activator by cultured corneal epithelial cells during differentiation and wound closure. Exp. Eye Res. 42, 417-31. Christman, J. K., Silverstein, S. C. and Acs, G. {1977). Plasminogen activators. Res. Monogr. Cell Tiss. Physiol. 2, 91-149. Dano, K., Andreasen, P.A., Grondahl-Hansen, J., Kristensen, P., Nielsen, L.S. and Skriver, L. (1985). Plasminogen activators and cancer. Adv. Cancer Res. 44, 139-266. Fujikawa, L. S., Foster, C. S., Gibson, J. K. and Colvin, R. B. (1984). Basement membrane components in healing rabbit corneal epithelial wounds: immunofiuorescence and ultrastructural studies. J. Cell Biol. 98, 128-38. Harnisch, J.-P. and Sinha, P.K. (1985). Fibronectin: Eine BehandlungsmSglichkeit therapieresistenter Hornhautulcera. Klin. Mbl. Augenheilk. 187, 53-6. Lantz, E. and Pandolfi, M. (1986). Fibrinolysis in cornea and conjunctiva: evidence of two types of activators. Albrecht yon Graefe's A~h. Clin. Exp. Ophthalmol. 224, 393-6. Nishida, T., Ohashi, Y., Awata, T. and Manabe, R. (1983). Fibronectin : a new therapy for corneal trophic ulcer. Arch. Ophthalmol. 101, 1046 8. Pandolfi, M. and Astrup, T. (1967). A histochemical study of the fibrinolytic activity. Cornea, conjunctiva and lacrimal gland. Arch. Ophthalmol. 77, 258--64. Pandolfi, M. and Lantz, E. (1979). Partial purifcation and characterization of keratokinase, the fibrinolytic activator of the cornea. Exp. Eye Res. 29, 563-71. Salonen, E.-M., Zitting, A. and Vaheri, A. (1984). Laminin interacts with plasminogen and its tissue type activator. FEBS Letters 172, 29-32. Salonen, E.-M., Saksela, O., Vartio, T., Vaheri, A., Nielsen, L. and Zeuthen, J. (1985). Plasminogen and its tissue-type activator bind to immobilized fibronectin. J. Biol. Chem. 260, 12302-7. Salonen, E.-M., Tervo, T., TSrm~:, E., Tarkkanen, A. and Vaheri, A. (1987). Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy. Acta Ophthalmol. 65, 3-12. Tervo, T., Salonen, E.-M. and Vaheri, A. (1986). A new therapy to promote corneal healing. Invest. Ophthalmol. Vis. Sci. (Suppl.) 27, 53.
PA AND PAI IN CORNEAL WOUND
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