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Tear Hepatocyte Growth Factor (HGF) Availability Increases Markedly after Excimer Laser Surface Ablation
Exp. Eye Res. (1997) 64, 501–504
Tear Hepatocyte Growth Factor (HGF) Availability Increases Markedly after Excimer Laser Surface Ablation T I M O T E R V Oa, M I N N A V E S A L U O M Aa, G R E G L. B E N N E T Tb, R A L P H S C H W A L Lb, M A R C O H E L E N Ac Q I A N W A L I A N Gc S T E V E N E. W I L S O Nc * a
Department of Ophthalmology, University of Helsinki, Helsinki, Finland, b Genentech, Inc., South San Francisco, CA, c Eye Institute and the Department of Cell Biology, The Cleveland Clinic Foundation, Cleveland, OH, U.S.A. (Received Columbia 29 August 1996 and accepted in revised form 14 August 1996) Tear cytokines and growth factors are likely to modulate the wound healing process following corneal epithelial injury. Hepatocyte growth factor (HGF) is a paracrine mediator of epithelial proliferation, motility, and differentiation that is produced by keratocytes and the lacrimal gland. Tear samples were collected preoperatively and one, two, and seven days postoperatively in eyes undergoing excimer laser surface ablation [photorefractive keratoplasty (PRK) or phototherapeutic keratoplasty (PTK)]. Tear HGF concentration was measured with a sensitive ELISA assay, Tear HGF production was calculated using the tear flow rate in the collection capillary and HGF concentration. Although the instantaneous concentration of HGF in tears decreased significantly in the days following PRK, a large increase in tear flow resulted in a marked increase in HGF bioavailability. The heparin-binding characteristics of HGF would result in increased binding to glycosaminoglycans and other heparin-like matrix components and, therefore, increased growth factor availability to the cognate receptor. This is the first report documenting changes in tear film HGF production. HGF may have an important function in maintenance and wound healing of the ocular surface epithelium since HGF is present in the normal tear film and the HGF secretion rate increases markedly in parallel with aqueous tear production following corneal surgical injury. # 1997 Academic Press Limited Key words: hepatocyte growth factor ; cornea ; wound healing ; lacrimal gland ; tears ; photorefractive keratectomy.
1. Introduction Cytokines, growth factors, other tear components, and receptors expressed by responsive cells are likely to contribute to normal maintenance of the ocular surface and to corneal wound healing after injury (Wilson, 1991 ; Wilson, Lloyd and Kennedy, 1991 ; van Setten et al., 1989 ; Tervo K et al., 1990 ; Tsutsumi, Tsutsumi and Takami, 1988 ; Ohashi et al., 1989 ; Ubels, Foley and Rismondo, 1986 ; van Setten and Schultz, 1994). These components may be derived from the lacrimal gland, conjunctival vessels, corneal cells, conjunctival cells, and other sources. Studies on the production and function of individual growth factors are likely to yield important insights that will eventually lead to strategies for modulating the wound healing process after corneal surgery. The role of hepatocyte growth factor (HGF) and HGF receptor in corneal maintenance and wound healing has been an area of intense investigation. HGF, also known as the scatter factor, is best characterized as a classical paracrine mediator of stromal-epithelial interactions secreted by fibroblast cells to modulate the functions of epithelial cells in numerous tissues * For correspondence at : Eye Institute}A31, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, U.S.A. Presented in part at the Association for Research in Vision and Ophthalmology annual meeting Ft Lauderdale, FL, April 1996.
0014-4835}97}04050104 $25.00}0}ey96226
(Nakamura et al., 1989 ; Matsumoto et al., 1991 ; Rubin et al., 1991 ; Montesano et al., 1991 ; Wilson et al., 1994a). HGF and HGF receptor are expressed in corneal cells (Wilson et al., 1993, Li et al., 1996) and HGF has been shown to modulate proliferation, motility, and differentiation of corneal epithelial cells (Wilson et al., 1994b). Recently, we have shown that HGF is also produced in lacrimal gland fibroblast cells (Li et al., 1996). In addition to having local effects on epithelial cells within the lacrimal tissue, HGF collects in the ducts and becomes a component in tears. In the present study, changes in tear HGF concentration and production were monitored in human eyes following excimer laser surface ablation. 2. Materials and Methods Nineteen eyes from 19 patients undergoing excimer laser surface photoablation [14 photorefractive keratectomy (PRK) for myopia and five for phototherapeutic keratectomy (PTK)] were included in the study. Immediately prior to medication or other preoperative manipulations excess tears were cleared by first gently absorbing them from the lid margin with a tissue wipe. A 10 to 30 µl sample of tears was collected with a calibrated, polished glass capillary. Care was taken to minimally stimulate the ocular surface. The time required to collect the sample was recorded and the rate of tear flow was determined as # 1997 Academic Press Limited
502
previously described (Tervo et al., 1994a ; Tervo et al., 1994b). The tear sample was centrifuged at 12 000 g to remove any cells or debri and the supernatant immediately transferred to a cryovial and maintained at ®72 °C to ®85 °C until analysis. Similarly, tear samples were collected on the first, second, and seventh day following PRK. Each eye received Liquifilm-FML twice a day and chloramphenicol ointment (Parke-Davis) once or twice a day during the postoperative interval that tears were collected. In addition, each patient received oral diclotenac 25 mg two to three times per day for the first two days after surgery. No medications were given within two hours of tear collection at the postoperative examinations. The HGF-specific antibody used to perform the ELISA assay and the ELISA method have been previously reported (Li et al., 1996). Briefly, all of the samples were evaluated simultaneously using 96-well Maxisorb microtiter plates (Nunc, Rosklide, Denmark) coated with A 3.1.2 anti-HGF monoclonal antibody. After blocking and washing plates, 100 µl per well standard HGF or serially diluted tear samples, samples, starting at 1 : 10, were added and incubated overnight. After washing the plates, 100 µl per well biotinylated sheep anti-HGF were added and incubated for 2 hr. One hundred microliters per well horseradish peroxidase-conjugated streptavidin (Zymed, South San Francisco, CA, U.S.A.) were added to washed plates and incubated for 30 minutes. After washing, 100 µl per well ELAST4 biotinyl-tyramide solution (DuPont NEN, N. Billerica, MA, U.S.A.) were added. After a 15 minute incubation, plates were washed, and 100 µl per well horseradish peroxidase-conjugated streptavidin were again added and incubated for 30 minutes. After washing the plates, 100 µl per well 3, 3, 5, 5«tetramethylbenzidine substrate (Kirkegaard and Perry, Gaithersburg, MD, U.S.A.) were added. Color development was stopped after 5 minutes with 100 µl per well of 1 phosphoric acid. Plates were read at 450 nm on an automated plate reader. Data was reduced by a Parameter curve fitting program. The sensitivity of the HGF ELISA was approximately 150 pg ml−". The rate of HGF production in pg min−" was determined by multiplying the tear flow in µl min−" by the HGF concentration in pg µl−". Variations were expressed as the mean³the standard error of the mean. Statistical significance was determined with the Mann-Whitney U-test and P ! 0±05 was considered statistically significant. 3. Results Several patients had one or more tear samples with HGF concentrations below the sensitivity of the assay and these samples were excluded from statistical analysis. Mean and ... values for tear flow rate (µl min−"), HGF concentration (pg ml−"), and HGF production (pg min−") are provided in Table I for the
T. T E R V O E T A L.
preoperative, one day postoperative, two day postoperative, and seven day postoperative examinations. Percent change in tear HGF production relative to the preoperative level for each postoperative day is also provided. Percent change in HGF production compared with the preoperative level for individual eyes on each day after surgery are provided in Table I. There was no difference in the increased tear HGF production response between eyes that underwent PRK and those that had PTK. Mean percent change in tear HGF production for the postoperative visits are provided in Table I. Mean HGF production was markedly increased on the first and second postoperative days and the difference compared with the preoperative value was statistically significant (P ! 0±001). In this relatively small study, a correlation between the levels of HGF in tears and the clinical rate of healing of the epithelium after surgery has not detected. The epithelium in all eyes healed within three days of surgery. Discussion The results of this study demonstrate that there is a marked increase in tear HGF availability in the majority of human eyes following corneal epithelial and stromal wounding. Tear HGF production may also have been increased in eyes with HGF concentrations below the sensitivity of the ELISA assay, but a more sensitive ELISA would be needed to analyse these samples. Even though the instantaneous concentration of HGF in the tears was actually decreased on the first and second postoperative day in most patients, marked increases in the flow of tears resulted in a large increase in total tear HGF availability. There is likely to be a large increase in the bioavailability of HGF for binding to HGF receptors on the surface of corneal epithelial cells since HGF is a heparin-binding growth factor that adheres to glycosaminoglycans and other components in basement and cell membranes (Nakamura et al., 1989 ; Matsumoto et al., 1991 ; Rubin et al., 1991 ; Montesano et al., 1991). Once bound to these low affinity receptors the HGF should be available for binding to the high affinity cognate HGF receptor. The possibility that HGF in tears binds to some soluble factor or factors that limit its bioavailability cannot be excluded, although soluble receptors for HGF have not been identified. What is the source of the increased tear HGF production following corneal wounding ? It has recently been demonstrated that HGF production is increased in keratocytes following corneal epithelial wounding (Li et al., 1996). It is likely that IL-1 released from corneal epithelial cells with injury increases HGF mRNA transcription in keratocytes (unpublished data). Since the epithelial barrier function is lost during the early stages following excimer laser surface ablation because of an epithelial defect of size lasting for the first two to three days, it is likely
TEAR HGF PRODUCTION FOLLOWING EXCIMER LASER SURFACE ABLATION
that the keratocytes are one source of the increased tear HGF production. We have, however, also demonstrated that HGF is produced by the lacrimal gland (Li et al., 1996). Thus, lacrimal gland HGF production could also be increased, although the decrease in HGF concentration would indicate that the increase is less than for the aqueous component of tears (Table I). Further study will be needed to determine whether HGF mRNA and}or protein production is increased in the lacrimal gland following ocular surface injury. What function is served by the increased HGF that is available to the epithelial cells during the early postoperative period following corneal wounding ? HGF has been shown to increase proliferation, increase motility, and inhibit differentiation of human corneal epithelial cells (Li et al., 1996). Conversely, HGF appears to have no effect on the stromal fibroblast cells (Li et al., 1996). Thus, it is likely that HGF modulates processes that contribute to epithelial wound healing following corneal injury. The relationship between HGF and epidermal growth factor (van Setten et al., 1989 ; Tervo et al., 1990), transforming growth factor alpha (van Setten and Schultz, 1994), and other tear components following corneal epithelial wounding has yet to be determined. Could exogenous topical HGF be of therapeutic benefit to facilitate corneal epithelial wound healing ? The large increases that occur in tear HGF production in most patients following corneal injury suggest that this is unlikely to be the case. It is possible, however, that deficient HGF production could be detected in some patients with persistent epithelial defects. Any studies to determine the efficacy of HGF in stimulating corneal epithelial wound healing should concentrate on the latter group of patients. Prediction of the potential therapeutic effects of individual growth factors is difficult, however, without a more complete understanding of the complex interactions that occur between the numerous growth factors, cytokines, and
503
receptors expresses by corneal and lacrimal gland cells. Acknowledgement Supported in part by US Public Health Service grant EY10056 from the National Eye Institute, National Institutes of Health, Bethesda, Maryland, The Cleveland Clinic Foundation, The Mary and Georg Ehrnrooth Foundation, The Finnish Eye and Tissue Bank Foundation, and The Friends of Blind, Finland.
References Li, Q., Weng, J., Bennett, G. L., Schwall, R., Wang, Z-F., Tabor, T., Kim, J., Hargrave, S., Cuevas, K. H. and Wilson, S. E. (1996). Hepatocyte Growth Factor (HGF) and HGF receptor protein in lacrimal gland, tears, and cornea. Invest. Ophthalmol. Vis. Sci. 37, 727–39. Matsumoto, K., Hashimoto, K., Yoshikawa, K. and Nakamura, T. (1991). Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor. Exp. Cell Res. 96, 114–20. Montesano, R., Matsumoto, K., Nakamura, T. and Orci, L. (1991). Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67, 901–8. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K. and Smizu, S. (1989). Molecular cloning and expression of hepatocyte growth factor. Nature 342, 440–3. Ohashi, Y., Motokura, M., Kinoshita, Y., Mano, T., Wananabe, H., Kinoshita, S., Manabe, R., Oshiden, K. and Yanaihara, C. (1989). Presence of epidermal growth factor in human tears. Invest. Ophthalmol. Vis. Sci. 30, 1879–82. Rubin, J. S., Chan, A. M-L., Bottaro, D. P., Burgess, W. H., Taylor, W. G., Cech, A. C., Hirschfield, D. W., Wong, J., Miki, T., Finch, P. W. and Aaronson, S. A. (1991). A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor. Proc. Natl. Acad. Sci. 88, 415–9. Tervo, T., Honkanen, N., Van Setten, G. B., Virtanen, T., Tarkkanen, A. and Ha$ rko$ nen, M. (1994b). A rapid fluorometric assay for tear fluid plasmin activity. Cornea 13, 148–55.
504
Tervo, K., van Setten, G. B., Virtanen, I., Tarkkanen, A. and Tervo, T. (1990). The lacrimal gland is the source of epidermal growth factor in tear fluid—an immunochemical study in rats. Invest. Ophthalmol. Vis. Sci. 31 (Suppl), 45. Tervo, T., Virtanen, T, Honkanen, N., Ha$ rho$ nen, M. and Tarkkanen, A. (1994a). Tear fluid plasmin activity after excimer laser photorefractive keratectomy. Invest. Ophthalmol. Vis. Sci. 35, 3045–50. Tsutsumi, O., Tsutsumi, A. and Takami, O. (1988). Epidermal growthfactor-like, corneal wound healing substance in mouse tears. J. Clin. Invest. 81, 1067–71. Ubels, J. L., Foley, K. M. and Rismondo, V. (1986). Retinol secretion by the lacrimal gland. Invest. Ophthalmol. Vis. Sci. 27, 1261–6. van Setten, G. and Schultz, G. (1994). Transforming growth factor-alpha is a constant component of human tear fluid. Graefes Arch. Clin. Exp. Ophthalmol. 232, 523–6. van Setten, G-B., Viinikka, L., Tervo, T., Pesonen, K., Tarkkanen, A. and Perheentupa, J. (1989). Epidermal growth factor is a constant component of normal human tear fluid. Graefe’s Arch. Ophthalmol. 227, 184–7.
T. T E R V O E T A L.
Wilson, S. E. (1991). Lacrimal gland epidermal growth factor production and the ocular surface. Am. J. Ophthalmol. 6, 763–5. Wilson, S. E., Lloyd, S. A. and Kennedy, R. H. (1991). Epidermal growth factor messenger RNA production in human lacrimal gland. Cornea 10, 519–24. Wilson, S. E., He, Y. G., Weng, J., Zieske, J. D., Jester, J. V. and Schultz, G. S. (1994a). Effect of epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, on proliferation, motility and differentiation of human corneal epithelial cells. Exp. Eye Res. 59, 665–78. Wilson, S. E., Walker, J. W., Chwang, E. L. and He, Y. G. (1993). Hepatocyte growth factor, keratinocyte growth factor, their receptors, fibroblast growth factor receptor2, and the cells of the cornea. Invest. Ophthalmol. Vis. Sci. 34, 2544–61. Wilson, S. E., Weng, J., Chwang, E., Gollahon, L., Leitch, A. M. and Shay, J. W. (1994b). Hepatocyte Growth Factor (HGF), Keratinocyte Growth Factor (KGF), and their Receptors in Human Breast Cells and Tissues : alternative HGF and KGF receptors. Mol. Cell. Biol. Res. 40, 337–50.
Tear Hepatocyte Growth Factor (HGF) Availability Increases Markedly after Excimer Laser Surface Ablation T I M O T E R V Oa, M I N N A V E S A L U O M Aa, G R E G L. B E N N E T Tb, R A L P H S C H W A L Lb, M A R C O H E L E N Ac Q I A N W A L I A N Gc S T E V E N E. W I L S O Nc * a
Department of Ophthalmology, University of Helsinki, Helsinki, Finland, b Genentech, Inc., South San Francisco, CA, c Eye Institute and the Department of Cell Biology, The Cleveland Clinic Foundation, Cleveland, OH, U.S.A. (Received Columbia 29 August 1996 and accepted in revised form 14 August 1996) Tear cytokines and growth factors are likely to modulate the wound healing process following corneal epithelial injury. Hepatocyte growth factor (HGF) is a paracrine mediator of epithelial proliferation, motility, and differentiation that is produced by keratocytes and the lacrimal gland. Tear samples were collected preoperatively and one, two, and seven days postoperatively in eyes undergoing excimer laser surface ablation [photorefractive keratoplasty (PRK) or phototherapeutic keratoplasty (PTK)]. Tear HGF concentration was measured with a sensitive ELISA assay, Tear HGF production was calculated using the tear flow rate in the collection capillary and HGF concentration. Although the instantaneous concentration of HGF in tears decreased significantly in the days following PRK, a large increase in tear flow resulted in a marked increase in HGF bioavailability. The heparin-binding characteristics of HGF would result in increased binding to glycosaminoglycans and other heparin-like matrix components and, therefore, increased growth factor availability to the cognate receptor. This is the first report documenting changes in tear film HGF production. HGF may have an important function in maintenance and wound healing of the ocular surface epithelium since HGF is present in the normal tear film and the HGF secretion rate increases markedly in parallel with aqueous tear production following corneal surgical injury. # 1997 Academic Press Limited Key words: hepatocyte growth factor ; cornea ; wound healing ; lacrimal gland ; tears ; photorefractive keratectomy.
1. Introduction Cytokines, growth factors, other tear components, and receptors expressed by responsive cells are likely to contribute to normal maintenance of the ocular surface and to corneal wound healing after injury (Wilson, 1991 ; Wilson, Lloyd and Kennedy, 1991 ; van Setten et al., 1989 ; Tervo K et al., 1990 ; Tsutsumi, Tsutsumi and Takami, 1988 ; Ohashi et al., 1989 ; Ubels, Foley and Rismondo, 1986 ; van Setten and Schultz, 1994). These components may be derived from the lacrimal gland, conjunctival vessels, corneal cells, conjunctival cells, and other sources. Studies on the production and function of individual growth factors are likely to yield important insights that will eventually lead to strategies for modulating the wound healing process after corneal surgery. The role of hepatocyte growth factor (HGF) and HGF receptor in corneal maintenance and wound healing has been an area of intense investigation. HGF, also known as the scatter factor, is best characterized as a classical paracrine mediator of stromal-epithelial interactions secreted by fibroblast cells to modulate the functions of epithelial cells in numerous tissues * For correspondence at : Eye Institute}A31, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, U.S.A. Presented in part at the Association for Research in Vision and Ophthalmology annual meeting Ft Lauderdale, FL, April 1996.
0014-4835}97}04050104 $25.00}0}ey96226
(Nakamura et al., 1989 ; Matsumoto et al., 1991 ; Rubin et al., 1991 ; Montesano et al., 1991 ; Wilson et al., 1994a). HGF and HGF receptor are expressed in corneal cells (Wilson et al., 1993, Li et al., 1996) and HGF has been shown to modulate proliferation, motility, and differentiation of corneal epithelial cells (Wilson et al., 1994b). Recently, we have shown that HGF is also produced in lacrimal gland fibroblast cells (Li et al., 1996). In addition to having local effects on epithelial cells within the lacrimal tissue, HGF collects in the ducts and becomes a component in tears. In the present study, changes in tear HGF concentration and production were monitored in human eyes following excimer laser surface ablation. 2. Materials and Methods Nineteen eyes from 19 patients undergoing excimer laser surface photoablation [14 photorefractive keratectomy (PRK) for myopia and five for phototherapeutic keratectomy (PTK)] were included in the study. Immediately prior to medication or other preoperative manipulations excess tears were cleared by first gently absorbing them from the lid margin with a tissue wipe. A 10 to 30 µl sample of tears was collected with a calibrated, polished glass capillary. Care was taken to minimally stimulate the ocular surface. The time required to collect the sample was recorded and the rate of tear flow was determined as # 1997 Academic Press Limited
502
previously described (Tervo et al., 1994a ; Tervo et al., 1994b). The tear sample was centrifuged at 12 000 g to remove any cells or debri and the supernatant immediately transferred to a cryovial and maintained at ®72 °C to ®85 °C until analysis. Similarly, tear samples were collected on the first, second, and seventh day following PRK. Each eye received Liquifilm-FML twice a day and chloramphenicol ointment (Parke-Davis) once or twice a day during the postoperative interval that tears were collected. In addition, each patient received oral diclotenac 25 mg two to three times per day for the first two days after surgery. No medications were given within two hours of tear collection at the postoperative examinations. The HGF-specific antibody used to perform the ELISA assay and the ELISA method have been previously reported (Li et al., 1996). Briefly, all of the samples were evaluated simultaneously using 96-well Maxisorb microtiter plates (Nunc, Rosklide, Denmark) coated with A 3.1.2 anti-HGF monoclonal antibody. After blocking and washing plates, 100 µl per well standard HGF or serially diluted tear samples, samples, starting at 1 : 10, were added and incubated overnight. After washing the plates, 100 µl per well biotinylated sheep anti-HGF were added and incubated for 2 hr. One hundred microliters per well horseradish peroxidase-conjugated streptavidin (Zymed, South San Francisco, CA, U.S.A.) were added to washed plates and incubated for 30 minutes. After washing, 100 µl per well ELAST4 biotinyl-tyramide solution (DuPont NEN, N. Billerica, MA, U.S.A.) were added. After a 15 minute incubation, plates were washed, and 100 µl per well horseradish peroxidase-conjugated streptavidin were again added and incubated for 30 minutes. After washing the plates, 100 µl per well 3, 3, 5, 5«tetramethylbenzidine substrate (Kirkegaard and Perry, Gaithersburg, MD, U.S.A.) were added. Color development was stopped after 5 minutes with 100 µl per well of 1 phosphoric acid. Plates were read at 450 nm on an automated plate reader. Data was reduced by a Parameter curve fitting program. The sensitivity of the HGF ELISA was approximately 150 pg ml−". The rate of HGF production in pg min−" was determined by multiplying the tear flow in µl min−" by the HGF concentration in pg µl−". Variations were expressed as the mean³the standard error of the mean. Statistical significance was determined with the Mann-Whitney U-test and P ! 0±05 was considered statistically significant. 3. Results Several patients had one or more tear samples with HGF concentrations below the sensitivity of the assay and these samples were excluded from statistical analysis. Mean and ... values for tear flow rate (µl min−"), HGF concentration (pg ml−"), and HGF production (pg min−") are provided in Table I for the
T. T E R V O E T A L.
preoperative, one day postoperative, two day postoperative, and seven day postoperative examinations. Percent change in tear HGF production relative to the preoperative level for each postoperative day is also provided. Percent change in HGF production compared with the preoperative level for individual eyes on each day after surgery are provided in Table I. There was no difference in the increased tear HGF production response between eyes that underwent PRK and those that had PTK. Mean percent change in tear HGF production for the postoperative visits are provided in Table I. Mean HGF production was markedly increased on the first and second postoperative days and the difference compared with the preoperative value was statistically significant (P ! 0±001). In this relatively small study, a correlation between the levels of HGF in tears and the clinical rate of healing of the epithelium after surgery has not detected. The epithelium in all eyes healed within three days of surgery. Discussion The results of this study demonstrate that there is a marked increase in tear HGF availability in the majority of human eyes following corneal epithelial and stromal wounding. Tear HGF production may also have been increased in eyes with HGF concentrations below the sensitivity of the ELISA assay, but a more sensitive ELISA would be needed to analyse these samples. Even though the instantaneous concentration of HGF in the tears was actually decreased on the first and second postoperative day in most patients, marked increases in the flow of tears resulted in a large increase in total tear HGF availability. There is likely to be a large increase in the bioavailability of HGF for binding to HGF receptors on the surface of corneal epithelial cells since HGF is a heparin-binding growth factor that adheres to glycosaminoglycans and other components in basement and cell membranes (Nakamura et al., 1989 ; Matsumoto et al., 1991 ; Rubin et al., 1991 ; Montesano et al., 1991). Once bound to these low affinity receptors the HGF should be available for binding to the high affinity cognate HGF receptor. The possibility that HGF in tears binds to some soluble factor or factors that limit its bioavailability cannot be excluded, although soluble receptors for HGF have not been identified. What is the source of the increased tear HGF production following corneal wounding ? It has recently been demonstrated that HGF production is increased in keratocytes following corneal epithelial wounding (Li et al., 1996). It is likely that IL-1 released from corneal epithelial cells with injury increases HGF mRNA transcription in keratocytes (unpublished data). Since the epithelial barrier function is lost during the early stages following excimer laser surface ablation because of an epithelial defect of size lasting for the first two to three days, it is likely
TEAR HGF PRODUCTION FOLLOWING EXCIMER LASER SURFACE ABLATION
that the keratocytes are one source of the increased tear HGF production. We have, however, also demonstrated that HGF is produced by the lacrimal gland (Li et al., 1996). Thus, lacrimal gland HGF production could also be increased, although the decrease in HGF concentration would indicate that the increase is less than for the aqueous component of tears (Table I). Further study will be needed to determine whether HGF mRNA and}or protein production is increased in the lacrimal gland following ocular surface injury. What function is served by the increased HGF that is available to the epithelial cells during the early postoperative period following corneal wounding ? HGF has been shown to increase proliferation, increase motility, and inhibit differentiation of human corneal epithelial cells (Li et al., 1996). Conversely, HGF appears to have no effect on the stromal fibroblast cells (Li et al., 1996). Thus, it is likely that HGF modulates processes that contribute to epithelial wound healing following corneal injury. The relationship between HGF and epidermal growth factor (van Setten et al., 1989 ; Tervo et al., 1990), transforming growth factor alpha (van Setten and Schultz, 1994), and other tear components following corneal epithelial wounding has yet to be determined. Could exogenous topical HGF be of therapeutic benefit to facilitate corneal epithelial wound healing ? The large increases that occur in tear HGF production in most patients following corneal injury suggest that this is unlikely to be the case. It is possible, however, that deficient HGF production could be detected in some patients with persistent epithelial defects. Any studies to determine the efficacy of HGF in stimulating corneal epithelial wound healing should concentrate on the latter group of patients. Prediction of the potential therapeutic effects of individual growth factors is difficult, however, without a more complete understanding of the complex interactions that occur between the numerous growth factors, cytokines, and
503
receptors expresses by corneal and lacrimal gland cells. Acknowledgement Supported in part by US Public Health Service grant EY10056 from the National Eye Institute, National Institutes of Health, Bethesda, Maryland, The Cleveland Clinic Foundation, The Mary and Georg Ehrnrooth Foundation, The Finnish Eye and Tissue Bank Foundation, and The Friends of Blind, Finland.
References Li, Q., Weng, J., Bennett, G. L., Schwall, R., Wang, Z-F., Tabor, T., Kim, J., Hargrave, S., Cuevas, K. H. and Wilson, S. E. (1996). Hepatocyte Growth Factor (HGF) and HGF receptor protein in lacrimal gland, tears, and cornea. Invest. Ophthalmol. Vis. Sci. 37, 727–39. Matsumoto, K., Hashimoto, K., Yoshikawa, K. and Nakamura, T. (1991). Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor. Exp. Cell Res. 96, 114–20. Montesano, R., Matsumoto, K., Nakamura, T. and Orci, L. (1991). Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67, 901–8. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K. and Smizu, S. (1989). Molecular cloning and expression of hepatocyte growth factor. Nature 342, 440–3. Ohashi, Y., Motokura, M., Kinoshita, Y., Mano, T., Wananabe, H., Kinoshita, S., Manabe, R., Oshiden, K. and Yanaihara, C. (1989). Presence of epidermal growth factor in human tears. Invest. Ophthalmol. Vis. Sci. 30, 1879–82. Rubin, J. S., Chan, A. M-L., Bottaro, D. P., Burgess, W. H., Taylor, W. G., Cech, A. C., Hirschfield, D. W., Wong, J., Miki, T., Finch, P. W. and Aaronson, S. A. (1991). A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor. Proc. Natl. Acad. Sci. 88, 415–9. Tervo, T., Honkanen, N., Van Setten, G. B., Virtanen, T., Tarkkanen, A. and Ha$ rko$ nen, M. (1994b). A rapid fluorometric assay for tear fluid plasmin activity. Cornea 13, 148–55.
504
Tervo, K., van Setten, G. B., Virtanen, I., Tarkkanen, A. and Tervo, T. (1990). The lacrimal gland is the source of epidermal growth factor in tear fluid—an immunochemical study in rats. Invest. Ophthalmol. Vis. Sci. 31 (Suppl), 45. Tervo, T., Virtanen, T, Honkanen, N., Ha$ rho$ nen, M. and Tarkkanen, A. (1994a). Tear fluid plasmin activity after excimer laser photorefractive keratectomy. Invest. Ophthalmol. Vis. Sci. 35, 3045–50. Tsutsumi, O., Tsutsumi, A. and Takami, O. (1988). Epidermal growthfactor-like, corneal wound healing substance in mouse tears. J. Clin. Invest. 81, 1067–71. Ubels, J. L., Foley, K. M. and Rismondo, V. (1986). Retinol secretion by the lacrimal gland. Invest. Ophthalmol. Vis. Sci. 27, 1261–6. van Setten, G. and Schultz, G. (1994). Transforming growth factor-alpha is a constant component of human tear fluid. Graefes Arch. Clin. Exp. Ophthalmol. 232, 523–6. van Setten, G-B., Viinikka, L., Tervo, T., Pesonen, K., Tarkkanen, A. and Perheentupa, J. (1989). Epidermal growth factor is a constant component of normal human tear fluid. Graefe’s Arch. Ophthalmol. 227, 184–7.
T. T E R V O E T A L.
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