Expression of GPR-68 in Human Corneal and Conjunctival Epithelium. Possible indicator and mediator of attrition associated inflammation at the ocular surface.

INTRODUCTION: Attrition and osmotic stress have been identified as major forces in the pathophysiology of dry eye. Impaired tolerance to mechano-transduction in the presence of insufficient lubrication has been associated with disturbances of ocular surface homeostasis and encouragement of inflammatory reactions, challenging the usual regulatory coping mechanisms. In spite of the probable link between enhanced attrition and secondary inflammation, the key mediators driving the vicious cycle of severe dry eye disease have not yet been identified. The goal of this study was therefore to investigate human corneal and conjunctival epithelium for the presence of the G protein-coupled receptor GPR-68. This protein had most recently been shown to be not only chemically activated but also mechanically, possibly through attrition. METHODS: De-identified sections of human cornea and conjunctiva were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohistochemical methods. Results Specific staining for G-protein-coupled receptor 68 (GPR68) was observed in all samples of the cornea throughout the epithelial layers of the corneal epithelium, most prominently in the area of the wing cells and the basement membrane. Even in the conjunctiva, specific staining for GPR-68 was found. DISCUSSION: The detection of G protein-coupled receptor GPR-68 in human corneal and conjunctival epithelium raises the question of its function and purpose. The mechanical activation of GPR68 in situations with enhanced friction and attrition could modify various cellular functions and possibly jeopardize normal inflammatory homeostasis at the ocular surface. Accordingly, decreased lubrication in dry eye disease could result in activation of GPR-68. This could lead to secondary inflammation, initially in the epithelium and surrounding stroma. Continuous mechanical stress could result in chronic inflammation, also reaching deeper structures of the cornea, possibly making GPR-68 an important actor in the vicious cycle of dry eye disease. CONCLUSION: G protein-coupled receptor GPR-68, sensitive to flow and mechanic stimulation, is present in the human corneal epithelium and conjunctiva. Decreased lubrication and increased attrition, accompanied by sensations typical for dry eye, might lead to local inflammation. It is possible that subtle signs of conjunctival, and later corneal, surface damage in the context of these sensations could be a better indicator of the need for and success of therapy than the clinical signs of dry eye disease alone, at least in the early stages of the disease. Inhibition of G protein-coupled receptor GPR-68 could represent a new strategy in the treatment of dry eye disease.

GPR-68 in human lacrimal gland. Detection and possible role in the pathogenesis of dry eye disease.

INTRODUCTION: In parallel to ocular surface disease in dry eye there is often a dysfunctionality of the lacrimal gland apparatus. The functionality of the lacrimal gland is of major importance for maintenance of ocular surface integrity and health, even in conditions of enhanced stimulation and secretion requirements. Such enhanced secretion demands can push the lacrimal gland to its limits, with maximized tear fluid secretion and increased flow through the lacrimal ducts. The goal of this study was to investigate whether G protein-coupled receptor GPR-68 is present in the lacrimal gland, as this protein has recently been shown to be sensitive to flow rate and osmolarity. METHODS: For this purpose, de-identified sections of human lacrimal gland tissue were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohistochemistry. RESULTS: Specific staining was detected in the acini and ducts of human lacrimal gland. In the ducts, the specific staining was found around the lumen of the ducts. In the acini, the specific staining was observed more towards the lumen but also intercellularly between the acinar cells. DISCUSSION: The detection of G protein-coupled receptor GPR-68 in the lacrimal gland, especially around the lumen of the ducts, raises the question about its function and purpose. Activation of GPR68 leads to modification of various cell functions and is associated with regulation of inflammation. Accordingly, enhanced, secretion-induced, augmentation of flow might exert fluid flow stress on the ducts and acini. This might lead to transient, localized activation of GPR-68 and secondary inflammation within the gland. Depending on the intensity, continuity or repetitive nature of the stimuli, exhaustion of the lacrimal gland secretion capacity might follow, and chronicity of the inflammation in the parenchyma as well as around the ducts might be a consequence. CONCLUSION: G protein-coupled receptor GPR-68, sensitive to flow, is present in the human lacrimal gland. Increased flow, triggered by sensations such as are typical for dry eye, might lead to local inflammation. It is possible that these sensations might serve as a better indicator for the need and success of therapy than the clinical signs of dry eye disease, at least in the early stages of the disease.

Detection of TSG-6-like protein in human corneal epithelium. Simultaneous presence with CD44 and hyaluronic acid.

INTRODUCTION: Tumor necrosis factor-inducible gene 6 protein (TSG-6) is member of the hyaluronan-binding protein family (hyaladherins) to which CD44 also belongs. Inflammatory mediators such as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) stimulate TSG-6 production. Recently, however, externally applied TSG-6 has been shown to be effective in the treatment of inflammatory dry eye. On the other hand, it is still unknown whether TSG-6 is naturally present in human corneal epithelium. MATERIAL AND METHODS: Corneal sections of 15 eyes enucleated for posterior segment uveal melanoma were immunohistochemically stained for hyaluronic acid (HA), CD44, and TSG-6. RESULTS: Throughout the corneal epithelium of all sections, CD44 and hyaluronic acid were detected most intensely in the basal epithelial layer. Whereas the presence of HA was intense even in the cytoplasm of the cells, CD44 was located predominantly at the cell membranes. The intensity of the specific staining decreased towards the surface, where CD44 was barely detectable. Hyaluronic acid was, on the other hand, detectable in the extracellular matrix and cells, even at the surface. TSG-6 like immunoreactivity was detected in all sections in a pattern similar to CD44 but much more distinct and intense, with a marked localization in the cell membranes and intercellular spaces, i.e., extracellular matrix. TSG-6 like immunoreactivity was clearly detectable through all cell layers of the corneal epithelium. All control sections were negative. DISCUSSION: Tumor necrosis factor-inducible gene 6 (TSG-6)- like protein is present in human corneal epithelium. It might be a natural component of this tissue which is constantly exposed and mechanically traumatized, and displays localization with similarities to that of CD44. The immunohistological detection of HA as major component of the ECM and epithelial tissue only confirms the results of earlier studies. However, the simultaneous presence and colocalization of CD44 and TSG-6, both HA-binding proteins, requires further investigation of the individual role, regulation and interaction of this system. CONCLUSION: The detection of TSG-6 in human corneal epithelium in the absence of inflammation underlines the importance of normal mechanical forces on the gene expression and regulation of this protein in ocular surface tissues. Given the relationship between inflammation and the protein, TSG-6 may be a major unknown and underestimated player in the regulation of the inflammation encountered in the presence of ocular surface desiccation and dry eye disease.

Osmokinetics: A new dynamic concept in dry eye disease.

INTRODUCTION: Tear fluid osmolarity has been increasingly accepted as an accessible parameter in the diagnosis of ocular surface and dry eye disease. After having been proposed as the gold standard, recent results have put this into question. However, the most recent guidelines for dry eye disease identify specific values of osmolarity as thresholds to help to differentiate between various stages of severity of ocular surface disease. The limits of this approach were investigated to propose a new concept, that of osmokinetics. MATERIALS AND METHODS: Available data on tear fluid osmolarity in normal and diseased eyes were compared. The possibility of normo-osmolar dry eye was investigated and repeated measurements of osmolarity performed. RESULTS: The currently applied static model of a threshold value of osmolarity for diagnosing dry eye disease is apparently insufficient. Not only does it not take into account normo-osmolar dry eye, but it also applies too much significance to a single parameter. Instead, it was found that there is a daily variation in osmolarity (DVO), which appears to be higher in eyes with tear film deficiencies than in healthy eyes. DISCUSSION: Tear film osmolarity does vary considerably throughout the day. Its value should be considered in a kinetic model taking into account the dynamics of osmolarity changes moreso than the current static model. The terms of osmotic stress and diurnal variation of osmolarity were found to offer a more physiological understanding of osmolarity. CONCLUSION: A more dynamic model for osmolarity is presented in which not the value itself but the daily variation of osmolarity is identified. It is suggested that the amplitude of change in osmolarity over the course of a day or even shorter time periods could play a decisive role as a stress factor for the surface cells. The varying osmolar stress could be one of the key mechanisms leading to the cell death, inflammation, apoptosis, and goblet cell disappearance as observed in dry eye disease. Perhaps it is the mean osmolarity level at which these changes occur together with the magnitude of DVO which could identify the level of severity of dry eye disease.

Expression of tenascin and cellular fibronectin in the rabbit cornea after anterior keratectomy. Immunohistochemical study of wound healing dynamics.

Anterior keratectomy (AKE) was done on rabbits, and the appearance of immunohistochemically demonstrable tenascin (TN) or cellular fibronectin (cFN) was studied at different times (5 min to 14 months) after the operation. The substance TN was first observed 12 hr after wounding in the posterior stroma; cFN appeared with the same localization 12 hr later. During postoperative week 1, both TN and cFN immunoreactions shifted to more anterior parts of the cornea, and 9 days after wounding, they were localized in the most anterior part of the stroma only. Thereafter the reactions gradually decreased in intensity but still were visible 3 months after AKE. No reaction for TN or cFN was present 14 months postoperatively.

Detection of transforming growth factor-alpha mRNA and protein in rat lacrimal glands and characterization of transforming growth factor-alpha in human tears.

PURPOSE: To assess whether the lacrimal gland is a possible site of synthesis of transforming growth factor-alpha (TGF-alpha) and to characterize TGF-alpha biochemically in human tears. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to analyze rat lacrimal glands for the presence of TGF-alpha mRNA. Specific monoclonal antibodies were used to localize TGF-alpha immunohistochemically in lacrimal gland tissue of rats. Human tears were analyzed for immunoreactive TGF-alpha protein using a specific radioimmunoassay, and the molecular weight of TGF-alpha in tears was characterized by Western blot analysis. RESULTS: RT-PCR amplification of rat lacrimal gland RNA generated a band of the predicted 492 base pairs for TGF-alpha mRNA. Immunohistochemical staining of rat lacrimal gland localized TGF-alpha protein to lacrymocytes constituting acini but not to interacinar and intraacinar ducts of lacrimal glands. Western blot analysis of human tears detected a single band at MWt 16,000. Logit transformation of radioimmunoassay data for tears and TGF-alpha standard generated parallel displacement lines, indicating the presence of immunoreactive TGF-alpha levels in human tears with an average concentration of 100 +/- 20 pg/ml (mean +/- SEM). CONCLUSIONS: Rat lacrymocytes synthesize TGF-alpha mRNA and protein, and human tears contain immunoreactive TGF-alpha, suggesting that the lacrimal gland may be an exocrine source for TGF-alpha in tears. The single MWt 16,000 form of TGF-alpha in human tears appears to be generated by an unusual proteolytically processing of the pro-TGF-alpha transmembrane precursor protein.

Beta-amyloid protein protein precursor expression in lacrimal glands and tear fluid.

PURPOSE: Proteases in tear fluid play important roles in the regulation of corneal wound healing. Inhibitors of proteolytic activity are major modulators of the associated events. Although it is known that various enzyme inhibitors exist in tear fluid, it is not known whether certain isoforms of the beta-amyloid protein precursor (beta-APP), a potent inhibitor of serine proteases, are present in tear fluid. The purpose of this study was to investigate whether beta-APP can be detected in human tear fluid and, if so, to determine the isoform composition and cellular origin. METHODS: Tear fluid was collected from healthy volunteers. The beta-APP was identified and characterized by immunoblotting using antibodies specific for domains of the beta-APP. The protein was characterized further by ion exchange chromatography. Expression of the beta-APP gene was studied using in situ hybridization and RNA-RNA solution hybridization assay. RESULTS: beta-APP with protease inhibitory properties was identified in all samples of human tear fluid. Immunologic analysis revealed that it had been processed proteolytically before secretion. Gene expression studies showed that the beta-APP gene was expressed in lacrimal glands, particularly in acinar cells. The gene transcript almost exclusively corresponded to beta-APP containing the protease inhibitor insert. CONCLUSIONS: beta-APP is expressed in lacrimal glands and subsequently is secreted into tear fluid. Because the bulk of the beta-APP contained the protease inhibitor insert, the authors propose that beta-APP is an important regulator of proteolysis in tear fluid and that possibly it plays a role in the events associated with corneal wound healing. This suggests a novel physiological function of beta-APP in addition to those previously described-regulation of blood coagulation and cell growth.

Inhibition of pseudomonal ulceration in rabbit corneas by a synthetic matrix metalloproteinase inhibitor.

PURPOSE: To evaluate the effect of the synthetic matrix metalloproteinase inhibitor, Galardin, on proteases produced by Pseudomonas aeruginosa (PA) and on a rabbit model of Pseudomonas keratitis. METHODS: Protease activities of culture broths from Pseudomonas strains PA-28 and W-186 were characterized in vitro by gelatin zymography and by digestion of Azocasein in the presence and absence of Galardin and the serine protease inhibitor, aprotinin. In a noninfectious in vivo experiment, sterile PA culture broth from W-186 was injected intrastromally into rabbit corneas that were treated topically with Galardin or vehicle, then evaluated clinically and histologically. In an infectious in vivo experiment, rabbit corneas were injected with washed PA-28, then treated topically with Galardin or vehicle and clinically scored. RESULTS: Gelatin zymography of culture broth from W-186 and PA-28 detected two proteases that were both inhibited by Galardin. Galardin reduced the digestion of Azocasein by both PA culture broths by 99%, whereas aprotinin did not significantly reduce the protease activity of PA-28 conditioned broth. Intrastromal injection of sterile W-186 culture broth caused rapid corneal destruction that was prevented by topical treatment with Galardin. Intrastromal injection of washed PA-28 bacteria resulted in progressive corneal melting that was significantly (P < 0.005) delayed, but ultimately not prevented, by topical treatment with Galardin. CONCLUSIONS: Pseudomonal protease activity in culture broth consisted predominantly of metalloproteinases and were effectively inhibited by Galardin in vitro. Topical treatment with Galardin prevented destruction of rabbit corneas by bacterial products present in culture broth, and it delayed corneal destruction after injection of PA bacteria. Galardin may be a useful adjuvant when corneal destruction proceeds despite prompt antibiotic treatment.

Levels of transforming growth factor alpha (TGF-alpha) in human cerebrospinal fluid.

In this study, we investigated cerebrospinal fluid of patients with various neurological symptoms for the presence of transforming growth factor alpha (TGF-alpha). 41 samples of cerebrospinal fluid were collected by lumbar puncture performed routinely due to the clinical suspicion of neurological disease from 22 females (age 15-80 years, median 42 years) and from 19 males (age 18-82 years, median 48 years). A highly sensitive and specific radioimmunoassay was used to determine the concentration of TGF-alpha in the samples. The detection limit of the assay was about 200 pg TGF-alpha. There was no cross-reactivity to human EGF. We showed CSF indeed does contain TGFalpha. As TGF-alpha was detected in all 41 samples investigated, this growth factor appears to be a constant component of CSF. The mean concentration was 5.5 ng TGF-alpha (S.D. +/- 2.7 pg/ml, range 1.1 to 13.9 pg/ml). There was no significant correlation between TGF-alpha concentration in CSF and age (r = -0.006) and there was no significant difference between females (mean 5.8+/-3.10 pg/ml) and males (mean 5.2+/-1.96 pg/ml). No diagnosis was over represented in patients with TGF-alpha concentrations above or below 1 S.D. off the mean. However, highest concentrations of TGF-alpha were found in the group of patients with peripheral neurological sensory dysfunctions and polyneuropathy. We conclude that TGF-alpha is not only a constant component of human cerebrospinal fluid in adults but could also be significantly involved in the pathophysiology of various neurological diseases. The earlier hypothesis that TGF-alpha could mainly have a role in brain development needs hence to be re-evaluated.

Comparison of corneal epithelial wound healing after photorefractive keratectomy in the rabbit with two types of excimer lasers.

PURPOSE: To evaluate the differences in epithelial wound healing following photorefractive keratectomy when performed with the Summit UV 200 LA and the VISX 20/20 excimer lasers. METHODS: Sixteen New Zealand rabbits were divided into two groups. One group was treated with the Summit laser and the other group treated with the VISX 20/20 laser. The treatment consisted of a -6.00 diopter photorefractive keratectomy with a 5-mm diameter treatment zone. Epithelial wound healing was followed by photography at 4 hour intervals for 64 hours. The length of the wound edge and the size, shape, and closure time of the wound were measured. RESULTS: The median wound edge length at 4 hours was 18.3 mm for the Summit laser and 16.7 mm for the VISX laser. The median wound size at 4 hours was 22.0 mm2 for the Summit and 21.2 mm2 for the VISX. The median wound closure time was 53.4 hours for the Summit laser and 54.0 hours for the VISX laser. CONCLUSION: There was no statistically significant difference in the epithelial healing of rabbit corneal wounds created by photorefractive keratectomy when performed with two current ophthalmic excimer lasers, the Summit UV 200 LA and the VISX 20/20.

Integrins in human corneal epithelium.

Monoclonal antibodies (Mabs) were used for immunohistochemical location of integrin beta- (beta 1,3, and 4) and alpha subunits (alpha 1-6 and alpha v) in the epithelium of both normal and tissue-cultured human cornea. Immunoreaction for the beta 1 integrin subunit was the most intense at the membranes of the basal epithelial cells and weaker at the superficial cell membranes. Anti-beta 4 immunofluorescence appeared in the basal part of the epithelium only, apposing the basement membrane. Both anti-alpha 2 and anti-beta 3 Mabs showed an immunoreaction with distribution similar to the beta 1 integrin subunit. Immunoreaction for the alpha 6 integrin subunit resembled the distribution of the beta 4 subunit. Anti-alpha v showed a faint immunoreaction at the basal and lateral aspects of the basal cell layer. Antisera against beta 3, alpha 1, alpha 4, and alpha 5 integrin subunits showed no specific reactions. The present results suggest that both the normal and tissue-cultured human corneal epithelium contain alpha 2 beta 1, alpha 3 beta 1, and probably also alpha v beta 1 and alpha 6 beta 4 integrin dimers or complexes. We discuss their role as possible receptors for some known ligands.

Distribution of integrins alpha 6 and beta 4 in the rabbit corneal epithelium after anterior keratectomy.

Integrins are heterodimeric cell-surface receptor glycoproteins involved in cell-matrix and also in cell-cell interactions. The alpha 6 beta 4 integrin heterodimer has been shown to be a component of the hemidesmosome. In response to wounding, hemidesmosomes are disassembled, the epithelium migrates to cover the denuded area, and eventually the hemidesmosomes reappear. In the present investigation the distribution of the integrin alpha 6 and beta 4 subunits after anterior keratectomy was studied by indirect immunohistochemistry. Labeling for the alpha 6 subunit was observed around the entire cell surface, right to the leading edge. The immunoreaction for the beta 4 subunit was confined to the basal cell membrane facing the basement membrane as in the normal cornea. Cells at the leading edge of the migrating epithelium did not show any labeling for beta 4. Patchy labeling for beta 4 was first observed in the region midway between the wound margin and the leading edge. Because integrins are only expressed as heterodimers, the alpha 6 subunit may be complexed with the beta 1 subunit, instead of with beta 4, at the leading edge of the migrating epithelium. We also suggest that this alpha 6 beta 1 heterodimer may play a role in the reformation of the adhesion complex.

A rapid fluorometric assay for tear fluid plasmin activity.

A rapid (5- to 10-min), sensitive (detection limit 0.6 IU/L), and moderately specific fluorometric plasmin assay for small volume tear fluid samples was developed. Addition of albumin (up to 0.1% final concentration) to the assay buffer improved the sensitivity of the test so that plasmin activity in healthy controls could be detected. pH in the reaction buffer was 8.0, Michaelis-Menten constant for the substrate, H-D-Val-Leu-Lys.7-amido-4-methyl-coumarin (AMC), was 0.28 mM, and final substrate concentration in the reaction buffer was 1 mM. Intra- and interassay imprecisions were 1.6 and 4.4%, respectively at a plasmin level of 10 IU/L. Tear fluid flow was significantly higher in the patients than in the healthy controls, and this dilatory effect must be considered when using plasmin determination for diagnostic purposes. This effect was counteracted by correcting the plasmin activity values by tear fluid flow. Plasmin flux is plasmin activity (microIU) secreted in units of time (min). This parameter showed highly significant differences between the patients and controls. All patients with microbial keratitis, corrosive trauma, ocular trauma, herpetic infection, and other diseases showed highly significant elevation of plasmin flux compared with controls. The highest plasmin flux values (several hundredfold that of controls) were recorded in patients with severe corneal ulcers. Few patient samples showed some involvement of other proteases, which were not inhibited by aprotinin.

Basic fibroblast growth factor in human saliva: detection and physiological implications.

After the detection of epidermal growth factor and transforming growth factor alpha in various body fluids and human saliva the current study aimed to investigate the presence of basic fibroblast growth factor (bFGF) in human saliva. Basic FGF is stimulating the proliferation of cells of mesodermal and neuroectodermal origin and is highly angiogenetic. After ELISA technique was established, saliva was collected from eight healthy individuals. Run in duplicate, 14 (87.5%) of the 16 samples investigated contained measurable amounts of bFGF. In the samples containing bFGF the concentration varied between 0.1 pg/mL and 8.4 pg/mL (mean concentration, 3.8 pg/mL; SD, 3.5). There was no correlation between age and sex and bFGF concentrations. It is therefore concluded that bFGF is present in human saliva and may even constitute a constant component. The physiological importance of this finding is discussed.

Epidermal growth factor in human tear fluid: increased release but decreased concentrations during reflex tearing.

To further clarify the role of epidermal growth factor (EGF) in the physiology and pathophysiology of the ocular surface the effects of reflex tearing on concentrations of EGF in tear fluid were studied. Tear fluid samples were collected with glass capillaries before (basal samples, 59 eyes, 30 individuals) and during reflex tearing (stimulated samples, n = 212, 40 eyes, 20 individuals). The rate of tear fluid flow in the capillaries (TFFc) was measured. The concentrations of human EGF (hEGF) was determined by time-resolved immunofluorometric assay (TR-IFMA). The first basal samples contained higher concentrations of hEGF than the samples from the contralateral eyes (n = 28) collected thereafter (p less than 0.05). The basal samples from 40 eyes contained a significantly higher mean concentration of hEGF (8466 pg/ml) than did the stimulated samples (n = 212, 2763 pg/ml); p less than 0.001). The mean TFFc increased from 63 nl/s to 506 nl/s during reflex tearing (p less than 0.001) and the amount of hEGF released from 567 fg/s (n = 40) to 1400 fg/s (n = 212; p less than 0.001). Basal samples from females contained higher concentrations of hEGF than did those from males. The maintenance of the hEGF concentration at a certain level and the increased amount of hEGF released into the tear fluid during reflex tearing suggests continuous release of hEGF into tear fluid from the lacrimal gland.

Ocular disease leads to decreased concentrations of epidermal growth factor in the tear fluid.

The concentration of epidermal growth factor (EGF) in tear fluid (TF) was recently shown to decrease with increasing tear fluid flow (TFF). The purpose of the present study was to clarify the effects of ocular surface disease on the TF EGF concentrations. Tear fluid samples (n = 243) were collected from diseased eyes by means of blunted glass capillaries. The time of collection was measured for each sample, and the tear fluid flow in the capillaries (TFFc) was calculated. The concentration of human EGF (hEGF) was determined using a time-resolved immunofluorometric assay (TR-IFMA). For statistical analysis diagnosis-dependent multigrouping was performed and the data of the patient groups were compared to the data for a control group. The control material consisted of 271 TF samples collected from healthy eyes before (n = 59) and after stimulation of reflex tearing (n = 212). It was shown that TF specimens of patients (n = 243) contained significantly (p less than 0.001) less EGF (mean 952 pg/ml) than the TF of healthy control individuals before (n = 59 samples; mean 6589 pg/ml) or after stimulation of reflex tearing (n = 212 samples; mean 2762 pg/ml). The EGF concentration of every patient group was significantly lower than that found in the TF of control individuals both before and during reflex tearing (p less than 0.001). The rate of EGF released with TF during collection did not differ significantly between the various groups of patients or from that released with the TF of normal individuals before induction of reflex tearing.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmin and plasminogen activator activities in tear fluid during corneal wound healing after anterior keratectomy.

Plasmin can degrade fibronectin and laminin, two important components of the extracellular matrix facilitating cell sliding and healing following a wound. In this study we investigated the relationship between the tear fluid level of plasmin and plasminogen activator and the healing of a corneal wound. Anterior keratectomy (AKE) was performed for seven rabbits (11 eyes). Eight eyes were rewounded after re-epithelialization. Tear fluid samples were collected with capillaries before wounding and during wound healing. Plasmin and plasminogen activator (PA) activities were determined using radial caseinolysis procedures. After AKE the plasmin concentrations increased rapidly, from a mean (+/- SEM) of 3.9 +/- 0.9 micrograms/ml to a mean of 37.9 +/- 7.8 micrograms/ml (p less than 0.01), and decreased during wound healing. Rewounding also resulted in an increase in plasmin concentration in the tear fluid (from a mean of 2.9 +/- 0.6 micrograms/ml to a mean of 5.0 +/- 1.1 micrograms/ml; p greater than 0.05). The PA activity showed an inverse trend as it decreased after AKE from a mean of 2.0 +/- 0.6 IU/ml to a mean of 0.3 +/- 0.1 IU/ml (p less than 0.001). During wound healing and re-epithelialization, the PA activity increased again, to 2.1 +/- 0.3 IU/ml (p less than 0.001). Abrasion of the newly grown epithelium in eight eyes caused a second elevation of PA activity which was not significant. This study demonstrates a close association between the healing of corneal wounds and changes in the plasmin and PA activities in tear fluid. Determination of the activity of these enzymes may therefore be useful for monitoring corneal wound healing.

Growth factors in human tear fluid and in lacrimal glands.

EGF has been shown to be a constant component of human tear fluid. Its concentration depends on the actual tear fluid flow, as shown for other proteins secreted by the lacrimal gland. This organ has also been considered to be the origin of tear fluid EGF and immunohistochemical evidence for this hypothesis was found. During corneal disease the concentration of EGF in tear fluid considerably decreases to levels even lower than those found during short time stimulation of reflex tearing. Other members of the EGF family, such as TGF-alpha, have considerable similarity with the EGF molecule and even bind to the same receptor. Currently it is thought that TGF-alpha may be, in certain phases of cell life, even more important in the regulation of cell metabolism than EGF. In the present study we have investigated the presence of TGF-alpha in tear fluid and the lacrimal gland. The initial results presented here, show for the first time that TGF-alpha like EGF, seems to be constant component of human tear fluid and to originate, at least partially, from the lacrimal gland.

Vascular endothelial growth factor (VEGF) in normal human corneal epithelium: detection and physiological importance.

PURPOSE: Corneal neovascularization is a major challenge following chemical burns and corneal inflammation. The factors triggering corneal neovascularization involve various growth factors. In the release and control of these factors the regenerating tissue plays a decisive role. Only recently has vascular endothelial growth factor been shown to be involved in the basic events of retinal neovascularization. From the cornea current knowledge allows only for the statement that corneal epithelium could be able to produce vascular endothelial growth factor. The present study was designed to show the presence of vascular endothelial growth factor-like substances in the corneal epithelium of the normal eye in vivo. METHODS: Using specific antibodies to the N-terminus of human vascular endothelial growth factor indirect immuno-histochemistry was applied to sections of normal human corneal epithelium and to five sections of enucleated human eyes with a morphologically normal anterior segment. RESULTS: In all sections specific staining for vascular endothelial growth factor was found over the entire epithelium. Staining was most intense in the basal layer of epithelial cells. Only a weak staining was detectable in the cell layers closer to the surface. Here, however, the samples of corneal epithelium having been subject to traumatic erosion showed a slightly more intense staining than the sections from the undisturbed corneal tissue of whole globe sections. CONCLUSION: It was shown that vascular endothelial growth factor-like protein is present in the human corneal epithelium. Observed differences in the staining pattern could suggest that the expression of vascular endothelial growth factor might be enhanced due to ocular surface trauma. It is suggested that corneal epithel vascular endothelial growth factor might be an important factor in the cascade leading to the onset of corneal neovascularization.