Involvement of pigment epithelium-derived factor, docosahexaenoic acid and neuroprotectin D1 in corneal inflammation and nerve integrity after refractive surgery.

Alterations in corneal innervations result in impaired corneal sensation, severe dry eye and damage to the epithelium that may in turn lead to corneal ulcers, melting and perforation. These alterations can occur after refractive surgery. We have discovered that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA or the docosanoid bioactive neuroprotectin D1 (NPD1)) induces nerve regeneration after corneal surgery that damages the stromal nerves. We found that PEDF is released from corneal epithelial cells after injury, and when DHA is provided to the cells it stimulates the biosynthesis of NPD1 by an autocrine mechanism. The combination of PEDF plus DHA also decreased the production of leukotriene B4 (LTB4), a neutrophil chemotactic factor, thereby decreasing the inflammation induced after corneal damage. These studies suggest that PEDF plus DHA and its derivative NPD1 hold promise as a future treatment to restore a healthy cornea after nerve damage.

HGF- and KGF-induced activation of PI-3K/p70 s6 kinase pathway in corneal epithelial cells: its relevance in wound healing.

In this study we have investigated the involvement of PI-3K and its downstream target p70 S6K in the signaling response of corneal epithelial cells after HGF and KGF stimulation. HGF induced three- to five-fold increase in PI-3K activity in 5-10 min, whereas KGF stimulation resulted in two- to three-fold increase in activity in 2-10 min. Both growth factors also caused the phosphorylation of p70 S6K and stimulation of its activity. HGF increased p70 S6K activity by 300% and KGF by about 200%. Protein kinase C (PKC) activator TPA also induced the phosphorylation of p70 S6K. Both the PI-3K inhibitor wortmannin and PKC inhibitor calphostin C blocked the phosphorylation of p70 S6K mediated by the growth factors. However, the mitogen-activated protein kinase (p42/44 MAPK) cascade inhibitor PD98059 had no effect on p70 S6K activation. Furthermore, HGF and KGF increased the rate of corneal epithelial wound healing in an organ culture model, and wortmannin and rapamycin (the p70 S6K inhibitor) blocked corneal epithelial wound healing promoted by the growth factors. These studies suggest that PI-3K and p70 S6K are important signal transducers in the stimulation of corneal epithelial cells by HGF and KGF. PKC is involved in the PI-3K-dependent activation of p70 S6K but not MAPK. Inhibition of wound closure by PI-3K and p70 S6K inhibitors suggests these enzymes play a significant role in corneal wound repair stimulated by HGF and KGF.

Corneal stimulation of MMP-1, -9 and uPA by platelet-activating factor is mediated by cyclooxygenase-2 metabolites.

PURPOSE: This study was undertaken to evaluate the significance of cyclooxygenase-2 (COX-2) activity on urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs)-1 and -9 induction in cornea following platelet-activating factor (PAF) treatment. METHODS: Corneal organ cultures were pre-treated with increasing concentrations of COX-2-specific inhibitors NS398 or nimesulide prior to PAF stimulation. To determine the effect of exogenous prostaglandins (PGs) on uPA, MMP-1 and MMP-9 levels, corneas were pre-treated with COX-2 inhibitors followed by the addition of 2.5 microM PGD2, PGE2 or PGF2alpha. The levels of uPA and MMP-9 were assayed by casein and gelatin zymography, respectively. MMP-1 levels were determined by Western Blot analysis. RESULTS: The increase in uPA, MMP-9 and MMP-1 levels detected in corneal organ cultures treated with 100 nM cPAF was blocked by 5 microM NS398 and 10 microM nimesulide, concentrations at which these inhibitors selectively inhibit COX-2 activity. Furthermore, pre-incubation with COX-2 inhibitors, followed by supplementation with PGD2, PGE2 or PGF 2alpha, increases uPA, MMP-9 and MMP-1 levels in corneas similar to and in some cases greater than that produced by cPAF treatment alone. CONCLUSIONS: During corneal injury and inflamation, PAF is an important factor in the activation of proteolytic cascades, which could lead to corneal epithelial defects and ultimately ulceration. One important goal in treating these defects is to modulate the activity of enzymes that destroy the extracellular matrix. Our results suggest that COX-2 induction following PAF stimulation and subsequent eicosanoid release may play a crucial role in the induction of uPA, MMP-1 and MMP-9 enzymes. Specific COX-2 inhibition could therefore block the actions of PAF when inflammation is sustained.

Platelet-activating factor (PAF) enhances apoptosis induced by ultraviolet radiation in corneal epithelial cells through cytochrome c-caspase activation.

PURPOSE: To examine the role of platelet-activating factor (PAF) on apoptosis of corneal epithelial cells exposed to radiation. METHODS: Rabbit corneal epithelial (RCE) and human corneal epithelial (HCE) cells were exposed to UVC radiation and then to carbamyl PAF (cPAF) for different increments of time. The PAF antagonist BN50739 was added 30 min before cPAF. The caspase inhibitors Ac-DEVD-CHO and Ac-YVAD-CHO were added 1 h before, and the phospholipase A(2) (PLA(2)) inhibitor MAFP was added 3 h before UVC irradiation. FITC-dUTP TUNEL and DAPI staining were performed to assess the percentage of apoptotic cells. DNA ladder analysis was used to investigate apoptosis induced by different intensities of UVC (50-600 J/m(2)) with or without cPAF. Caspase activation and release of cytochrome c from mitochondria to cytosol were determined by Western blot. RESULTS: While only 2.7% of RCE cells were DAPI positive in controls incubated for 12 h, 44% of cells were stained positive 4 h after irradiation; these values increased to 63% in the presence of cPAF. Cells incubated with cPAF alone were similar to controls. TUNEL staining and DNA laddering showed also increased in apoptosis after PAF treatment of UV-irradiated cells and BN50739 blocked the effect of cPAF. cPAF increased caspase-3 activation induced by UV irradiation in HCE cells. Cytochrome c release from mitochondria to cytosol was observed 30 min after irradiation. cPAF almost doubled the release of cytochrome c at 30 min and 1 h. Here, too, BN50739 blocked the PAF effect. No release of cytochrome c by PAF was seen in non-irradiated cells, even at higher concentrations. MAFP caused a decrease in cytochrome c release from UV-treated cells, and caused an even greater inhibition of cytochrome c release in cells stimulated with PAF. CONCLUSIONS: PAF increases RCE and HCE apoptosis caused by UV irradiation by stimulating PLA(2), producing an early release of cytocrome c from mitochondria and activating caspase-3 by a receptor-mediated mechanism. This accelerating effect of PAF on the apoptotic cascade only occurred when corneal epithelial cells had been previously damaged by UV radiation.

Increased platelet-activating factor receptor gene expression by corneal epithelial wound healing.

PURPOSE: Platelet activating factor (PAF) is a potent inflammatory mediator the synthesis of which increases in the cornea after injury. The effects of PAF are mediated by receptors (PAF-R), which are present in target cells. This study was undertaken to investigate the effects of wound healing, PAF, and growth factors on modulating PAF-R mRNA levels in corneal epithelial cells. METHODS: Cultures of rabbit corneal epithelial (RCE), rabbit limbal epithelial (RLE), rabbit corneal fibroblast (RCF), and rabbit corneal endothelial (RCEn) cells, as well as rabbit corneal keratocytes (RCKs) were used. For the in vivo wound-healing experiments, a 7-mm central corneal deepithelialization was performed in anesthetized rabbits. For the in vitro experiments, wounded rabbit corneas were maintained in organ culture. Corneas were stimulated with 120 nM PAF or preincubated with PAF antagonists, cyclohexamide (CHX) or actinomycin D (AcD) before adding PAF. RCE cells were stimulated with transforming growth factor (TGF)-beta1, -beta2, and, -beta3, basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF); and hepatocyte growth factor (HGF). Total RNA was isolated and PAF-R expression evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis, and quantitative RT-PCR. RESULTS: PAF-R mRNA was expressed in RCE, RLE, and RCEn cells and RCKs, but not in RCFs. After epithelial injury, PAF-R expression increased from 2.5 to 4 times, both in vitro and in vivo. Addition of cPAF further stimulated PAF-R gene expression in epithelium, which was abolished by PAF antagonists. Quantitative RT-PCR revealed that PAF stimulated PAF-R mRNA threefold after injury. The induction of PAF-R by its agonist required previous injury and was inhibited by AcD but not by CHX. Treatment of RCE cells with TGF-beta1, -beta2, or -beta3, HGF, and KGF increased mRNA in PAF-R; however, bFGF had no effect. CONCLUSIONS: Corneal injury produces changes in PAF-R mRNA expression. Whereas stroma fibroblastic cells lost the PAF-R gene expression found in keratocytes, corneal epithelial injury upregulated PAF-R mRNA. These results suggest that activation of selective growth factors and increases in PAF synthesis after injury stimulate PAF-R gene transcription and constitute important feedback mechanisms needed to maintain the inflammatory process and regulate epithelial wound healing.

Phosphatidylinositol 3-kinase in bovine lens and its stimulation by insulin and IGF-1.

PURPOSE: To identify and characterize phosphatidylinositol 3-kinase (PI-3K) in the lens and to study its involvement as a signal mediator in lens epithelial cells exposed to insulin and insulin-like growth factor (IGF)-1, which are known to induce lens epithelial cell proliferation and differentiation into fiber cells. METHODS: Concentric fiber cell layers from single bovine lens were prepared by dissolution in buffer. PI-3K activity in capsule-epithelium and fiber cell layers was determined after immunoprecipitation with antibodies against p85, the regulatory subunit of PI-3K. High-performance liquid chromatography on an ion exchange column (Partisil-SAX; Whatman, Maidstone, United Kingdom) was used to identify PI-3K reaction products. Cultured bovine lens epithelial cells were stimulated with insulin or IGF-1, and PI-3K activity was determined after immunoprecipitation with antibody against phosphotyrosine. Association of p85 with other proteins after stimulation was determined in anti-p85 immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis using anti-phosphotyrosine antibody. RESULTS: PI-3K activity was found in both lens epithelial cells and fiber cells. The highest specific activity was found in the capsule-epithelium, but there was considerable activity in other fiber cell layers. Insulin and IGF-1 stimulated the PI-3K activity in epithelial cells in culture by more than 100%, and activation of the enzyme resulted in tyrosine phosphorylation of the p85 subunit. After stimulation, the p85 subunit of PI-3K was associated with 100- and 180-kDa tyrosine phosphorylated proteins. CONCLUSIONS: The activation of PI-3K and its association with specific tyrosine-phosphorylated proteins may be important in insulin and IGF-1 signal transduction in lens epithelial cells. The presence of significant PI-3K activity throughout the lens further suggests that this signal transduction enzyme is sustained in fiber cells.

Corneal epithelial wound healing increases the expression but not long lasting activation of the p85alpha subunit of phosphatidylinositol-3 kinase.

PURPOSE: Corneal epithelial wound healing is a complex process involving several growth factors whose interaction with tyrosine kinase receptors (RTK) leads to the recruitment of enzymes coupled to second messengers that propagate and amplify growth factor-induced signals inside the cells. Phosphatidylinositol-3 kinase (PI-3K) is one such enzyme. Here we have investigated changes in PI-3K activity and expression during re-epithelialization after in vivo and in vitro corneal injury. METHODS: For the in vivo model, epithelium was collected from rabbit corneas at different stages of wound healing after complete de-epithelialization. For in vitro studies, after 7 mm central scrape wounds were applied, rabbit corneas were maintained in organ culture. Immunoprecipitation and Western blot using anti-p85alpha antibodies were employed to determine PI-3K activity and expression of the p85alpha regulatory subunit of PI-3K. Two specific PI-3K inhibitors, Wortmannin and LY 294002 were used to study the effect of PI-3K activity on corneal epithelial wound healing. RESULTS: Two to four days after in vivo corneal epithelial wound healing, there was a 6-8 fold increase in the expression of the p85alpha subunit of PI-3K. By 8 days, the expression of p85alpha was similar to non-injured tissue. Increased expression of the 85kDa protein was observed mainly in the membrane fraction. Similarly, the expression of PI-3K was increased 24h after injured corneas were maintained in organ culture. Increase of p85alpha was confined to the wound region and surrounding area. No concomitant increase in PI-3K activity was observed in any of the wound models. Forty-eight hours after the central injury, Wortmannin and LY294002 inhibited wound healing by about 50%. CONCLUSIONS: Association of most of the increased p85alpha with the membrane fraction and no detectable increase in PI-3K activity during corneal re-epithelialization indicates that PI-3K activation is transitory. The results also suggest a mechanism of down regulation of the enzyme to avoid uncontrollable growth and cellular hypertrophy after growth factor stimulation during wound healing.

PAF binding to a single receptor in corneal epithelium plasma membrane.

PURPOSE: To study the binding characteristics and the expression of platelet-activating factor receptors (PAF-R) in corneal epithelium to elucidate the site of action of PAF. METHODS: Binding of [3H]PAF was investigated in subcellular fractions of the epithelia of bovine corneas and in membranes from cultured rabbit corneal epithelial cells. Dose-response inhibition curves of [3H]PAF-specific binding were generated using increasing concentrations of several PAF-R antagonists. RNA from rabbit corneal epithelial cells was probed for PAF-R expression by reverse transcription-polymerase chain reaction (RT-PCR) with specifically designed degenerated primers. RESULTS: Scatchard analysis showed a high-affinity binding site in bovine and rabbit corneal epithelium. The dissociation constant (Kd) and the maximum binding sites (Bmax) in a bovine membrane preparation and similar rabbit fraction were 0.77+/-0.03 nM and 180+/-21 femtomoles/mg protein and 4.3 nM and 1.3 picomoles/mg protein, respectively. Specific PAF-binding sites were found in bovine preparations enriched in plasma membranes with a Kd = 69.6 pM and Bmax = 80 femtomoles/mg protein; no specific binding was found in nuclei or microsomal fractions. RT-PCR of rabbit corneal epithelium generated a single product of the predicted size (478 bp). The deduced amino acid sequence of the purified PCR product was 87% homologous to human PAF-R. The hetrazepines BN 50727 and BN 50730 and the PAF structural analogues CV 3988 and CV 6209 competitively inhibited [3H]PAF binding to corneal epithelium with similar potency. WEB 2086 BS was two orders of magnitude less active in antagonizing PAF binding. CONCLUSIONS: Corneal epithelium contains a single population of receptors localized in the plasma membrane. PAF antagonists exert their actions by blocking this PAF-R. The partial sequence deduced in rabbit corneal PAF-R show a higher homology to the human PAF-R.

Selective changes in protein kinase C (PKC) isoform expression in rabbit corneal epithelium during wound healing. Inhibition of corneal epithelial repair by PKCalpha antisense.

Protein kinase C (PKC) isoforms display different sensitivities to modulators, tissue specificities and subcellular localizations. PKCalpha increases during rabbit corneal epithelial wound healing. Here we report differential expression of PKC isoforms in the cornea of rabbits at 1, 2, 4 and 8 days during re-epithelization. Cytosolic, membrane and detergent-insoluble fractions from epithelium were analysed by Western blot using monoclonal antibodies against the different PKC isoforms. We have identified PKCalpha, gamma, epsilon, mu and iota. PKCalpha and gamma were expressed only in the cytosolic fraction, with the expression of PKCalpha markedly increasing 4 days after injury. Corneas cultured in the presence of rabbit-specific PKCalpha antisense showed a greater than 50% inhibition of wound closure, compared to controls. The PKCepsilon and mu were expressed in the soluble, as well as in the membrane fraction. Additionally, 12% of PKCmu was found attached to the detergent insoluble fraction. The expression of the membrane-bound PKCepsilon and mu isoforms decreased between 1 and 2 days following injury. Only 10% of the PKCiota expressed in corneal epithelium was membrane bound, but between 4 and 8 days after de-epithelization, the expression in this fraction increased three-fold. Our results suggest that changes in the expression and distribution within the various fractions of selective isoforms of PKC after injury could be involved in events leading to wound healing and that PKCalpha is a key modulator in rabbit corneal wound repair.

Platelet-activating factor induces cyclooxygenase-2 gene expression in corneal epithelium. Requirement of calcium in the signal transduction pathway.

PURPOSE: To investigate the effect of the inflammatory mediator platelet-activating factor (PAF) in the induction of the inducible prostaglandin H synthase-cyclooxygenase-2 (COX-2) gene expression in corneal epithelium. METHODS: Rabbit corneas were incubated in organ culture with or without carbamyl PAF (cPAF, 100 nM). The effects of PAF antagonist BN50730 (10 microM), protein synthesis inhibitor cycloheximide (CHX; 30 micrograms/ml), RNA synthesis inhibitor actinomycin D (50 micrograms/ml), and tumor promoter phorbol ester (TPA); (100 nM) were tested. Total RNA for corneal epithelium was analyzed by Northern blot analysis using mouse COX-2 cDNA fragments labeled with 32P as probes. Western blots were performed using mouse monoclonal antibodies. Primary cultures of rabbit corneal epithelium were loaded with the fluorescent dye fluo-3 AM and changes in intracellular calcium concentration [Ca2+]i were analyzed by laser scanning confocal microscopy. RESULTS: Platelet-activating factor induction of COX-2 expression was detectable by Northern blot analysis at 2 hours, peaked at 4 hours, and remained increased for as long as 8 hours. At 16 hours, there was a marked increase in COX-2 expression. The effect was abolished by the PAF antagonist. TPA also induced COX-2 gene expression. Neither PAF-nor TPA-induced expression was inhibited by CHX. In a Ca(2+)-free medium, there was a 50% inhibition of COX-2 gene induction by PAF. The calcium ionophore A23187 also caused an increase in expression of COX-2 messenger RNA; this did not occur in Ca(2+)-free medium. Confocal microscopy imaging showed that after the addition of PAF, there was a transient increase in [Ca2+]i in corneal epithelial cells that peaked between 30 and 60 seconds. The increase was inhibited in the presence of BN50730 or in a Ca(2+)-free medium. A23187 also caused a transient increase in [Ca2+]i that was not altered in cells previously treated with PAF or BN50730. CONCLUSIONS: PAF may enhance prostaglandin synthesis in the corneal epithelium by increasing COX-2 gene expression. This increase is by means of transcriptional activation of the gene and results in increased COX-2 protein formation. Influx of Ca2+ due to PAF stimulation is required to induce the COX-2 gene. A PAF antagonist abolishes all PAF effects and could be of therapeutic value by modulating ocular inflammation at the level of COX-2 gene expression.