Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor.

To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture. Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator. The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.

Chandrasekher G ，Ma X ，Lallier TE ，Bazan HE ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

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

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. 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. 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. 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.

Ma X ，Bazan HE 《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

Hepatocyte growth factor and keratinocyte growth factor regulation of epithelial and stromal corneal wound healing.

To investigate the effects of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) on early wound healing in the corneal epithelium and stroma. Cell and Molecular Biology Unit, Department of Optometry and Vision Sciences, Cardiff University, and the Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom. Corneal keratocyte cell cultures and wounded corneal organ cultures (both maintained in serum-free conditions) were treated with 0.1 to 100 ng/mL of HGF or KGF for up to 5 days. Cell cultures were assessed for proliferation, migration, and differentiation into myofibroblasts. Organ cultures were used to evaluate the effect of HGF and KGF on reepithelialization following a wound, epithelial morphology and stratification, keratocyte numbers directly beneath the wounded area, and differentiation into myofibroblasts. The 2 growth factors had opposite effects on the rate of reepithelialization, with HGF delaying and KGF accelerating epithelial coverage of the wound. Morphologic assessment showed that both growth factors affected the stratification and differentiation of the epithelium. Both factors stimulated proliferation of keratocytes in serum-free cell culture, although neither induced the appearance of myofibroblasts. This was in contrast to wounded organ cultures treated with 100 ng/mL HGF, in which large numbers of myofibroblasts were observed under the wound. Control corneas and those receiving KGF contained very few myofibroblasts. Keratocyte repopulation of the denuded area under the wound was enhanced in the presence of HGF but decreased in response to KGF. Hepatocyte growth factor and KGF appeared to have potent and often opposite effects on epithelial and stromal cells following a wound. Hepatocyte growth factor was more detrimental than KGF, resulting in an aberrant epithelium and mass differentiation of keratocytes into myofibroblasts. Inhibition of HGF may be an appropriate therapeutic intervention in the case of persistent epithelial defects and to prevent fibrosis following a corneal stromal wound such as can occur after refractive surgery.

Carrington LM ，Boulton M 《JOURNAL OF CATARACT AND REFRACTIVE SURGERY》

Corneal wound healing is modulated by topical application of amniotic fluid in an ex vivo organ culture model.

The purpose of this study was to evaluate the effects of topical human amniotic fluid (HAF) and equine amniotic fluid (EAF) on corneal reepithelialization and stromal wound healing. New Zealand white rabbit corneas (n=52) were placed in an ex vivo air-interface organ culture. An 8.5mm-diameter mark in the center of the cornea was produced with a hand trephine to select the area for epithelial scraping. A number 15 surgical blade was used to remove the epithelial layer within the demarcated area in a standardized fashion. The corneas were assigned to one of four treatment groups (n=8): fetal bovine serum (FBS), HAF, EAF, and a control group that was exposed to phosphate buffer solution (PBS). Corneal epithelial defects were imaged every 8 h for 72 h after the application of a 30 microl drop of 0.015% fluorescein. Five corneas of each treatment group were used for histology, proliferation, and apoptosis assay at 72 h after the epithelial defect was created. There was no significant difference in the mean rate of closure of the corneal epithelial defect between FBS treated corneas and controls (P>0.06). The mean epithelial defect area (MEDA) was significantly smaller in the EAF group as compared to control corneas at 24 h (P=0.016), 40 h (P=0.032), 64 h (P=0.008) and 72 h (P=0.007) following epithelial scrape. The MEDA in the HAF group was significantly smaller at 16 h (P=0.008), 64 h (P=0.0072), and 72 h (P=0.016) compared to the control group. The MEDA in the HAF and EAF groups was smaller at all time points as compared to the FBS group, but the difference was not significant. At histology, the mean keratocyte density was significantly higher in the anterior stroma in the HAF (P<0.001) and EAF groups (P=0.001) as compared to control group. The number of BrdU positive keratocytes was significantly higher in the superficial and deep stromal sub-areas in the HAF group as compared to control (P<0.001 and P=0.002, respectively). EAF and FBS treated corneas also showed a higher number of BrdU positive cells compared to control, but this difference was not significant. Finally, we did not observe any difference in the amount of TUNEL positive keratocytes among the different groups. Our data indicates that the topical application of HAF and EAF is associated with accelerated reepithelialization in this cornea organ culture model. Similarly, corneal keratocyte density appears to be less affected after epithelial injury using this treatment.

Castro-Combs J ，Noguera G ，Cano M ，Yew M ，Gehlbach PL ，Palmer J ，Behrens A ... -  《EXPERIMENTAL EYE RESEARCH》

Platelet-activating factor induces the expression of metalloproteinases-1 and -9, but not -2 or -3, in the corneal epithelium.

The inflammatory mediator platelet-activating factor (PAF) induces the expression of interstitial collagenase (metalloproteinase-1) messenger RNA in rabbit corneal epithelium. In this study, the authors investigated the effect of PAF on gene expression and protein activity of other matrix metalloproteinases (MMPs) in the cornea. Rabbit corneas were incubated in an organ culture with 100 nM of cPAF (a nonhydrolyzable PAF analog), PAF, or lyso-PAF, an inactive metabolite of PAF. In some experiments, the corneas were preincubated for 1 hour with 10 microM BN50730, a PAF antagonist, before cPAF was added to the medium. Corneal epithelial cells and/or conditioned medium were collected at different times for analysis. Also, in vivo experiments were done by injecting 2 micrograms of cPAF intrastromally into rabbit eyes and collecting the epithelium 8 hours later for study. Northern blot analysis and zymography were performed to determine the mRNA abundance and/or enzyme activity of 92 kd gelatinase (MMP-9), 72 kd gelatinase (MMP-2), and stromelysin (MMP-3). The activity of MMP-1 was tested by collagenase assays. cPAF induced the expression of MMP-9 mRNA, but not MMP-3 mRNA. The message was induced at 4 hours and remained elevated at 48 hours, with a peak at 36 hours. In corneas preincubated with BN50730, MMP-9 mRNA activation by cPAF was inhibited. In vivo injection of cPAF also induced the expression of MMP-9. Furthermore, cPAF increased MMP-9 activity in the epithelial cells and in the conditioned media. The effect was blocked by BM50730. cPAF did not affect MMP-2 activity. Finally, cPAF also increased MMP-1 collagenolytic activity of the corneal epithelium, which was blocked by the PAF antagonist. These results suggest a novel mechanism by which PAF activates MMPs. The lipid mediator selectively enhances the expression of MMP-1 and MMP-9 in rabbit corneal epithelium. This activation by PAF may be involved in the remodeling mechanisms of the cornea after injury and, when overexpressed, may lead to the formation of corneal ulcers. Specific PAF antagonists could therapeutically deter corneal ulcer formation and facilitate corneal wound healing.

Tao Y ，Bazan HE ，Bazan NG 《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》