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》

IL-1 upregulates keratinocyte growth factor and hepatocyte growth factor mRNA and protein production by cultured stromal fibroblast cells: interleukin-1 beta expression in the cornea.

To determine whether interleukin 1 beta (IL-1 beta) messenger RNA (mRNA) and protein were expressed in corneal cells and to examine the effects of IL-1 alpha and IL-1 beta on the expression of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) mRNAs and proteins in corneal stromal fibroblasts. IL-1 beta mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). IL-1 beta protein was detected by immunohistologic tests. Changes in the expression of HGF and KGF mRNAs and proteins in response to stimulation of cultured corneal stromal fibroblasts with IL-1 alpha and IL-1 beta were monitored by Northern and Western blotting, respectively. IL-1 beta mRNA is expressed in human primary cultured corneal epithelial, stromal fibroblast, and endothelial cells. IL-1 beta protein was detected in epithelium and endothelium in fresh frozen human and rabbit corneal tissue. Little, if any, IL-1 beta was detected in the unwounded corneal stroma. IL-1 alpha and IL-1 beta at 10 ng/ml upregulated the levels of HGF and KGF mRNAs and proteins in cultured human corneal fibroblasts. IL-1 alpha and IL-1 beta may serve as key modulators in an epithelial-stromal regulatory loop in the cornea. These data and previously published observations support the hypothesis that corneal epithelial wounding releases IL-1 alpha and IL-1 beta from epithelial cells; these cytokines in turn upregulate HGF and KGF mRNA and protein levels in keratocytes, and HGF and KGF released by the keratocytes modulate healing of the wounded corneal epithelial cells by regulating proliferation, motility, and differentiation.

Weng J ，Mohan RR ，Li Q ，Wilson SE ... -  《CORNEA》

Platelet-activating factor (PAF) induces corneal neovascularization and upregulates VEGF expression in endothelial cells.

Platelet-activating factor (PAF) is a potent proinflammatory mediator that accumulates in the cornea after injury and induces the expression of genes related to inflammation and wound healing. The current study was conducted to investigate the direct effect of PAF on corneal neovascularization and on the expression of angiogenic growth factors in vascular endothelial cells. Pellets containing carbamyl-PAF (cPAF) were implanted in corneas of wild-type or PAF-receptor (PAF-R)-knockout mice, and the progression of angiogenesis was monitored by microscope. In some experiments, mice were treated with a daily intraperitoneal injection of the PAF-R antagonist LAU8080. Migration assays of human umbilical cord vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs) were performed in a Boyden chamber after addition of various concentrations of cPAF or bovine fibroblast growth factor (FGF-2). Cell proliferation was assessed by fluorescence-binding assay in the presence of cPAF or FGF-2 for 8 days. Vascular endothelial growth factor (VEGF) and FGF-2 expression was studied by RT-PCR and Northern- and Western-blot analyses in cells stimulated with cPAF at different concentrations and for different times. Six days after cPAF pellet implantation, there were new vessels growing from the limbus to the center of the cornea. The PAF-induced neovascularization was significantly reduced in PAF-R-knockout mice and in mice treated with the PAF antagonist. cPAF added to the lower well of the Boyden chamber produced a dose-dependent migration of HUVECs and HMVECs that was inhibited in cells preincubated with LAU8080 or with a VEGF-blocking antibody. In contrast, cPAF did not stimulate proliferation of endothelial cells. cPAF induced VEGF mRNA and protein expression but not FGF-2 expression in HUVECs and HMVECs. PAF stimulates corneal neovascularization by a receptor-mediated mechanism. Induction of VEGF expression and stimulation of vascular endothelial cell migration are initial events in PAF-promoted corneal angiogenesis.

Ma X ，Ottino P ，Bazan HE ，Bazan NG ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

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》

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》