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》

Platelet-activating factor induces the gene expression of TIMP-1, -2, and PAI-1: imbalance between the gene expression of MMP-9 and TIMP-1 and -2.

Previous studies in the laboratory have shown that platelet-activating factor (PAF), a potent inflammatory mediator that accumulates rapidly in the cornea after an injury, stimulates the expression of urokinase (uPA) and matrix metalloproteinase-1 (MMP-1) and -9 (MMP-9). Tissue inhibitors of MMPs (TIMPs) and plasminogen activator inhibitor (PAI-1) are produced in conjunction with these enzymes and are important regulators of their activity. Here, the authors investigated how PAF affects the expression of PAI-1, TIMP-1 and -2 relative to that of uPA, MMP-1, and -9 in rabbit corneal epithelial cells. Rabbit corneas were incubated in MEM medium containing 100 nM cPAF. To block the effects of PAF in some studies, corneas were preincubated for 1 hr in the presence of the PAF antagonist BN50730 (10 microM). At several time intervals, mRNA was extracted from epithelial cells and the levels of gene expression for the enzymes and their inhibitors were determined by real-time PCR. All quantitations were normalized to the 18s rRNA values (endogenous control) and changes in gene expression were reported as fold increase relative to untreated controls. PAF produced a 20-fold increase in the gene expression of PAI-1 at 8 hr, while similar fold increases in uPA mRNA expression occurred at 2 hr. PAF treatment also stimulated the expression of TIMP-1 and -2 genes, with a six-fold increase in TIMP-1 expression occurring at 36 hr and a four-fold increase in TIMP-2 expression at 24 hr. Maximal induction of MMP-1 and -9 mRNA, on the other hand, occurred at 4 and 8 hr, respectively. Induction of MMP-1 gene expression was similar to that of its inhibitors TIMP-1 and -2, while MMP-9 mRNA induction exceeded that of these inhibitors by 100-fold. The PAF-induced expression of PAI-1, TIMP-1 and -2 mRNAs was abolished by pre-treatment with BN50730. These data indicate that PAF activates the gene expression of TIMP-1, -2, and PAI-1 in corneal epithelium by a receptor-mediated mechanism. Furthermore, PAF induced overexpression of MMP-9 mRNA relative to that of TIMP-1 and -2, suggesting an imbalance between the expression of this proteolytic enzyme and its inhibitors, which may contribute to changes in the wound-healing process and ultimately lead to corneal ulcer development.

Ottino P ，Taheri F ，Bazan HE 《EXPERIMENTAL EYE RESEARCH》

PAF-induced furin and MT1-MMP expression is independent of MMP-2 activation in corneal myofibroblasts.

Corneal stromal myofibroblasts express the platelet-activating factor (PAF) receptor, but its role is unclear. In the present study, the effect of PAF on induction of metalloproteinases (MMPs) was investigated. Rabbit corneal myofibroblasts were identified by immunodetection of alpha-smooth muscle (alpha-SM)-actin. MT1-MMP, MMP-2, MMP-9, and tissue inhibitor of matrix metalloproteinase (TIMP)-2 were detected by immunofluorescence. Cells were treated with 100 nM cPAF, with or without the PAF antagonist BN 50730 or the furin inhibitor nona-D-arg-NH(2). Gene-expression levels for furin, urokinase plasminogen activator, MMP-2, MMP-9, MT1-MMP, and TIMP-2 were determined by real-time PCR. Protein expression was assessed by Western blot. MMP-2 and -9 activity was determined by gelatin zymography. Active MT1-MMP levels were measured by ELISA. cPAF triggered significantly increased MT1-MMP, MMP-2, MMP-9, and TIMP-2 mRNA expression, followed by increased active MT1-MMP protein expression at 12 hours, whereas TIMP-2 protein increased at 24 hours. PAF also induced furin gene expression, followed by increased protein expression. Nona-D-arg-NH(2) blocked cPAF induction of MT1-MMP activity. PAF-treated myofibroblasts showed increased active MMP-9 protein, but unchanged MMP-2 activity. Pretreatment with BN 50730 blocked PAF-induced transcription and translation of these proteins. PAF, through a receptor-mediated mechanism, induces a specific pattern of furin, MMP, and TIMP-2 expression in corneal myofibroblasts. MMP-2 activity was unchanged by PAF treatment. These results suggest that in response to the inflammatory mediator PAF, induction of MT1-MMP is independent of MMP-2 activity in corneal myofibroblasts. Thus, PAF-mediated changes in extracellular matrix composition surrounding the myofibroblasts could be important in regulating the corneal scarring process. Moreover, PAF antagonists could be useful in maintaining corneal transparency.

Ottino P ，He J ，Axelrad TW ，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》

Platelet-activating factor enhances urokinase-type plasminogen activator gene expression in corneal epithelium.

To determine whether platelet-activating factor (PAF), a lipid mediator that is accumulated in the cornea after alkali burn, induces the gene expression of urokinase-type plasminogen activator (uPA) in the corneal epithelium. Possible signaling mechanisms of uPA gene induction by PAF also were examined. Rabbit corneas were cultured with or without PAF. One hour before stimulation, PAF antagonists or other modulators were added to PAF. In some experiments, the corneas were permeabilized to introduce guanosine triphosphate analogs into the corneal epithelial cells. Corneal epithelia were then harvested for Northern blot analysis, nuclear runoff transcription assay, and zymography. Platelet-activating factor induced uPA mRNA expression in the corneal epithelium. New protein synthesis was not required for the induction of uPA mRNA. The induction was at the level of transcription as shown by nuclear runoff assays. Additionally, both actinomycin D and alpha-amanitin inhibited the increase in uPA mRNA by PAF. The message was translated into protein, which was secreted into the conditioned medium. An antagonist with high affinity for intracellular PAF binding sites (BN 50730) inhibited uPA gene expression and cellular secretion of the protein. The effect of PAF was not mediated by G proteins and was independent of protein kinase C- and cyclic adenosine monophosphate-dependent signal transduction pathways. Okadaic acid increased the expression of uPA and, at longer times, augmented the effect of PAF, suggesting that a signaling pathway that requires phosphorylation is involved in activated uPA mRNA synthesis. After corneal injury and inflammation, PAF may be an important initiator of the proteolytic cascade, leading to epithelial defects and corneal ulceration. Antagonists of PAF could be useful in the prevention of these diseases.

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