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》

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》

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》

Effect of epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, on proliferation, motility and differentiation of human corneal epithelial cells.

Wilson SE ，He YG ，Weng J ，Zieske JD ，Jester JV ，Schultz GS ... -  《EXPERIMENTAL EYE RESEARCH》

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》