Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

Stromal-epithelial interactions in the cornea

Stromal-epithelial interactions are key determinants of corneal function. Bi-directional communications occur in a highly coordinated manner between these corneal tissues during normal development, homeostasis, and wound healing. The best characterized stromal to epithelial interactions in the cornea are mediated by the classical paracrine mediators hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). HGF and KGF are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions, of epithelial cells. Other cytokines produced by keratocytes may also contribute to these interactions. Epithelial to stromal interactions are mediated by cytokines, such as interleukin-1 (IL-1) and soluble Fas ligand, that are released by corneal epithelial cells in response to injury. Other, yet to be identified, cytokine systems may be released from the unwounded corneal epithelium to regulate keratocyte viability and function. IL-1 appears to be a master regulator of corneal wound healing that modulates functions such as matrix metalloproteinase production, HGF and KGF production, and apoptosis of keratocyte cells following injury. The Fas/Fas ligand system has been shown to contribute to the immune privileged status of the cornea. However, this cytokine-receptor system probably also modulates corneal cell apoptosis following infection by viruses such as herpes simplex and wounding. Pharmacologic control of stromal-epithelial interactions appears to offer the potential to regulate corneal wound healing and, possibly, treat corneal diseases in which these interactions have a central role.     

Differential expression analysis by gene array of cell cycle modulators in human corneal epithelial cells stimulated with epidermal growth factor (EGF), hepatocyte growth factor (HGF), or keratinocyte growth factor (KGF)

PURPOSE: To identify and differentiate cell cycle and differentiation genes that are up-regulated or down-regulated in human corneal epithelial cells in response to alternative epithelium-modulating cytokines epidermal growth factor (EGF), hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF). METHODS: Primary cultures human corneal epithelial cell (HCE) were treated with 25 ng/ml of EGF, 25 ng/ml HGF, 25 ng/ml KGF, or vehicle for 8 hours. Complementary DNA (cDNA) probes were synthesized from total cellular RNA isolated from the HCE cells. The cDNA probes were hybridized to the Atlas human cell cycle/differentiation array membrane. RNAse protection assay was used to confirm up-regulation of the serine/threonine-protein kinase PITALRE gene by EGF, KGF, and HGF. RESULTS: The expression of one hundred and eleven cell cycle and differentiation genes was monitored with the gene array system. It was found that these epithelial cell-modulating cytokines shared similar effects on some of the cell cycle and differentiation genes that were monitored, but had specific effects on some cytokines. Up-regulation of PITALRE gene expression was confirmed using RNAse protection assay. CONCLUSION: EGF, HGF and KGF had differential effects on cell cycle- and differentiation-related gene expression in corneal epithelial cells. For example, all three mitogenic growth factors up-regulated the expression of cyclin D1 (BCL-1 oncogene) and serine/threonine-protein kinase PITALRE in the primary cultured human corneal epithelial cells. However, EGF and KGF, but not HGF, up-regulated expression of the E2F-1 pRB-binding protein gene. Thus, while these three epithelial mitogens have similar effects on many genes that were analyzed, important differences were noted that may relate to differing effects of these growth factors on corneal epithelial cells. Studies to analyze the significance of the identified differences among these growth factors are in progress.     

Effect of exogenous keratinocyte growth factor on corneal epithelial migration after photorefractive keratectomy

PURPOSE: To investigate the effect of topical keratinocyte growth factor (KGF) on wound healing after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom, St. Erick's Eye Hospital, Stockholm, Sweden, and the University of Regensberg, Regensberg, Germany. METHODS: In a placebo-controlled trial, 24 New Zealand white female rabbits were divided into 3 equal groups. Group 1 (n=8) had myopic PRK (6.0 diopters [D]) using the Technolas 217z laser (Bausch & Lomb). Group 2 and Group 3 had myopic LASIK (6.0 D) with a flap depth of 140 microm and 180 microm, respectively. Topical KGF (20 microg/mL) was administered to half the treated eyes in each group intraoperatively and postoperatively; the other half received placebo eyedrops. Epithelial closure, corneal haze, and keratocyte activation in the rabbit eyes were analyzed and compared with those in placebo-controlled eyes for 5 weeks postoperatively. RESULTS: In Group 1, the mean reepithelialization after PRK was 0.10 mm2/h +/- 0.02 (SD) in the KGF group and 0.33 +/- 0.05 mm2/h in the control group (P=.001). There was no significant difference in the mean backscatter between the KGF eyes (154 +/- 45.95) and the control eyes (141 +/- 38.45) after PRK (P=.42). Histology revealed reduced epithelial cell layers in the KGF group and comparable keratocyte density as in the control group. In Groups 2 and 3, there was no significant difference in backscatter, epithelial layers, and keratocyte density between KGF and control eyes after LASIK. CONCLUSIONS: Topical KGF (20 microg/mL) delayed reepithelialization after PRK. It had no effect on stromal wound healing in LASIK eyes with an intact epithelial barrier.     

Hepatocyte growth factor/scatter factor in the eye

Hepatocyte growth factor, also known as scatter factor (HGF/SF) is a multipotential cytokine which can produce a range of responses in target cells and its influence in the eye in health and disease is just beginning to be appreciated. Usually HGF/SF is synthesised by mesenchymally derived cells and targets and signals epithelial cells in a paracrine manner via their c-Met surface receptor. However, there is growing evidence for the existence of autocrine loops in a number of cell systems prominent among which are ocular cells such as the corneal endothelium, the lens epithelium, the retinal pigment epithelium (RPE) and others. Marked cellular proliferation is stimulated when activated HGF/SF is exposed to hepatocytes, renal epithelium, melanocytes and vascular endothelial cells but it is often a poor mitogen for other cell types. In target cells the cytokine promotes other bioactions such as junctional breakdown, shape change, cell scattering, directional and nondirectional migration, cell survival, invasive behaviour and/or tubule formation. These activities seem to depend on HGF/SF linking with the c-Met receptor and pathways to stimulate the various types of cytokine/receptor response are being unravelled at the present time. In corneal wound healing, HGF/SF is produced by stromal keratocytes and targets the repairing epithelium. HGF/SF is a constituent of tears, aqueous humour and vitreous humour at levels above that found in plasma although it is not clear how much is activated. Aqueous HGF/SF may well influence lens epithelial, corneal endothelial and trabecular meshwork cell survival. Vitreous levels of HGF/SF are elevated in proliferative vitreoretinopathy (PVR), where a target cell is the RPE and in proliferative diabetic retinopathy (PDR) where HGF/SF has been shown to be a major angiogenesis factor. Finally HGF/SF may be involved in the metastatic spread of tumour cells from uveal melanomata and in the formation of vascular channels in these tumours.     

Phosphorylation of MAP kinase in corneal epithelial cells during wound healing

PURPOSE: To investigate the role of mitogen-activated protein kinase (MAPK), such as p44/42 MAPK, p38 MAPK and stress-activated protein kinase (SAPK), in corneal epithelial cells during the wound healing process. METHODS: A single non-penetrating incision was produced on rat cornea. Then the corneal wound healing process was observed with an immunocytochemical technique using specific antibodies reacting only with phosphorylated p44/42 MAPK, p38 MAPK or SAPK. Cell lysates of corneal epithelial cells in rabbits stimulated with keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were processed for Western blot using antibodies to phosphorylated p44/42 MAPK. RESULTS: Maximum activation of p44/42 MAPK was observed in wing and basal cells at wounded regions in rat cornea at 1 hour after the incision. Activation of p44/42 MAPK was still detected in all basal and wing cells at wounded regions at up to 24 hours when the incisions were completely closed, and then receded to normal intensity after 7 days. Neither p38 MAPK nor SAPK were activated during the wound healing process. Western blot analysis of cultured corneal epithelial cells in rabbits showed phosphorylation of p44/42 MAPK after 30 minutes in response to KGF and HGF, whereas non-activated p44/42 MAPK was ordinarily detected even at the absence of KGF or HGF. CONCLUSIONS: These results demonstrate that p44/42 MAPK is activated during the corneal wound healing process and suggest that KGF and HGF play an important role in initiation of cell migration and proliferation in the initial wound healing process by activating p44/42 MAPK.     

Interleukin-1 and Transforming Growth Factor Beta: Commonly Opposing,but Sometimes Supporting,Master Regulators of the Corneal Wound Healing Response to Injury

PurposeInterleukin (IL)-1α/IL-1β and transforming growth factor (TGF)β1/TGFβ2 have both been promoted as “master regulators” of the corneal wound healing response due to the large number of processes each regulates after injury or infection. The purpose of this review is to highlight the interactions between these systems in regulating corneal wound healing.MethodsWe conducted a systematic review of the literature.ResultsBoth regulator pairs bind to receptors expressed on keratocytes, corneal fibroblasts, and myofibroblasts, as well as bone marrow-derived cells that include fibrocytes. IL-1α and IL-1β modulate healing functions, such as keratocyte apoptosis, chemokine production by corneal fibroblasts, hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) production by keratocytes and corneal fibroblasts, expression of metalloproteinases and collagenases by corneal fibroblasts, and myofibroblast apoptosis. TGFβ1 and TGFβ2 stimulate the development of myofibroblasts from keratocyte and fibrocyte progenitor cells, and adequate stromal levels are requisite for the persistence of myofibroblasts. Conversely, TGFβ3, although it functions via the same TGF beta I and II receptors, may, at least in some circumstances, play a more antifibrotic role—although it also upregulates the expression of many profibrotic genes.ConclusionsThe overall effects of these two growth factor-cytokine-receptor systems in controlling the corneal wound healing response must be coordinated during the wound healing response to injury or infection. The activities of both systems must be downregulated in coordinated fashion to terminate the response to injury and eliminate fibrosis.Translational RelevanceA better standing of the IL-1 and TGFβ systems will likely lead to better approaches to control the excessive healing response to infections and injuries leading to scarring corneal fibrosis.     

Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the growth factors such as epidermal growth factor and hepatocyte growth factor (HGF), but not keratinocyte growth factor (KGF), regulate PKCalpha activation to promote epithelial wound healing [Sharma, G.D., Ottino, P., Bazan, H.E.P., 2005. Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase C alpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis. J. Biol. Chem. 280, 7917--924]. Protein kinase C alpha (PKCalpha) and protein kinase C epsilon (PKCvarepsilon) are two differentially regulated isoenzymes. While PKCalpha requires Ca(2+) for its activation, PKEvarepsilon is Ca(2+) independent. However, growth factor-induced activation of these enzymes and their specific regulation of epithelial migration and proliferation have not been explored. In the present study, we overexpressed PKCvarepsilon fused to green fluorescent protein to examine its translocation in real-time to the plasma membrane in living human corneal epithelial cells. Stimulation with HGF and KGF demonstrated translocation of PKCvarepsilon to the plasma membrane. Because HGF activates both PKCs, this growth factor was used to stimulate wound healing. PKCalpha or PKCvarepsilon-genes were knocked down individually without affecting the basal expression of the other PKC isoforms. Gene knockdown of PKCalpha significantly inhibited HGF-stimulated proliferation of human corneal epithelial cells. In contrast, PKCvarepsilon-gene-silencing severely impaired the HGF-stimulated migratory ability of human corneal epithelial cells. When migrating epithelial cells in the cornea wound bed after injury were transfected with specific PKCalpha- or PKCvarepsilon-siRNA, there was a significant delay in wound healing. Corneal wound healing stimulated with HGF in similar conditions was also inhibited. On the other hand, overexpression of PKCalpha or PKCvarepsilon-genes fused with green fluorescent protein in migrating corneal epithelium accelerated repair of the epithelial defect. Our findings demonstrate that PKCalpha and PKCvarepsilon modulate different stages of wound healing stimulated by HGF and contribute to epithelial repair by playing selective regulatory roles in epithelial proliferation and migration, both crucial to corneal wound healing.     

角质细胞生长因子促进角膜上皮损伤修复的研究

目的寻找促进角膜上皮损伤修复，治疗持续性角膜上皮缺损的有效方法。方法用３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入及液体闪烁技术，观察不同浓度的外源性角质细胞生长因子（ｋｅｒａｔｉｎｏｃｙｔｅｇｒｏｗｔｈｆａｃｔｏｒ，ＫＧＦ）对体外培养的人角膜上皮细胞生长的影响，由此推算出有效滴眼液浓度并应用于兔眼。用计算机图形分析系统计算角膜上皮愈合速率；用光镜和电镜评估愈合的质量。结果０．１～１００ｎｇ／ｍｌＫＧＦ有明显促进体外培养的人角膜上皮细胞生长的作用（增长率为２７．６６％～７６．７３％），且呈剂量依赖性（ｒ＝０．９２３３，Ｐ＜０．００１）。１μｇ／ｍｌＫＧＦ滴兔眼，加速了角膜上皮损伤修复（愈合速率，ＫＧＦ组为１．７７±０．２３ｍｍ２／ｈ，与对照组１．４９±０．２４ｍｍ２／ｈ比较，Ｐ＜０．０５）。结论外源性ＫＧＦ对体外培养的人角膜上皮细胞有明显的促生长作用，其滴眼液有加速兔眼角膜上皮创伤修复的作用。     

血小板活化因子致角膜上皮伤口迟愈合的实验研究

目的　利用兔去上皮角膜模型 ,研究血小板活化因子 (PAF)对角膜伤口愈合的作用及其分子生物学机制。方法　离体角膜上做正中直径 7mm圆形去上皮角膜伤口。去上皮角膜分为 3组 ,即对照、PAF及BN (PAF拮抗剂 )组 ,培养 4 8h后 ,行角膜上皮染色观察伤口愈合状况。分别对兔角膜上皮 (RCE)和角膜基质 (RCK)细胞进行体外传代培养 ,RCE和RCK细胞经PAF和 (或 )BN处理 ,培养 2 4h ,提纯RNA。应用RT PCR及核酸杂交技术分别检测肝细胞生长因子 (HGF)、角质形成生长因子 (KGF)及表皮生长因子 (EGF)基因在RCK和RCE细胞及HGF受体 (HGF R)基因在RCE细胞中的表达强度。分别应用CyQUANT荧光结合和Boyden小房技术检测PAF对RCE细胞黏附、增殖和迁徙的影响。结果　PAF (10 0nmol/L )明显抑制角膜上皮伤口愈合 ,4 8h对照、PAF和BN组角膜上皮未愈合面积经电脑图像分析分别为 :(6 0± 1.5 )U、(5 8 0± 7 0 )U和 (5 0± 1 0 )U。PAF明显增强RCE细胞黏附作用 ,对照、PAF和BN组每 96孔板贴附细胞数荧光光度平均值分别为 :36 96± 372、790 8± 6 71和 3487± 32 4。RT PCR结果显示 :PAF使HGFmRNA在RCK的表达强度降低 4 .1倍 ,同时明显减弱HGF R在RCE细胞中的表达 ,核酸杂交实验证实PCR结果。结论PAF明显增强RCE细胞的黏附作用 ,     

Growth factor mRNA and protein in preserved human amniotic membrane

PURPOSE: To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). METHODS: RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80 degrees C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2) in preserved human corneas and in AM both with and without amniotic epithelium. RESULTS: RT-PCR revealed that human AM expresses mRNA for EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. CONCLUSIONS: Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.     

The effects of growth factors and conditioned media on the proliferation of human corneal epithelial cells and keratocytes

Background: As growth factors play an important role in epithelial wound repair, we evaluated the effect of exogenous growth factors in the presence and absence of corneal epithelial and keratocyte conditioned medium on human corneal epithelial cell and keratocyte proliferation. • Methods: Preconfluent cultures of human corneal epithelial cells or stromal keratocytes were exposed to varying concentrations of EGF, TGF-β or bFGF in the presence or absence of human corneal epithelial or stromal keratocyte conditioned medium. Cell numbers were determined after 48 h incubation. RIA and ELISA were used to quantify the levels of EGF, TGF-β and bFGF in conditioned media. • Results: EGF and bFGF increased, while TGF-β decreased, the proliferation of both cell types in a dose- dependent manner. Epithelial cell conditioned medium inhibited, and keratocyte conditioned medium stimulated, the proliferation of both cell types. The proliferative effects of EGF, TGF-β and bFGF in the presence of keratocyte conditioned medium were additive for both cell types. By contrast, the addition of exogenous growth factors was unable to overcome the inhibitory potential of epithelial conditioned medium. Both conditioned media contained significant levels of bFGF, but TGF-β levels in epithelial conditioned medium were up to 5 times greater than that in keratocyte conditioned medium. • Conclusions: The results indicate that corneal cells maintain tissue homeostasis and modulate the wound healing response via paracrine/autocrine pathways. Received: 6 February 1997 Revised version received: 2 April 1997 Accepted: 10 April 1997     

The effects of growth factors and conditioned media on the proliferation of human corneal epithelial cells and keratocytes

• Background: As growth factors play an important role in epithelial wound repair, we evaluated the effect of exogenous growth factors in the presence and absence of corneal epithelial and keratocyte conditioned medium on human corneal epithelial cell and keratocyte proliferation. • Methods: Preconfluent cultures of human corneal epithelial cells or stromal keratocytes were exposed to varying concentrations of EGF, TGF-β or bFGF in the presence or absence of human corneal epithelial or stromal keratocyte conditioned medium. Cell numbers were determined after 48 h incubation. RIA and ELISA were used to quantify the levels of EGF, TGF-β and bFGF in conditioned media. • Results: EGF and bFGF increased, while TGF-β decreased, the proliferation of both cell types in a dosedependent manner. Epithelial cell conditioned medium inhibited, and keratocyte conditioned medium stimulated, the proliferation of both cell types. The proliferative effects of EGF, TGF-β and bFGF in the presence of keratocyte conditioned medium were additive for both cell types. By contrast, the addition of exogenous growth factors was unable to overcome the inhibitory potential of epithelial conditioned medium. Both conditioned media contained significant levels of bFGF, but TGF-β levels in epithelial conditioned medium were up to 5 times greater than that in keratocyte conditioned medium. • Conclusions: The results indicate that corneal cells maintain tissue homeostasis and modulate the wound healing response via paracrine/autocrine pathways.     

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

PURPOSE: To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

TGFbeta induced myofibroblast differentiation of rabbit keratocytes requires synergistic TGFbeta,PDGF and integrin signaling

There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFbeta). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFbeta. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFbeta1 stimulated cell cycle entry of serum-free cultured rabbit corneal keratocytes. AntiPDGF treatment also markedly reduced the TGFbeta1-induced intracellular actin filament re-organization, fibronectin fibril assembly, and focal contact formation as well as reducing by 80% the expression of alpha-smooth muscle (alpha-SM) specific isoform of actin characteristic of myofibroblast differentiation. Although PDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFbeta1 as did quiescent keratocytes. Furthermore, blocking TGFbeta1 induction of myofibroblast differentiation with the Arg-Gly-Asp containing peptide, GRGDdSP, for 3 days followed by allowing progression of myofibroblast differentiation by removing GRGDdSP did not change the temporal response or tyrosine phosphorylation cascade (2-72 hr) leading to myofibroblast differentiation. Nor did PDGF treatment of keratocytes reverse the RGD blockade of TGFbeta1 induced myofibroblast differentiation. Overall these cumulative findings indicate that myofibroblast differentiation in the rabbit corneal keratocyte requires synergistic growth factor/integrin signaling involving TGFbeta, PDGF, and the fibronectin receptor. Additionally, the similar TGFbeta1 temporal response of PDGF-stimulated compared to nai;ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

Expression of basic fibroblast growth factor in rabbit corneal alkali wounds in the presence and absence of granulocytes

PURPOSE: To study the expression of basic fibroblast growth factor (bFGF) in the early phases of corneal wound healing in the presence or absence of granulocytes. METHODS: A central penetrating corneal alkali wound was inflicted to one eye in each of 14 rabbits under general anaesthesia. Subsequently, seven of the rabbits were given fucoidin i.v. for 36 hours in order to block the selectins on the vascular endothelium, thus preventing blood granulocytes from entering the tissues. Then, corneas were prepared, stained for bFGF and evaluated by light microscopy. RESULTS: Whereas normal corneal epithelium expressed bFGF weakly, conjunctival epithelium did so strongly, particularly the goblet cells. The corneal endothelium showed medium staining, while keratocytes and vascular endothelial cells did not consistently express bFGF. After 36 hours of wound healing, a marked up-regulation of bFGF expression was observed in the corneal epithelial and endothelial cells, as well as in the keratocytes, that were migrating into the wound. No other changes were noted. None of these features were modulated when granulocyte emigration was prevented by fucoidin administration. CONCLUSIONS: The difference in bFGF expression between the corneal and conjunctival epithelium suggests a role for this growth factor in the barrier function at the limbus. Moreover, the specific presence of bFGF in cells migrating into the wound indicates the participation of bFGF in corneal wound healing. Expression of bFGF was independent of granulocytes.     

Differential effects of epidermal growth factor and interleukin 6 on corneal epithelial cells and vascular endothelial cells.

PURPOSE: Epidermal growth factor (EGF) and interleukin 6 (IL-6) stimulate corneal epithelial wound healing. When applied to the cornea, these cytokines act on various types of cells and therefore may induce corneal neovascularization. We investigated the effects of EGF and IL-6 on cell proliferation and cell migration in rabbit corneal epithelial cells and human umbilical vein endothelial cells (HUVECs). METHODS: Corneal epithelial cells or HUVECs were cultured with EGF or IL-6 in the presence of 1% fetal bovine serum, and the number of cells were counted, or the radioactivity of [3H]thymidine-incorporated cells was measured. Monolayered cultured corneal epithelial cells or HUVECs were mechanically wounded, and then the cells were cultured with serum-free basal medium containing EGF or IL-6. After 12 or 24 h, the wounded area was measured. Corneal blocks were cultured with serum-free TC-199 medium containing EGF or IL-6 for 24 h, and then the length of the path of the corneal epithelium was measured. RESULTS: Estimated cell count and [3H]thymidine uptake showed that EGF stimulated cell proliferation in both corneal epithelial cells and HUVECs in a dose-dependent manner. In contrast, IL-6 did not affect cell proliferation in either cell type. Furthermore, EGF also stimulated cell migration by increasing the monolayer and organ-culture system in both cells in a dose-dependent fashion. However, IL-6 stimulated cell migration only in corneal epithelial cells and not in HUVECs. CONCLUSION: These results demonstrated that EGF stimulated cell proliferation and migration in both corneal epithelial cells and HUVECs. In contrast, IL-6 stimulated only corneal epithelial cell migration and did not affect cell proliferation in either cell type or cell migration in HUVECs. These results suggest that, when applied to the cornea, EGF might induce corneal neovascularization, and IL-6 probably would not.     

Involvement of S100A4 in stromal fibroblasts of the regenerating cornea

PURPOSE. S100A4 is a member of the S100 family of calcium-binding proteins. Members of the S100 family have been implicated in a variety of cellular events, including growth, signaling, differentiation, and motility. It has been suggested that S100A4 modulates cell shape and motility by interacting with components of the cytoskeleton. In the present study, the distribution patterns of S100A4 were investigated in normal and regenerating mouse corneas. METHODS. Rabbit cDNA libraries were prepared from cultures of corneal fibroblasts. S100A4 was identified as the most abundant message present. Expression of S100A4 in the cornea was determined using Northern blot analysis, in situ hybridization, and immunohistochemistry. Distribution patterns of S100A4 in primary corneal fibroblast cultures treated with either FGF-2/heparin or TGFbeta1 were analyzed by immunofluorescence. RESULTS. S100A4 mRNA was rarely detected in keratocytes or epithelial cells of the normal rabbit cornea. Likewise, S100A4 antigen was not found in normal mouse corneas. However, after removal of the corneal epithelium, fibroblasts are activated and had readily detectable S100A4 expression 6 days after wounding. In the in vitro equivalent of activated keratocytes, cultured rabbit corneal fibroblasts, S100A4 was restricted to the cytoplasm. In contrast, in cultures treated with TGFbeta1, which induces a myofibroblast phenotype, more than 90% of the cells showed a nuclear localization of S100A4. CONCLUSIONS. The findings show that S100A4 is expressed in the keratocyte phenotypes that appear in stromal tissue of corneas recovering from damage, the fibroblasts, and myofibroblasts. Its expression and distinct subcellular redistribution patterns suggest that S100A4 may be involved in the interconversions that occur between keratocytes, fibroblasts, and myofibroblasts during corneal wound healing.