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.     

Donor age and gestational age influence on growth factor levels in human amniotic membrane

Acta Ophthalmol. 2010: 88: e211–e216

Abstract.

Purpose: Amniotic membrane (AM) is used as a biomaterial for reconstruction in ocular surface surgery. This study investigated the influence of interdonor variations and processing and preservation procedures applied to the AM on growth factors and protein levels. Methods: Samples of human AM from thirteen donors were analysed. Collected donor data were age, parity and gestational age. Total protein amount was measured in extracts of intact AM nonpreserved, lyophilized and cryopreserved, at ?80°C and in liquid nitrogen. An enzyme‐linked immunosorbent assay (ELISA) was used to assay growth factors protein levels for epidermal growth factor, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), transforming growth factor beta1 (TFG‐β₁) and nerve growth factor (NGF). Univariate and multivariate statistical analyses were used to study the influence of the preservation method applied and interdonor variations on growth factors levels. Results: We detected important variations in growth factors and protein concentrations between samples from different donors. Total protein amount, bFGF, HGF, KGF and TGF‐β₁ showed lower levels in samples from donors with higher gestational ages and donor ages, for all groups. Conclusion: The variability in the biochemical composition of AM from different donors is considerable, and it is related with donor factors as donor age and gestational age. As AM biochemical composition has a role in its therapeutic effects, these variations could affect the clinical results of amniotic membrane transplantation and must be taken into account in donor selection processes.     

The Changes of TGF—α,TGF—β1 and Basic FGF Messenger RNA Expression i

OBJECTIVE: To study the mechanism of haze formation and investigate the expression changes of transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1(TGF-beta 1) and basic fibroblast growth factor (bFGF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK). METHODS: Sixteen white rabbits were randomly divided into 4 groups, and PRK was performed on each eye of 12 rabbits. The haze formation was examined under a slit-lamp microscope at the 1st, 2nd and 3rd month after PRK, and the expressions of TGF-alpha, TGF-beta 1 and bFGF mRNA were detected with in situ hybridization. RESULTS: The corneal haze formed at the 1st month after PRK. The most prominent haze formation was observed at the 2nd month, and declined gradually at the 3rd month after ablation. TGF-alpha mRNA expression was presented on the normal corneal epithelium and not on the corneal stroma. TGF-beta 1 and bGFG mRNA were expressed by both corneal epithelium and stroma. The capacities for cornea tissue expression of three growth factors mRNA increased after PRK, and the peaks appeared on the 1st, 2nd month. The extent for expressions of three growth factors related proportionally to the haze formation. CONCLUSION: Three growth factors took part in promoting corneal wound healing after PRK, and might contribute to corneal haze formation and development.     

Lacrimal gland HGF, KGF, and EGF mRNA levels increase after corneal epithelial wounding.

PURPOSE: To evaluate the effect of corneal epithelial wounding on lacrimal gland expression of hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and epidermal growth factor (EGF) in the rabbit model. METHODS: Rabbits had corneal epithelial scrape injuries, and the lacrimal gland was removed at different times after wounding. HGF, KGF, and EGF mRNA expression was examined by quantitative RNase protection assay. HGF, KGF, and EGF proteins were detected in rabbit lacrimal tissue using immunoprecipitation and western blot analysis. RESULTS: HGF mRNA and EGF mRNA were significantly increased in rabbit lacrimal gland tissue within 8 hours after corneal epithelial injury. The increase in KGF mRNA expression was small and reached significance I clay after corneal injury. Lacrimal gland expression peaked at 3 days after wounding for each growth factor mRNA, the same day, on average, that the epithelial defect healed. After the peak increase in expression, there was a progressive decline in expression of each growth factor mRNA, but production was still increased compared with prewound levels. HGF protein, KGF protein, and EGF proteins were detected in rabbit lacrimal gland tissue. CONCLUSIONS: Levels of HGF, KGF, and EGF mRNAs increase in rabbit lacrimal gland tissue in response to corneal epithelial wounding. The results of this study are consistent with the existence of a cornea-nervous system-lacrimal gland regulatory loop modulating expression of these growth factor mRNAs. The lacrimal gland is a likely source of increased HGF and EGF proteins detected in tears in previous studies.     

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.     

Detection of transforming growth factor-alpha messenger RNA and protein in human corneal epithelial cells.

Human corneal epithelial cells are normally shed from the apical surface and replaced primarily by mitosis of basal cells. Growth factors may regulate this process, but the sources for the growth factors have not been fully established. One potential source for growth factors is tear fluid, and epidermal growth factor (EGF) has been detected in the lacrimal gland and in tears. However, the hydrophilic structure and size of growth factors such as EGF may limit penetration to basal layers of intact epithelium. It is possible that turnover of basal human corneal epithelial cells might be regulated by growth factors acting by an autocrine mechanism. To determine if human corneal epithelial cells synthesize a potential autocrine growth factor, the authors analyzed human corneal epithelial cells for transforming growth factor-alpha (TGF-alpha) messenger RNA and protein, a growth factor that is structurally related to EGF and binds to the EGF receptor. Radioimmunoassay of human corneal epithelial cell cultures detected substantial levels of immunoreactive TGF-alpha (3 ng/10(6) cells). Immunohistochemical staining of human corneas also revealed the presence of immunoreactive TGF-alpha in the corneal epithelium. Northern hybridization with a 32P-labeled complementary DNA probe for TGF-alpha generated a single intense band at 4.4 kilobases, indicating the presence of TGF-alpha messenger RNA in cultured human corneal epithelial cells. These results support the hypothesis that normal turnover of corneal epithelium is controlled by the autocrine production of growth factors, such as TGF-alpha. Growth factors present in tears may function primarily as exocrine factors to stimulate healing of epithelial injuries after the epithelial barrier has been damaged.     

Corneal Neovascularization Suppressed by TIMP2 Released from Human Amniotic Membranes

Purpose: To investigate the effects of culture medium of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice. Methods: Culture medium of amniotic membrane was prepared by cultivating AM (with epithelium side up) in EGM basic medium for 3 days, and was collected separately to three groups, e.g. control (EGM only), AM with epithelium (AM) and AM without epithelium (De-AM). Corneal neovascularization was induced in mice by using micropocket assay with Hydron polymer pellets containing 100 ng bFGF. Migration and proliferation of human umbilical cord vein endothelial cells (HUVEC) were performed in Boyden chambers and by using the CyQUANT fluorescence binding assay respectively. The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP1, TIMP2) in culture medium were determined by ELISA assay. Results: CNV induced by bFGF was significantly suppressed by culture medium of amniotic membrane. When the medium was applied as an eyedrop     

Growth factor-induced proliferation in corneal epithelial cells is mediated by 12(S)-HETE

PURPOSE: Previous studies in our laboratory have shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a product of 12-lipoxygenase (12-LOX) activity, is the predominant metabolite formed in rabbit corneas after injury. The present study was undertaken to investigate the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) on 12-LOX expression and activity. We also investigated whether 12(S)-HETE mediated the growth factor-induced proliferation of corneal epithelial cells. METHODS: Rabbit corneas were stimulated with EGF, HGF, and KGF (10 ng ml(-1)) for different times. 12-LOX activity was assayed by incubating corneal microsomal preparations with radiolabeled arachidonic acid (AA) as substrate. For inhibitor studies, the microsomes were pretreated with 12-LOX-specific inhibitors baicalein (BC) or cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC). Lipid extracts were injected onto an Ultramex 5 microm C(18) column and radioactivity was monitored online by a Radiomatic Flo-One Beta detector. Stereochemical analysis of 12-HETE product was determined by chiral-phase HPLC. To evaluate the effects of growth factors on 12-LOX mRNA expression, mRNA was extracted at several time points (12, 24, 36, 48 hr) and subjected to real-time PCR. For 12-LOX protein expression, microsomal preparations from 24- and 48-hr incubations were analyzed by Western blot. In cell-proliferation studies, epithelial cells treated with EGF, HGF, or KGF for 24, 48, and 72 hr were measured with a CyQUANT cell-proliferation assay kit. To determine the role of growth factor-induced 12(S)-HETE synthesis on corneal epithelial cell proliferation, cells were pretreated with 12-LOX-specific inhibitors BC or CDC prior to growth-factor supplementation. RESULTS: Stimulation with EGF, HGF, or KGF for 12 hr induced 12-LOX mRNA expression in rabbit corneal epithelial cells. This gene induction was followed by an increase in protein expression at 24 and 48 hr and a marked increase in 12(S)-HETE synthesis when compared to untreated controls. At 24-hr incubations, KGF showed a greater capacity than did EGF and HGF to stimulate microsomal 12-LOX activity, while at 48 hr 12(S)-HETE synthesis was significantly greater in EGF-treated cells as compared to that of HGF- and KGF-treated cells. Pretreatment with 12-LOX inhibitors blocked the growth factor-induced increase in 12(S)-HETE synthesis. Stimulation with growth factors or 12(S)-HETE for 24, 48, and 72hr produced a significant increase in corneal epithelial proliferation, which was partially inhibited by pretreatment of cells with 12-LOX-specific inhibitors. CONCLUSION: These findings suggest that EGF, HGF, and KGF stimulate 12(S)-HETE production in rabbit corneal epithelial cells through gene induction of 12-LOX. Furthermore, 12(S)-HETE may play a role in regulating epithelial cell proliferation and the rate of corneal re-epithelialization following an injury.     

羊膜培养液抑制角膜新生血管的实验研究

目的　研究羊膜培养液对小鼠角膜新生血管的抑制作用及机制。方法　应用cornealmicropocket方法 ,将含 10 0ng碱性成纤维细胞生长因子 (bFGF)和 12 %hydronpolymer的缓释颗粒植入角膜层间 ,制备小鼠角膜新生血管模型。实验分对照组、羊膜组和去上皮羊膜组 ,每组 10只眼 ,收集各组培养液于模型制作当日起 ,每日滴眼 4次至术后 7d摄片观测角膜新生血管长入情况并处死动物。对脐静脉血管内皮细胞 (HUVEC)进行体外培养 ,并应用Boyden小房技术和荧光结合(CyQUANT)实验分别检测羊膜培养液对HUVEC细胞迁移和细胞增殖的影响。利用酶联免疫吸附(ELISA)法检测各组培养液中金属蛋白酶抑制剂 1(TIMP1)和 2 (TIMP2 )的含量。结果　羊膜培养液明显抑制由bFGF诱发的小鼠角膜新生血管的形成 ,对照组、羊膜组和去上皮羊膜组角膜新生血管面积分别是 (2 4 8± 0 76 )mm2 、(0 6 4± 0 5 2 )mm2 和 (1 96± 0 6 5 )mm2 。羊膜培养液明显抑制血管内皮细胞的迁移和增殖 ,而对照组和去上皮羊膜组对HUVEC细胞迁移无作用。羊膜培养液中TIMP2水平明显增高 ,而TIMP1的含量无变化。结论　羊膜培养液中TIMP1的含量未增加。而羊膜上皮产生或释放大量的TIMP2 ,抑制血管内皮细胞的迁移和增殖 ,其可能是羊膜培养液抑制角膜新生血管的