Daniels Julie T, Limb G Astrid, Saarialho-Kere Ulpu, Murphy Gillian, Khaw Peng T
Epithelial Repair and Regeneration Group, Wound Healing Research Unit, Division of Pathology, Institute of Ophthalmology, London, United Kingdom.
Invest Ophthalmol Vis Sci. 2003 Mar;44(3):1048-55. doi: 10.1167/iovs.02-0442.
To investigate the potential regulation of matrix metalloproteinases (MMPs) by hepatocyte growth factor (HGF), and to identify individual MMPs essential for migration of human corneal epithelial cells.
Migration of human corneal epithelial cells (HCECs) was measured with a colony dispersion assay in response to concentrations of HGF (0-50 ng/mL). MMP activity in the conditioned media collected from the dispersion assay was assessed by zymography. The broad-spectrum MMP inhibitor ilomastat (1-100 microM) or an MMP-9-neutralizing antibody (1-10 microg/mL) were included in the dispersion assay to determine their effects on HCEC migration. Immunocytochemistry and in situ hybridization were used to localize MMP-1 in HCECs in the colony dispersion assay and in a human ex vivo corneal wound-healing model, respectively. ELISA for MMP-1 was performed on conditioned medium from migrating HCECs. Neutralizing antibodies to MMP-1 and -9 were added to an in vitro scratch-wound model to assess the effect on HCEC healing.
HCEC migration (P < 0.05) and MMP-2 and -9 released into the medium increased in response to HGF in a dose-dependent manner up to 20 ng/mL. Broad-spectrum MMP inhibition significantly reduced HCEC migration (P < 0.05). In contrast, neutralization of MMP-9 increased migration (P < 0.05). MMP-1 was found in association with HCECs at the migratory leading edge in both the dispersion and the ex vivo wound-healing experiments, and was found to be stimulated above basal levels by HGF. Neutralization of MMP-1 significantly decreased (P < 0.05), whereas neutralization of MMP-9 significantly increased (P < 0.05), scratch-wound closure.
This study provided novel data regarding HCEC migration in response to HGF and highlighted the importance of MMPs, particularly MMP-1 in migration and possibly reepithelialization in vivo. MMP-9 and/or -2 may be released by HCECs to remodel matrix behind the leading migratory front. Studies such as this are essential to assist in the safe and efficacious design of MMP inhibitors for therapeutic use in the eye.
研究肝细胞生长因子（HGF）对基质金属蛋白酶（MMPs）的潜在调节作用，并确定对人角膜上皮细胞迁移至关重要的单个MMPs。
采用集落分散试验检测人角膜上皮细胞（HCECs）在不同浓度HGF（0 - 50 ng/mL）刺激下的迁移情况。通过酶谱法评估从分散试验收集的条件培养基中的MMP活性。在分散试验中加入广谱MMP抑制剂艾洛司他（1 - 100 microM）或MMP - 9中和抗体（1 - 10 microg/mL），以确定它们对HCEC迁移的影响。分别采用免疫细胞化学和原位杂交技术在集落分散试验和人角膜离体伤口愈合模型中定位HCECs中的MMP - 1。对迁移的HCECs的条件培养基进行MMP - 1的酶联免疫吸附测定（ELISA）。将MMP - 1和 - 9的中和抗体添加到体外划痕伤口模型中，以评估对HCEC愈合的影响。
在HGF浓度高达20 ng/mL时，HCEC迁移（P < 0.05）以及释放到培养基中的MMP - 2和 - 9呈剂量依赖性增加。广谱MMP抑制显著降低了HCEC迁移（P < 0.05）。相反，MMP - 9的中和增加了迁移（P < 0.05）。在分散试验和离体伤口愈合实验中，均在迁移前沿的HCECs中发现MMP - 1，且发现其受HGF刺激高于基础水平。MMP - 1的中和显著降低（P < 0.05），而MMP - 9的中和显著增加（P < 0.05）划痕伤口闭合。
本研究提供了关于HCECs对HGF反应性迁移的新数据，并突出了MMPs的重要性，特别是MMP - 1在体内迁移和可能的再上皮化中的重要性。MMP - 9和/或 - 2可能由HCECs释放以重塑迁移前沿后方的基质。此类研究对于协助安全有效地设计用于眼部治疗的MMP抑制剂至关重要。
