Neurotrophic factor modulation of corneal endothelium

角膜内皮的神经营养因子调节

• 批准号: 6790669
• 负责人: SHAY-WHEY M KOH
• 金额: $ 29.7万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6790669
• 关键词: SDS polyacrylamide gel electrophoresis; apoptosis; biological signal transduction; cAMP response element binding protein; corneal endothelium; cyclic AMP; cytoprotection; eye bank /preservation; eye transplantation; gel filtration chromatography; gene expression; high performance liquid chromatography; malonaldehyde; mass spectrometry; neurotrophic factors; oxidative stress; protein structure function; vasoactive intestinal peptide; western blottings

Success of corneal transplant is dependent on high corneal endothelial (CE) cell density maintained immediately after surgery and thereafter. CE cells die either by acute necrosis, which initiates detrimental inflammatory responses, or by a slow programmed, apoptotic death, which does not cause inflammation. We found that an aqueous humor neuropeptide, VIP, promotes CE cell survival under acute H2O2-induced oxidative stress and simultaneously stimulates CE cells to release a factor that promotes apoptosis among dying CE cells in corneoscleral explants. The VIP effects are associated with cAMP production, protein tyrosine phosphorylation, and activation of the cAMP- responsive- element binding protein (CREB), a key molecule in cell survival. CE cells express VIP protein and gene, which can be upregulated by the other CE cell autocrine trophic factor, CNTF, which in turn is released by CE cells surviving oxidative stress. Whereas CE cells in human donor corneas stored for transplant continue to express VIP mRNA and receptor for CNTF (CNTFRalpha) and VIP increases the density of CE cells of these corneas in long-term storage, CNTF- and CNTFRalpha-immunoreactivities are present in human aqueous humor. Our goal is to establish VIP and CNTF as CE cell trophic factors capable of: 1) prolonging the duration of preservation of corneas for transplant and 2) maintaining the integrity of transplanted corneas in the recipient eyes. Five aims to test hypotheses that: 1) VIP protection of CE cell against acute oxidative injury is associated with attenuation of lipid peroxidation; 2) VIP-released factor (VRF) switches necrosis to apoptosis in injured CE cells by attenuating H2O2-induced ATP depletion and mitochondrial potential dissipation and by augmentation of poly (ADP-ribose) polymerase cleavage, by using VRF that will be purified by sequential fast performance liquid chromatography from CE cell-conditioned medium and amino acid sequence analyzed; 3) VIP inactivates the pro-apoptotic BAD protein and up-regulates the anti-apoptotic protein Bcl-2 and the cytochrome C; 4) sublethal H2O2-injured CE cells release CNTF to promote own survival and that authentic CNTF and CNTFRalpha are in the aqueous humor of injured (enucleated) eyes; 5) CE cell CNTFRalpha lost during extensive eye bank storage can be restored to function to upregulate VIP mRNA by exogenous CNTFRalpha.

角膜移植的成功取决于手术后立即及其后维持的高角膜内皮（CE）细胞密度。 CE细胞要么通过急性坏死（引发有害的炎症反应）而死亡，要么通过缓慢的程序性细胞凋亡死亡（不会引起炎症）。我们发现房水神经肽 VIP 可促进 CE 细胞在 H2O2 诱导的急性氧化应激下存活，同时刺激 CE 细胞释放一种促进角巩膜外植体中垂死 CE 细胞凋亡的因子。 VIP 效应与 cAMP 产生、蛋白质酪氨酸磷酸化和 cAMP 反应元件结合蛋白 (CREB)（细胞存活的关键分子）的激活有关。 CE 细胞表达 VIP 蛋白和基因，该蛋白和基因可被另一种 CE 细胞自分泌营养因子 CNTF 上调，而 CNTF 又由在氧化应激中幸存的 CE 细胞释放。虽然储存用于移植的人类供体角膜中的 CE 细胞继续表达 VIP mRNA 和 CNTF (CNTFRα) 受体，并且 VIP 增加了长期储存中这些角膜的 CE 细胞的密度，但人房水中存在 CNTF 和 CNTFRα 免疫反应性。我们的目标是建立 VIP 和 CNTF 作为 CE 细胞营养因子，能够：1）延长移植角膜的保存时间，2）维持受体眼中移植角膜的完整性。五个目的是检验以下假设：1）VIP 对 CE 细胞免受急性氧化损伤的保护与脂质过氧化的减弱有关； 2) VIP 释放因子 (VRF) 通过减弱 H2O2 诱导的 ATP 消耗和线粒体电位耗散以及通过使用 VRF 增强聚 (ADP-核糖) 聚合酶裂解，将受损 CE 细胞的坏死转变为细胞凋亡，VRF 将通过连续快速高效液相色谱法从 CE 细胞条件培养基中纯化，并分析氨基酸序列； 3) VIP使促凋亡BAD蛋白失活并上调抗凋亡蛋白Bcl-2和细胞色素C； 4）亚致死的H2O2损伤的CE细胞释放CNTF以促进自身存活，并且真正的CNTF和CNTFRα存在于受伤（去核）眼睛的房水中； 5) 在大量眼库储存过程中丢失的CE细胞CNTFRalpha可以恢复到通过外源CNTFRalpha上调VIP mRNA的功能。