Neurotrophic factor modulation of corneal endothelium

角膜内皮的神经营养因子调节

• 批准号: 7096521
• 负责人: SHAY-WHEY M KOH
• 金额: $ 29万
• 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/7096521
• 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即刻和之后维持的高角膜内皮（CE）细胞密度。CE细胞通过急性坏死死亡，这引发了有害的炎症反应，或者通过缓慢的程序性凋亡死亡，这不会引起炎症。我们发现，一种房水神经肽，VIP，促进CE细胞的生存急性H2 O2诱导的氧化应激，同时刺激CE细胞释放一种因子，促进死亡的CE细胞在角巩膜外植体的细胞凋亡。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）的活化相关。CE细胞表达VIP蛋白和基因，其可以被另一种CE细胞自分泌营养因子CNTF上调，CNTF反过来又由在氧化应激中存活的CE细胞释放。而CE细胞在人类供体角膜储存移植继续表达VIP mRNA和受体的CNTF（CNTFR α）和VIP增加这些角膜的CE细胞的密度在长期储存，CNTF和CNTFR α免疫反应存在于人类的房水。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和2）维持受体眼中移植角膜的完整性。本研究旨在验证以下假设：1）VIP对CE细胞急性氧化损伤的保护作用与减轻脂质过氧化有关; 2）VIP释放因子（VRF）通过减弱H_2O_2诱导的ATP耗竭和线粒体电位耗散以及通过增加多聚腺苷酸（poly），使损伤的CE细胞的坏死转变为凋亡。（ADP-核糖）聚合酶切割，通过使用VRF，其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使促凋亡蛋白BAD失活，并上调抗凋亡蛋白Bcl-2和细胞色素C; 4）亚致死H2 O2损伤的CE细胞释放CNTF以促进自身存活，并且真正的CNTF和CNTFR α在损伤的房水中（摘除的）眼睛; 5）在大量眼库储存期间损失的CE细胞CNTFR α可以恢复到通过外源性CNTFR α上调VIP mRNA的功能。