Neurotrophic factor modulation of corneal endothelium

角膜移植的成功依赖于术后即刻和术后维持较高的角膜内皮细胞密度。CE细胞要么死于急性坏死，引发有害的炎症反应，要么死于缓慢的程序性、凋亡性死亡，不会引起炎症。我们发现房水神经肽VIP能促进CE细胞在急性H_2O_2诱导的氧化应激下存活，同时刺激CE细胞释放一种促进角巩膜外植体中死亡CE细胞凋亡的因子。VIP效应与cAMP的产生、蛋白酪氨酸的磷酸化以及cAMP反应元件结合蛋白(CREB)的激活有关，CREB是细胞存活的关键分子。CE细胞表达VIP蛋白和VIP基因，这种蛋白和基因可被CE细胞自分泌的营养因子CNTF上调，而CNTF则是CE细胞在氧化应激后释放出来的。供移植的人供体角膜中的CE细胞继续表达VIP mRNA和CNTF受体(CNTFRpha)，VIP在长期保存中增加了这些角膜CE细胞的密度，而CNTF和CNTFRpha免疫反应存在于人房水中。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)保持移植眼角膜的完整性。VIP对CE细胞急性氧化损伤的保护作用与减轻脂质过氧化作用有关；2)VIP释放因子(VIP-Related Function，VRF)通过减少H_2O_2诱导的ATP耗竭和线粒体电势的耗散，并通过增强聚(ADP-核糖)聚合酶的裂解作用，使损伤的CE细胞由坏死转为凋亡，VIP通过从CE细胞条件培养液中分离纯化的VIP，并对氨基酸序列进行分析；3)VIP失活促凋亡的BAD蛋白，上调抗凋亡蛋白Bcl2和细胞色素C的表达；4)H_2O_2亚致死损伤的CE细胞释放CNTF促进自身存活，且损伤(去核)眼房水中存在真实的CNTF和CNTFRα；5)在大量眼库储存过程中丢失的CE细胞CNTFRα可通过外源性CNTFRα恢复功能，上调VIP mRNA表达。

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.