Neurotrophic factor modulation of corneal endothelium

角膜移植的成功取决于术后和术后角膜内皮细胞密度的维持。CE细胞要么死于急性坏死，这引发了有害的炎症反应，要么死于缓慢的程序性凋亡，这不会引起炎症。我们发现房水神经肽VIP在急性h2o2诱导的氧化应激下促进CE细胞存活，同时刺激CE细胞释放一种促进角质层上皮细胞凋亡的因子。VIP效应与cAMP生成、蛋白酪氨酸磷酸化和cAMP响应元件结合蛋白（CREB）的激活有关，CREB是细胞存活的关键分子。CE细胞表达VIP蛋白和基因，而另一种CE细胞自分泌营养因子CNTF可以上调VIP蛋白和基因，CNTF在CE细胞氧化应激后释放。尽管储存用于移植的人供体角膜中的CE细胞继续表达VIP mRNA和CNTF受体（cntfrα），并且VIP增加了长期储存的这些角膜中CE细胞的密度，但在人房水中存在CNTF-和cntfrα免疫反应。我们的目标是建立VIP和CNTF作为CE细胞营养因子，能够：1)延长角膜移植保存时间，2)维持移植角膜在受体眼睛中的完整性。5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脂质过氧化的衰减有关；2) vip释放因子（VRF）通过减弱h2o2诱导的ATP耗竭和线粒体电位耗散，以及增强聚腺苷核糖（adp -核糖）聚合酶的裂解，在CE细胞条件培养基中通过序列快速高效液相色谱纯化VRF，并分析其氨基酸序列，从而在损伤CE细胞中转换坏死至凋亡；3) VIP使促凋亡BAD蛋白失活，上调抗凋亡蛋白Bcl-2和细胞色素C；4)亚致死h2o2损伤CE细胞释放CNTF促进自身存活，真实CNTF和cntfrα存在于损伤（去核）眼房水中；5)外源性cntfrα可恢复CE细胞在大量眼库储存过程中丢失的cntfrα上调VIP mRNA的功能。

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.