Corneal endothelial cell survival in human donor corneas for transplantation

描述（由申请人提供）：角膜内皮（CE）细胞丢失是角膜移植物生命周期中任何时间（可能迟至最初成功移植后5-10年）角膜移植物失败的关键风险因素。一种新的手术，后弹力层剥脱自动内皮角膜移植术（DSAEK），这是上级在许多方面优于传统的技术，需要广泛的操作角膜内皮，导致广泛的CE细胞损失。因此，在接受DSAEK的眼睛中，未来可能会发生晚期CE失败。以前，我们已经报道了如何CE自分泌营养因子，28个氨基酸的血管活性肠肽（VIP）维持角膜内皮的分化状态，包括CE细胞的大小，形状和保留，在原位在人供体角膜，而外源性VIP保护它免受氧化应激的杀伤作用。VIP增加CE细胞分化标记物N-钙粘蛋白（细胞-细胞粘附分子）和抗凋亡蛋白Bcl-2的合成。睫状神经营养因子（CNTF）也是CE细胞在氧化应激后释放的CE自分泌营养因子，可上调CE细胞中的内源性VIP。CNTF受体（CNTFR 1）在人供体角膜的CE细胞中表达，并在角膜储存期间逐渐丢失。通过将从研究CE细胞生理学中获得的这些知识应用于角膜移植中CE细胞存活的增强，这是我们的目标，我们还将验证这些知识的相关性。我们的具体目标是证明1。VIP处理新鲜解剖的人供体角膜储存前增加其CE细胞N-钙粘蛋白，Bcl-2，CNTFR 1，并保留在角膜储存水平，2。在微角膜刀切割为DSAEK储存的人供体角膜时的一种额外VIP处理带来了目的1中描述的有益效果，并减少了CE细胞凋亡和对DSAEK预切割角膜的损伤，3。在储存新鲜解剖的人供体角膜之前和在微角膜刀切割之前VIP处理产生用于DSAEK的上级的预切割角膜，证明在建立的体外内皮角膜移植模型中CE损伤降低，4。在已建立的体外内皮角膜移植模型中，术中VIP（或CNTF）治疗可降低CE损伤。 公共卫生相关性：角膜内皮（CE）细胞丢失是角膜移植物在移植物生命周期中任何时候失败的关键风险因素，可能迟至最初成功移植后5-10年。我们先前报道了CE自分泌营养因子血管活性肠肽（VIP）如何维持分化状态并促进角膜内皮细胞的存活。通过将这些知识应用于角膜移植中CE细胞存活的增强，这是我们的目标，我们也将验证这些知识的相关性。

DESCRIPTION (provided by applicant): Corneal endothelial (CE) cell loss is a critical risk factor in corneal graft failure at any time in the life of the graft, which can be as late as 5-10 years after an initially successful transplant. A new procedure, Descemet's stripping automated endothelial keratoplasty (DSAEK), which is superior to the traditional technique in many aspects, requires extensive manipulation of the corneal endothelium, resulting in extensive CE cell loss. Thus, future development of late CE failure in eyes that have received DSAEK is likely. Previously, We have reported how a CE autocrine trophic factor, the 28 amino-acid vasoactive intestinal peptide (VIP) maintains the differentiated state of the corneal endothelium, including CE cell size, shape, and retention, in situ in the human donor cornea, whereas exogenous VIP protects it against the killing effect of oxidative stress. VIP increases synthesis of the CE cell differentiation marker N-cadherin, the cell-cell adhesion molecule, and that of the anti-apoptotic protein Bcl-2. The endogenous VIP in CE cells is upregulated by ciliary neurotrophic factor (CNTF), which is also a CE autocrine trophic factor released by CE cells surviving the oxidative stress. The CNTF receptor (CNTFR1) is expressed in CE cells in human donor cornea and gradually becomes lost during corneal storage. By applying this knowledge gained from studying CE cell physiology to the enhancement of CE cell survival in corneal transplantation, which is our goal, we will also validate the relevance of this knowledge. Our specific aims are to demonstrate that 1. VIP treatment prior to storage of freshly dissected human donor corneas increases their CE cell N-cadherin, Bcl-2, CNTFR1, and retention levels in corneal storage, 2. One additional VIP treatment at the time of microkeratome cutting of the human donor corneas stored for DSAEK brings about beneficial effects described in aim 1 and decreased CE cell apoptosis and injury to precut corneas for DSAEK, 3. VIP treatments prior to storage of freshly dissected human donor corneas and prior to microkeratome cutting produce superior precut corneas for DSAEK demonstrating decreased CE damage in an established in vitro endothelial keratoplasty model, 4. Intra-operative VIP (or CNTF) treatment decreases CE damage in an established in vitro endothelial keratoplasty model. PUBLIC HEALTH RELEVANCE: Corneal endothelial (CE) cell loss is a critical risk factor in corneal graft failure at any time in the life of the graft, which can be as late as 5-10 years after an initially successful transplant. We reported previously how a CE autocrine trophic factor, vasoactive intestinal peptide (VIP) maintains the differentiated state and promotes survival of the corneal endothelium. By applying this knowledge to the enhancement of CE cell survival in corneal transplantation, which is our goal, we will also validate the relevance of this knowledge.