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失败的关键危险因素，在移植生命周期的任何时候，可能在最初成功移植后5-10年。Descemet剥离自动内皮角膜移植术（Descemet’s stripping automated endothelial keratplasty， DSAEK）是一种新的手术方法，它在许多方面都优于传统技术，但需要对角膜内皮进行大量的操作，导致大量CE细胞的丢失。因此，在接受DSAEK的眼睛中，晚期CE衰竭的未来发展是可能的。在此之前，我们已经报道了CE自分泌营养因子，28个氨基酸的血管活性肠肽（VIP）如何维持角膜内皮的分化状态，包括CE细胞的大小、形状和保留，在人供体角膜中原位，而外源性VIP保护它免受氧化应激的杀伤作用。VIP增加CE细胞分化标志物n -钙粘蛋白、细胞-细胞粘附分子和抗凋亡蛋白Bcl-2的合成。ciliary neurotrophic factor （CNTF）上调CE细胞内源性VIP， CNTF也是CE细胞在氧化应激后释放的一种CE自分泌营养因子。CNTF受体（CNTFR1）在人供体角膜的CE细胞中表达，并在角膜储存过程中逐渐丢失。通过将这些从研究CE细胞生理学中获得的知识应用于增强角膜移植中CE细胞的存活率，这是我们的目标，我们也将验证这些知识的相关性。我们的具体目标是证明1。在新鲜解剖的人供体角膜储存前进行VIP处理可增加角膜储存中的CE细胞n -钙粘蛋白、Bcl-2、CNTFR1和保留水平。在储存用于DSAEK的人供体角膜微角化体切割时进行额外的VIP处理，可带来目的1所述的有益效果，并减少CE细胞凋亡和对DSAEK预切角膜的损伤，3。在已建立的体外内皮角膜移植术模型中，在储存新鲜解剖的人类供体角膜和在微角膜切割之前进行VIP治疗，可为DSAEK提供更好的预切角膜，显示CE损伤减少。在已建立的体外内皮角膜移植术模型中，术中VIP（或CNTF）治疗可减少CE损伤。

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.