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，在移植生命的任何时候，移植失败可能晚至最初成功移植后5-10年。Descemet的剥离式自动内皮角膜移植(DSAEK)是一种新的手术方法，它在很多方面都优于传统的角膜移植技术，但它需要对角膜内皮进行广泛的操作，导致大量的CE细胞丢失。因此，接受过DSAEK治疗的患者未来可能发生晚期CE衰竭。此前，我们已经报道了由28个氨基酸组成的血管活性肠肽(VIP)是一种CE自分泌营养因子，它在人供体角膜中原位维持角膜内皮的分化状态，包括CE细胞的大小、形状和滞留，而外源性VIP则保护其免受氧化应激的杀伤作用。VIP促进CE细胞分化标记物N-钙粘附素、细胞间黏附分子和抗凋亡蛋白Bcl2的合成。睫状神经营养因子(CNTF)上调CE细胞内源性血管活性肠肽(VIP)，是CE细胞在氧化应激条件下释放的一种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，并在角膜保存过程中逐渐丢失。通过将研究CE细胞生理学所获得的知识应用于提高角膜移植中CE细胞的存活率，这也是我们的目标，我们也将验证这些知识的相关性。我们的具体目的是证明：1.在保存新鲜解剖的人供体角膜之前进行VIP处理增加了它们的CE细胞N-钙粘素、Bcl-2、CNTFR1和在角膜存储中的保留水平；2.在为DSAEK储存的人供体角膜的微角膜切割机切割时，另一种VIP处理产生了目标1中描述的有益效果，并减少了DSAEK预切角膜的CE细胞凋亡和损伤；3.VIP处理在保存新鲜解剖的人供体角膜之前和在微角膜切割术之前为DSAEK提供了更好的预切角膜，在建立的体外角膜移植模型中证明了CE损伤的减少。4.在已建立的体外角膜内皮移植模型中，术中应用VIP(或CNTF)可减少CE损伤。 公共卫生相关性：角膜内皮(CE)细胞丢失是角膜移植失败的关键风险因素，在移植生命的任何时候，移植失败的时间可能晚至最初成功移植后5-10年。我们之前报道了一种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。通过将这一知识应用于提高角膜移植中CE细胞的存活率，这也是我们的目标，我们也将验证这一知识的相关性。

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.