Corneal endothelial cell survival in human donor corneas for transplantation

描述（由申请人提供）：角膜内皮 (CE) 细胞损失是角膜移植失败的关键风险因素，在移植物生命周期的任何时候，这种失败可能会在最初成功移植后的 5-10 年发生。后弹力层剥离自动内皮角膜移植术 (DSAEK) 是一种新手术，在许多方面优于传统技术，但需要对角膜内皮进行大量操作，导致 CE 细胞大量丢失。因此，接受 DSAEK 的眼睛未来有可能出现晚期 CE 失败。此前，我们报道了CE自分泌营养因子，即28个氨基酸的血管活性肠肽（VIP）如何在人类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、形状和保留，而外源性VIP可以保护其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、细胞间粘附分子和抗凋亡蛋白 Bcl-2 的合成。 CE细胞中的内源性VIP被睫状神经营养因子(CNTF)上调，CNTF也是CE细胞在氧化应激中幸存后释放的CE自分泌营养因子。 CNTF 受体 (CNTFR1) 在人类供体角膜的 CE 细胞中表达，并在角膜保存过程中逐渐丢失。通过将从研究 CE 细胞生理学中获得的知识应用于提高角膜移植中 CE 细胞的存活率（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. 在储存新鲜解剖的人类供体角膜之前进行 VIP 治疗可增加其 CE 细胞 N-钙粘蛋白、Bcl-2、CNTFR1 和角膜储存中的保留水平，2. 在对为 DSAEK 储存的人类供体角膜进行微型角膜刀切割时进行另一项 VIP 治疗可带来目标 1 中所述的有益效果，并减少 CE 细胞凋亡和对 DSAEK 预切角膜的损伤，3. 储存前的 VIP 治疗新鲜解剖的人类供体角膜和在微型角膜刀切割之前产生用于 DSAEK 的优质预切角膜，证明在已建立的体外内皮角膜移植模型中减少了 CE 损伤，4. 术中 VIP（或 CNTF）治疗可在已建立的体外内皮角膜移植模型中减少 CE 损伤。 公共卫生相关性：角膜内皮 (CE) 细胞丢失是角膜移植物生命周期中任何时候失败的关键危险因素，最迟可能会在最初成功移植后 5-10 年发生。我们之前报道了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的存活。通过将这些知识应用于提高角膜移植中 CE 细胞的存活率（这是我们的目标），我们还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.