Efficacy of the Neuropeptide Alpha-Melanocyte Stimulating Hormone (α-MSH) in Promoting Survival of Corneal Endothelial Cells in Eye Banking and Transplantation

神经肽α-黑素细胞刺激激素（α-MSH）在眼库和移植中促进角膜内皮细胞存活的功效

• 批准号: 9762115
• 负责人: Reza Dana
• 金额: $ 29.55万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762115
• 关键词: Acute; Aging; Allogenic; Allografting; Antioxidants; Apoptosis; Apoptotic; Applications Grants; Area; Biology; Cell Count; Cell Death; Cell Density; Cell Survival; Cell Transplantation; Cell Transplants; Cells; Cessation of life; Clinical; Clinical Data; Collaborations; Cornea; Corneal Endothelium; Cytoprotection; Data; Diabetes Mellitus; Edema; Endothelial Cells; Endothelium; Eye; Eye Banks; Eye diseases; Graft Rejection; Graft Survival; Grant; Herpetic Keratitis; Human; Hydrogen Peroxide; Immune; Immunity; Inflammation; Inflammatory; Interferons; Investigation; Keratitis; Keratoplasty; Knock-out; Lead; Measurable; Mediating; Mediator of activation protein; Methodology; Monitor; Mus; Nerve; Nerve Tissue; Neuropeptide Receptor; Neuropeptides; Optisol; Outcome; Oxidants; Oxidative Stress; Penetrating Keratoplasty; Play; Postoperative Period; Supplementation; TNF gene; TdT-Mediated dUTP Nick End Labeling Assay; Testing; Thick; Tissue Donors; Tissue Grafts; Tissue Preservation; Tissues; Translating; Transplantation; Work; alpha-Melanocyte stimulating hormone; base; cell injury; cellular targeting; corneal epithelium; cytochrome c; cytokine; density; eye dryness; graft failure; immunoregulation; improved; melanocortin receptor; monolayer; nerve injury; nerve supply; oxidant stress; oxidative DNA damage; oxidative damage; premature; prospective; receptor expression; response; synergism; transplant model

Corneal endothelial cells (CEnC) are critical for maintaining corneal transparency. Many factors, including aging, oxidative stress, and inflammation have been implicated in CEnC damage. CEnC loss also complicates eye banking where prolonged storage can lead to progressive decline in CEnC density, which can lead to graft edema. CEnC damage is also the chief proximate cause of graft failure, whether immune-driven or not. Thus, cytoprotective strategies that enhance CEnC viability could have a major impact on (i) the quality and number of donor corneas available for transplantation, and (ii) graft outcomes. Clinical data have suggested a correlation between nerve density and CEnC numbers; numerous conditions, including diabetes, dry eye, and herpetic keratitis, which induce nerve damage, are also associated with measurable CEnC loss. Procurement of donor corneas for eye banking also requires severing donor tissue from nerves, and is associated with significant CEnC loss. Our preliminary data show (1) high constitutive expression of melanocortin receptor (MCR) for the neuropeptide alpha-melanocyte stimulating hormone (α-MSH) in both human and murine CEnC; (2) significant suppression of CEnC death induced by inflammatory cytokines or the oxidant hydrogen peroxide by α-MSH; and 3) decrease in eye banked CEnC loss when donor tissues are kept in medium supplemented with α-MSH. Based on these preliminary data, we hypothesize that α-MSH provides cytoprotection to CEnC from both oxidative stress and inflammatory cytokines, and thus predict that α-MSH promotes survival of CEnC (i) in cornea storage and (ii) after transplantation. To test this hypothesis, we will pursue two specific aims. In Aim 1, we evaluate the effect of α-MSH on CEnC loss in eye bank-stored corneas in collaboration with the Eversight Eye Bank. We will keep human donor corneas in either standard Optisol-GS medium (Aim 1A) or in medium supplemented with hydrogen peroxide to induce oxidant stress (Aim 1B) before adding α-MSH or control PBS. Tissues will be monitored and assessed prospectively using standard eye bank methodologies, and tests for CEnC viability (TUNEL assay and cytochrome c release) and oxidative DNA damage. In Aim 2, we will assess the effect of α-MSH on the function and survival of grafted CEnC in murine corneal grafting. To discriminate the potential immunomodulatory effect of α-MSH from its cytoprotective effect, α-MSH (or control treatment) will be used in syngeneic grafts in Aim 2A. Then, we will use a variety of MCR knockout combinations in hosts (Aim 2B) or donors (Aim 2C) to discriminate the cellular targets of α-MSH. This grant, which represents a new area of investigation for our lab, brings together synergy between our lab with core expertise in transplantation, with an investigative group that includes experts on CEnC (Dr. Jurkunas) and neuropeptide biology (Dr. Taylor). We aim to employ data generated in this exploratory grant to generate a more mechanistically focused R01 application whose results can then be employed by eye banks for optimized tissue preservation.

角膜内皮细胞（Corneal endothelial cells，CEnC）是维持角膜透明性的关键。许多因素，包括 衰老、氧化应激和炎症与CEnC损伤有关。CEnC损失也使 眼库，其中长时间储存可导致CEnC密度进行性下降，这可导致移植物 水肿CEnC损伤也是移植失败的主要近因，无论是否由免疫驱动。因此，在本发明中， 增强CEnC活力的细胞保护策略可能对（i）细胞的质量和数量产生重大影响。 可用于移植的供体角膜，和（ii）移植结果。临床数据表明， 神经密度和CEnC数量之间的相关性;许多疾病，包括糖尿病，干眼症， 诱导神经损伤疱疹性角膜炎也与可测量的CEnC损失相关。采购 用于眼库的供体角膜也需要从神经上切断供体组织， 严重的CEnC损失。我们的初步数据表明：（1）黑皮质素受体的高组成性表达 (MCR)对于人和鼠CEnC中的神经肽α-黑素细胞刺激激素（α-MSH）; (2)显著抑制炎性细胞因子或氧化剂过氧化氢诱导的CEnC死亡 3）当供体组织保存在补充α-MSH的培养基中时， α-MSH基于这些初步的数据，我们假设α-MSH提供细胞保护， CEnC从氧化应激和炎症细胞因子，从而预测α-MSH促进 CEnC的存活（i）在角膜储存中和（ii）在移植后。为了验证这一假设，我们将追踪两个 明确的目标。在目的1中，我们评估了α-MSH对眼库储存的角膜中CEnC损失的影响， 与Eversight Eye Bank合作。我们将在标准Optisol-GS中保存人类供体角膜 培养基（Aim 1A）或在补充有过氧化氢的培养基中诱导氧化应激（Aim 1B）， 加入α-MSH或对照PBS。将使用标准眼库前瞻性监测和评估组织 方法学和CEnC活力（TUNEL测定和细胞色素c释放）和氧化DNA检测 损害目的2：研究α-MSH对移植CEnC的功能和存活的影响。 角膜移植为了区分α-MSH的潜在免疫调节作用和细胞保护作用， α-MSH（或对照处理）将用于Aim 2A中的同基因移植物。然后，我们将使用各种MCR 在宿主（Aim 2B）或供体（Aim 2C）中的敲除组合以区分α-MSH的细胞靶。这 格兰特，这代表了我们实验室的一个新的研究领域，汇集了我们实验室与 移植的核心专业知识，包括CEnC专家（Jurkunas博士）和 神经肽生物学（泰勒博士）。我们的目标是利用这项探索性资助产生的数据， 更机械地聚焦R 01应用，其结果然后可以被眼库用于优化 组织保存