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(CENC)是维持角膜透明度的关键。许多因素，包括 衰老、氧化应激和炎症已被认为与CENC损伤有关。CENC的损失也使情况复杂化 长时间储存会导致CENC密度逐渐下降的眼部银行，这可能会导致移植 浮肿。CENC损伤也是移植物失败的主要直接原因，无论是否由免疫驱动。因此， 提高CENC活性的细胞保护策略可能对以下方面产生重大影响：(1)质量和数量 可供移植的供体角膜的数量，以及(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；多种情况，包括糖尿病、干眼和 引起神经损伤的疱疹病毒性角膜炎也与可测量的CENC丢失有关。采购 用于眼库的供体角膜移植还需要切断供体组织与神经，并与 CENC的重大损失。我们的初步数据显示：(1)黑素皮质素受体的高结构性表达 (Mcr)神经肽α-黑素细胞刺激素(α-msh)在人和小鼠CENC； (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 通过α-MSH；3)当供体组织保存在补充的培养液中时，眼库CENC损失减少 使用α-MSH。根据这些初步数据，我们推测α-msh对 氧化应激和炎性细胞因子，从而预测α-MSH促进 (I)角膜保存和(Ii)移植后存活。为了验证这一假设，我们将研究两个问题 明确的目标。在目标1中，我们评估了α-MSH对眼库储存的角膜中央细胞丢失的影响。 与Eversight Eye Bank合作。我们将人类供体角膜保存在两种标准的Optisol-GS中 介质(目标1A)或在添加过氧化氢的介质中诱导氧化应激(目标1B)之前 加入α-MSH或对照PBS。将使用标准眼库对组织进行前瞻性监测和评估 CENC活性的方法学和测试(TUNEL法和细胞色素c释放)和氧化DNA 损坏。在目的2中，我们将评估α-MSH对小鼠移植的CENC功能和存活的影响。 角膜移植。为了区分α-MSH潜在的免疫调节作用和其细胞保护作用， α-MSH(或对照治疗)将用于AIM 2A的同基因移植物。然后，我们将使用多种MCR 宿主(目标2B)或供体(目标2C)中的敲除组合，以区分α-MSH的细胞靶标。这 Grant代表了我们实验室的一个新的研究领域，它将我们的实验室与 移植方面的核心专门知识，调查小组包括CENC(Jurkunas博士)和 神经肽生物学(泰勒博士)。我们的目标是使用在此探索性拨款中生成的数据来生成 更机械地关注R01应用程序，其结果可被眼库用于优化 组织保存。