The role and mechanistic regulation of cPLA2alpha in eicosanoid biosynthesis and wound healing

cPLA2α在类二十烷酸生物合成和伤口愈合中的作用和机制调节

基本信息

批准号：
10318663
负责人：
CHARLES E. CHALFANT
金额：
$ 46.36万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10318663
关键词：
Ablation Age Aging Agonist Anabolism Anti-Inflammatory Agents Arachidonic Acids Binding Biochemical Biological Markers CRISPR/Cas technology Cells Complex Coupled Cytosolic Phospholipase A2 Data Dermal Development Dinoprostone Eicosanoid Production Eicosanoids Endothelium Enzymes Equilibrium Etiology Exhibits Fibroblasts Foundations Generations Genetic Genetically Engineered Mouse Human Hydroxyeicosatetraenoic Acids Immunoglobulin Class Switching Impaired wound healing In Vitro Inflammatory Knock-in Mouse Knock-out Knockout Mice Laboratories Link Lipids Lipoxygenase Liquid substance Mediating Metabolism Methods Modeling Molecular Molecular Biology Mus Outcome Pathology Patients Phase Phenotype Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phospholipase A2 Phospholipases A Physiological Physiology Plasma Population Pre-Clinical Model Process Production Prostaglandin-Endoperoxide Synthase Prostaglandins Regulation Role Signal Pathway Site Sphingolipids Technology Testing Tissues Variant ceramide 1-phosphate ceramide kinase chronic ulcer chronic wound decubitus ulcer genetic manipulation healing in vivo inhibitor/antagonist lipid mediator lipidomics migration mortality mouse model multidisciplinary mutant neutrophil non-healing wounds novel patient population phosphatidylinositol phosphate, PtdIns(4,5)P2 pre-clinical response skin ulcer small molecule small molecule inhibitor translational therapeutics wound wound healing

项目摘要

In recent years, new technological advancements in small molecule analyses (e.g., lipidomics) have identified a biochemical manifestation of impaired wound healing: the development of an imbalance between pro- and anti-inflammatory eicosanoids1-9. The synthesis of eicosanoids begins with the initial rate-limiting step, the generation of arachidonic acid (AA) via the activity of a phospholipase A2 (PLA2)10-12. One of the major PLA2s involved in this initial step is group IVA cytosolic PLA2 (cPLA2α)10-12, which the Chalfant laboratory demonstrated is activated by direct binding to the sphingolipid, ceramide-1-phosphate (C1P)13-19. Employing newer lipidomic technology, we discovered that C1P is temporally regulated and specifically increases in the inflammatory phase of human wound healing5. To evaluate C1P-induced eicosanoids in wound healing, we created a knock-in mouse with the C1P site in cPLA2 ablated (KI). Our preliminary data show that KI mice, unlike the wild-type (WT) and cPLA2 knockout (KO) mice, exhibit dramatically enhanced wound healing. These beneficial effects were linked to the loss of inflammatory prostaglandins (e.g., cyclooxygenase (COX)-derived PGE2) and increased production of specific lipid mediators (i.e., lipoxygenase (LOX)-derived 5-HETE), which induced significantly accelerated migration of dermal fibroblasts and neutrophils. Importantly, in an initial study, we also found that high levels of 5-HETE in wound fluid from human pressure ulcers are linked to a better healing outcome. Thus, a balancing act between LOX- and COX-derived lipid mediators is critical in the wound healing process. Initial mechanistic studies also showed that relevant cellular phenotypes and variant production of eicosanoid classes observed in KI cells are linked to a differential cellular localization of the C1P-ablated mutant cPLA2 via association with PIP2. The findings provide a foundation for the premise that, when cPLA2 is unable to bind C1P, the enzyme becomes free to associate with other lipid regulators (e.g., PIP2) that drive the production of specific LOX-derived eicosanoids (e.g., 5- HETE). This mechanism is supported by our preliminary in vitro studies showing that C1P blocks the activation of cPLA2α by PIP2. As LOX and COX products are both cPLA2-dependent, but temporally contrast in their biosynthesis20.21, our data suggest that an overlooked complexity in cPLA2 regulation exists in response to inflammatory agonists. Thus, we hypothesize that the enhanced wound healing of pressure ulcers will reflect a novel “lipid-class switch” producing pro-healing eicosanoids involving the complex, antagonistic regulation of cPLA2 by C1P and PIP2 metabolism. We also hypothesize that aging humans, who display ineffective wound healing, will have ulcerative wounds lacking these pro-healing lipid mediators, and a lipid signature will act as biomarker of healing outcome. To test these hypotheses, we will employ a multi-disciplinary team, novel genetic mouse models, and “state of the art” lipidomics and molecular biology technologies to explore the underlying mechanisms and bioactive lipids associated with aging and the non-healing of ulcerative wounds.

近年来，小分子分析（例如脂质组学）的新技术进步确定了伤口愈合受损的生化表现：之间的不平衡的发展促炎和抗炎类二十烷酸1-9 类二十烷酸的合成始于初始限速步骤，通过磷脂酶 A2 (PLA2)10-12 的活性产生花生四烯酸 (AA)，这是主要的 PLA2 之一。参与这一初始步骤的是 IVA 组细胞溶质 PLA2 (cPLA2α)10-12，Chalfant 实验室证明了这一点通过直接结合鞘脂、神经酰胺-1-磷酸 (C1P)13-19 来激活。技术，我们发现C1P受到时间调节，并且在炎症阶段特别增加为了评估 C1P 诱导的类二十烷酸在伤口愈合中的作用，我们创建了一只基因敲入小鼠。我们的初步数据显示，与野生型 (WT) 和 cPLA2 中的 C1P 位点被消融的小鼠不同。 cPLA2 敲除 (KO) 小鼠表现出显着增强的伤口愈合效果。炎症性前列腺素（例如环氧合酶 (COX) 衍生的 PGE2）的损失和产量的增加特定的脂质介质（即脂氧合酶（LOX）衍生的 5-HETE），其诱导显着加速重要的是，在初步研究中，我们还发现高水平的真皮成纤维细胞和中性粒细胞的迁移。人类压疮伤口液中的 5-HETE 与更好的愈合效果有关，因此需要平衡。 LOX 和 COX 衍生的脂质介质之间的作用在伤口愈合过程中至关重要。初步的机制研究还表明，相关的细胞表型和变异产生在 KI 细胞中观察到的类二十烷酸类别与 C1P 消除突变体的差异细胞定位有关 cPLA2 通过与 PIP2 关联，这些发现为以下前提提供了基础：当 cPLA2 无法发挥作用时。为了结合 C1P，该酶可以自由地与其他脂质调节剂（例如 PIP2）结合，从而驱动我们的初步支持了特定 LOX 衍生类二十烷酸（例如 5-HETE）的生产。体外研究表明，C1P 可以阻断 PIP2 对 cPLA2α 的激活，因为 LOX 和 COX 都是产物。 cPLA2α 依赖，但在其生物合成中存在时间对比20.21，我们的数据表明，一个被忽视的 cPLA2α 调节的复杂性存在于对炎症激动剂的反应中。压疮伤口的加速愈合将反映一种新型的“脂质类开关”，产生促愈合作用类二十烷酸涉及 C1P 和 PIP2 代谢对 cPLA2α 的复杂、拮抗调节。老年人的伤口愈合不良，会出现溃疡性伤口这些促进愈合的脂质介质和脂质特征将作为愈合结果的生物标志物来测试这些。假设，我们将采用多学科团队、新颖的基因小鼠模型和“最先进的” 脂质组学和分子生物学技术探索潜在机制和生物活性脂质与衰老和溃疡性伤口不愈合有关。