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

• 批准号: 10644976
• 负责人: CHARLES E. CHALFANT
• 金额: $ 45.72万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 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/10644976
• 关键词: Ablation; Acceleration; 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; 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; Therapeutic; Tissues; Variant; ceramide 1-phosphate; ceramide kinase; chronic wound; decubitus ulcer; genetic manipulation; healing; in vivo; inhibitor; lipid mediator; lipidomics; migration; mortality; mouse model; multidisciplinary; mutant; neutrophil; new technology; non-healing wounds; novel; patient population; phosphatidylinositol phosphate, PtdIns(4,5)P2; pre-clinical; response; skin ulcer; small molecule; small molecule inhibitor; wound; wound healing

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。类二十烷酸的合成从初始限速步骤开始， 通过磷脂酶 A2 (PLA2)10-12 的活性生成花生四烯酸 (AA)。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。采用更新的脂质组学 技术，我们发现C1P受到时间调节，并且在炎症阶段特别增加 人体伤口愈合5。为了评估 C1P 诱导的类二十烷酸在伤口愈合中的作用，我们创建了一只敲入小鼠 cPLA2α 中的 C1P 位点已被消融 (KI)。我们的初步数据表明，KI 小鼠与野生型 (WT) 和 cPLA2 敲除 (KO) 小鼠表现出显着增强的伤口愈合能力。这些有益的影响是相互关联的 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 真皮成纤维细胞和中性粒细胞的迁移。重要的是，在一项初步研究中，我们还发现高水平的 人类压疮伤口液中的 5-HETE 与更好的愈合效果有关。因此，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 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 生产特定的 LOX 衍生类二十烷酸（例如 5-HETE）。这一机制得到了我们初步的支持 体外研究表明，C1P 可阻断 PIP2 对 cPLA2α 的激活。由于 LOX 和 COX 产品都是 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。因此，我们假设 压疮伤口愈合的增强将反映一种新型的“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。我们也 假设伤口愈合不良的老年人将出现溃疡性伤口 这些促进愈合的脂质介质和脂质特征将充当愈合结果的生物标志物。为了测试这些 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。