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，小分子分析的新技术进步（例如，脂肪组学）具有 确定伤口愈合受损的生化表现： 促和抗炎类花生酸1-9。类花生酸的合成从初始速率限制步骤开始， 通过磷脂酶A2（PLA2）10-12的活性产生花生四烯酸（AA）。主要PLA2S之一 参与此初始步骤是IVA胞质PLA2组（CPLA2α）10-12，Chalfant实验室证明了这一点 通过直接结合与鞘脂，神经酰胺1-磷酸（C1P）13-19激活。采用较新的脂肪组 技术，我们发现C1P在炎症阶段受到暂时调节，并特别增加 人类伤口愈合5。为了评估伤口愈合中C1P诱导的eicosanoids，我们创建了一只敲击小鼠 与CPLA2消融（KI）中的C1P站点。我们的初步数据表明，与野生型（WT）和 CPLA2敲除（KO）小鼠，暴露于伤口愈合大大增强。这些有益效果与 炎症前列腺素的丧失（例如，环氧酶（COX）衍生的PGE2）并增加了产生 特定的脂质介质（即脂氧合酶（LOX）衍生的5-HETE）的大量，这诱导了显着加速 皮肤成纤维细胞和中性粒细胞的迁移。重要的是，在最初的研究中，我们还发现高水平 人体压力溃疡的伤口液中的5-HETE与更好的愈合结果有关。那，平衡 在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。 最初的机械研究还表明，相关的细胞表型和变体产生 在Ki细胞中观察到的类固醇类别与C1P驱动突变体的差异细胞定位有关 CPLA2通过与PIP2关联。这些发现为前提提供了基础，即当Cpla2无法 要结合C1P，该酶可以自由与其他脂质调节剂（例如PIP2）相关联 特定Lox衍生的类类（例如5- hete）的产生。我们的初步支持了这种机制 体外研究表明，C1P通过PIP2阻止CPLA2α的激活。由于Lox和Cox产品都是 CPLA2依赖性，但在其生物合成中暂时对比20.21，我们的数据表明一个被忽略的 CPLA2调节的复杂性响应炎症激动剂。那我们假设 压力溃疡的伤口愈合增强将反映出一种新型的“脂质级开关”，产生亲治疗 涉及C1P和PIP2代谢对CPLA2的复杂的拮抗调节的类固醇。我们也是 假设表现出无效伤口愈合的衰老人类会缺乏溃疡性伤口 这些亲愈合的脂质介质以及脂质签名将充当愈合结果的生物标志物。测试这些 假设，我们将采用一个多学科的团队，新型的遗传老鼠模型和“最新技术” 脂质组学和分子生物学技术探索潜在的机制和生物活性脂质 与衰老和溃疡性伤口的不愈合有关。