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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（如脂质组学）的新技术取得了进展