Interplay between hypoxia and oxidative phosphorylation in herpes stromal keratitis

疱疹性基质角膜炎中缺氧与氧化磷酸化之间的相互作用

• 批准号: 10357859
• 负责人: Susmit Suvas
• 金额: $ 40.99万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357859
• 关键词: Acetylcysteine; Antioxidants; Biological Assay; Blindness; Blood Vessels; CD147 antigen; CD4 Positive T Lymphocytes; Cell Respiration; Cell membrane; Cell physiology; Cells; Chemicals; Chronic; Cicatrix; Confocal Microscopy; Cornea; Corneal Neovascularization; Corneal Opacity; Development; Dimerization; Disease Management; Disease model; Enzymes; Epithelial Cells; Equilibrium; Eye Infections; Flow Cytometry; GLUT-3 protein; Gene Expression; Genes; Genetic Transcription; Glucose Transporter; Glycolysis; Goals; HK2 gene; Herpesvirus 1; Hypoxia; Hypoxia Inducible Factor; Immune; Immunomodulators; Impairment; Infection; Inflammation; Inflammatory; Keratitis; Knockout Mice; Knowledge; Lactate Transporter; Lesion; Link; Mediating; Metabolic; Metabolism; Mitochondria; Mus; Myeloid Cells; NADPH Oxidase; Neutrophilic Infiltrate; Nuclear; Oxidative Phosphorylation; Oxygen; Pathogenesis; Pathologic; Pharmaceutical Preparations; Pharmacology; Phenformin; Pimonidazole; Prevalence; Proteins; Publishing; Reactive Oxygen Species; Recurrence; Reporting; Research; Resolution; Respiration; Respiratory Burst; Role; SLC2A1 gene; Severities; Shapes; Signal Pathway; Signal Transduction; Stains; Tamoxifen; Testing; Tissues; Tumor-infiltrating immune cells; Up-Regulation; Vascular Endothelial Cell; Vascular blood supply; angiogenesis; bHLH-PAS factor HLF; chetomin; conditional knockout; diphenyleneiodonium; ebselen; efficacy evaluation; experimental study; functional outcomes; hypoxia inducible factor 1; inhibitor; innovation; neutrophil; novel; novel strategies; novel therapeutic intervention; prevent; response; transcription factor

Herpes stromal keratitis (HSK), a corneal chronic inflammatory condition that develops in response to recurrent corneal herpes simplex virus-1 infection, can cause permanent scarring and vision loss. Chronic inflammation often makes tissue hypoxic due to the lack of the blood vessel, reduced supply of blood, and a high metabolic demand of infiltrating immune cells. Development of hypoxia leads to the stabilization of hypoxia inducible factor (HIF), a transcription factor, which enhances the expression of glycolysis regulating genes and thereby promotes glycolytic metabolism. However, blocking of HIF signaling may promote oxidative phosphorylation to sustain the energy demand of inflammatory cells in inflamed tissue, suggesting an interplay between hypoxia and oxidative phosphorylation to prevent the resolution of an ongoing inflammation. Our preliminary results showed the development of hypoxia in corneas with HSK. Furthermore, the development of hypoxia was linked with the extent of neutrophils in HSV-1 infected corneas. When hypoxia associated signaling pathway PCR array and RT-qPCR were carried out on progressing HSK lesions, we detected an elevated expression of genes encoding key glycolytic enzymes, including PFKFB3 in infected corneas. PFKFB3 is a glycolytic activator, which is reported to enhance hemangiogenesis. We also found an increased level of expression of lactate transporters, MCT4 and MCT1, in corneas with HSK. RT-qPCR results were confirmed with confocal microscopy and flow cytometry. Our results also showed the nuclear localization of HIF-2 in epithelial cells, and HIF-1 in infiltrating neutrophils and CD4 T cells in HSK corneas. Interestingly, blocking of HIF dimerization, while using acriflavine, in HSV-1 infected mice exacerbated the corneal opacity, but decreased the hemangiogenesis. Our preliminary results and supportive published evidence together led us to hypothesize that neutrophils in HSK lesion shape the development of hypoxia resulting in the prevalence of HIF-regulated glycolytic metabolism that promotes HSK pathogenesis, and blockers of HIF, when given in association with inhibitors of mitochondrial respiration or reactive oxygen species (ROS) should reduce the severity of HSK. Three aims are proposed to test this hypothesis. Aim 1 will test the hypothesis that neutrophils in HSK lesion shape the development of hypoxia, and hypoxia enhances the severity of HSK. Aim 2 will test the hypothesis that inhibition of glycolytic activator PFKFB3, and the lactate transporter MCT4 and MCT1 protein reduces the severity of HSK. Aim 3 will test the hypothesis that blockers of HIF in association with an inhibitor of mitochondrial respiration or ROS will reduce the severity of HSK. Given the results from these studies, novel therapeutic approaches for treating HSK lesions may be developed. The knowledge gained from this application will provide innovative information that could be applicable to other ocular infection disease models.

疱疹性基质角膜炎（HSK）是一种角膜慢性炎症性疾病， 角膜单纯疱疹病毒-1感染，可导致永久性疤痕和视力丧失。慢性炎症 由于缺乏血管、血液供应减少和高代谢， 免疫细胞浸润的需求。缺氧的发展导致缺氧诱导因子的稳定 (HIF)，一种转录因子，其增强糖酵解调节基因的表达，从而促进糖酵解。 糖酵解代谢然而，阻断HIF信号传导可能促进氧化磷酸化以维持HIF-1 α的表达。 炎症组织中炎症细胞的能量需求，表明缺氧和氧化应激之间的相互作用。 在一些实施方案中，所述细胞可以通过磷酸化来防止正在进行的炎症的消退。初步结果显示， HSK角膜缺氧的发展。此外，缺氧的发展与 HSV-1感染角膜中嗜中性粒细胞的程度。当缺氧相关信号通路PCR阵列和 对进行性HSK病变进行RT-qPCR，我们检测到编码HSK的基因表达升高， 关键糖酵解酶，包括感染角膜中的PFKFB 3。PFKFB 3是一种糖酵解激活剂， 以增强血管生成。我们还发现乳酸转运蛋白MCT 4和 MCT 1，角膜HSK。用共聚焦显微镜和流式细胞术证实RT-qPCR结果。我们 结果还显示HIF-2 α在上皮细胞中的核定位，HIF-1 α在浸润的中性粒细胞中的核定位， HSK角膜中的CD 4 T细胞。有趣的是，在HSV-1感染的小鼠中，使用吖啶黄阻断HIF二聚化， 小鼠角膜混浊加重，但血管生成减少。我们的初步结果和 支持性的已发表的证据使我们假设HSK病变中的中性粒细胞塑造了HSK细胞的形态， 缺氧的发展导致HIF调节的糖酵解代谢的流行，促进HSK 发病机制和HIF阻断剂，当与线粒体呼吸抑制剂联合使用时， 活性氧（ROS）可降低HSK的严重程度。提出了三个目标来检验这一点 假说.目的1将验证HSK病变中中性粒细胞影响缺氧发展的假设， 缺氧可加重HSK的病情。目的2将检验抑制糖酵解激活剂 PFKFB 3和乳酸转运蛋白MCT 4和MCT 1蛋白降低了HSK的严重程度。目标3将测试 假设与线粒体呼吸或ROS抑制剂相关的HIF阻断剂将减少 HSK的严重性。鉴于这些研究的结果，治疗HSK病变的新治疗方法 可以开发。从这个应用程序中获得的知识将提供创新的信息， 适用于其它眼部感染疾病模型。