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: 10586030
• 负责人: Susmit Suvas
• 金额: $ 42.25万
• 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 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586030
• 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; Herpes stromal keratitis; Herpesvirus 1; Hypoxia; Hypoxia Inducible Factor; Immune; Impairment; Infection; Inflammation; Inflammatory; Knockout Mice; Knowledge; Lactate Transporter; Lesion; Link; Mediating; Metabolic; Metabolism; Mitochondria; Mus; Myeloid Cells; NADPH Oxidase; Neutrophil Infiltration; Nuclear; Oxidative Phosphorylation; Oxygen; Pathogenesis; Pathologic; Pharmaceutical Preparations; 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; Up-Regulation; Vascular Endothelial Cell; Vascular blood supply; angiogenesis; chetomin; conditional knockout; diphenyleneiodonium; ebselen; efficacy evaluation; experimental study; functional outcomes; immune cell infiltrate; immune modulating agents; inhibitor; innovation; neutrophil; novel; novel strategies; novel therapeutic intervention; pharmacologic; 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)，一种因复发而发生的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 常因血管缺乏、供血减少、代谢率高而使组织缺氧。 浸润免疫细胞的需求。缺氧的发生导致缺氧诱导因子的稳定 (HIF)，一种转录因子，可增强糖酵解调节基因的表达，从而促进 糖酵解代谢。然而，阻断 HIF 信号传导可能会促进氧化磷酸化以维持 发炎组织中炎症细胞的能量需求，表明缺氧和氧化之间存在相互作用 磷酸化以防止持续炎症的消退。我们的初步结果表明 HSK 导致角膜缺氧。此外，缺氧的发生与 HSV-1 感染的角膜中中性粒细胞的范围。当缺氧时相关信号通路PCR阵列和 对进展中的 HSK 病变进行 RT-qPCR，我们检测到编码基因的表达升高 关键的糖酵解酶，包括受感染角膜中的 PFKFB3。 PFKFB3 是一种糖酵解激活剂，据报道 以增强血管生成。我们还发现乳酸转运蛋白、MCT4 和 MCT1，在角膜中，带有 HSK。 RT-qPCR 结果通过共聚焦显微镜和流式细胞术得到证实。我们的 结果还显示，HIF-2α 定位于上皮细胞，HIF-1α 定位于浸润性中性粒细胞和 HSK 角膜中的 CD4 T 细胞。有趣的是，在 HSV-1 感染中使用吖啶黄来阻断 HIF 二聚化 小鼠的角膜混浊加剧，但血管生成减少。我们的初步结果和 已发表的支持性证据共同使我们推测 HSK 病变中的中性粒细胞塑造了 缺氧的发生导致 HIF 调节的糖酵解代谢的普遍存在，从而促进 HSK 发病机制和 HIF 阻断剂，当与线粒体呼吸抑制剂或抑制剂联合使用时 活性氧 (ROS) 应该可以减轻 HSK 的严重程度。提出了三个目标来测试这一点 假设。目标 1 将检验 HSK 病变中的中性粒细胞影响缺氧发展的假设，以及 缺氧会加剧 HSK 的严重程度。目标 2 将检验以下假设：糖酵解激活剂的抑制 PFKFB3 以及乳酸转运蛋白 MCT4 和 MCT1 蛋白可降低 HSK 的严重程度。目标 3 将测试 假设 HIF 阻断剂与线粒体呼吸或 ROS 抑制剂联合使用会减少 HSK 的严重程度。鉴于这些研究的结果，治疗 HSK 病变的新治疗方法 可能会被开发。从该应用程序中获得的知识将提供创新信息，这些信息可以 适用于其他眼部感染疾病模型。