Role of lipoxygenase pathway in early microvascular dysfunction during diabetic r

脂氧合酶途径在糖尿病患者早期微血管功能障碍中的作用

基本信息

批准号：
8633459
负责人：
Mohamed Al-Sayed Al-Shabrawey
金额：
$ 33.08万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8633459
关键词：
Acids Address Adhesions Age Agonist American Arachidonate 15-Lipoxygenase Arachidonic Acids Attenuated Blindness Blood Vessels Blood-Retinal Barrier Bone Marrow Transplantation Catalytic Domain Cells Cellular Stress Cellular Stress Response Clinical Data Development Diabetes Mellitus Diabetic Retinopathy Diabetic mouse Disease Dyslipidemias Elements Endoplasmic Reticulum Endothelial Cells Equilibrium Extravasation Eye Functional disorder Glucose Goals Growth Factor Hyperglycemia In Vitro Infiltration Inflammation Inflammatory Inflammatory Response Injection of therapeutic agent Knock-out Laboratories Leukocytes Leukostasis Light Lipids Lipoxygenase Mediating Microvascular Dysfunction Molecular Muller&apos s cell Mus NADPH Oxidase Neuroglia Organelles Oxidation-Reduction Oxidative Stress Pathogenesis Pathway interactions Permeability Phosphorylation Play Prevention Protein Tyrosine Kinase Protein Tyrosine Phosphatase Proteins Reactive Oxygen Species Retina Retinal Retinal Neovascularization Role Signal Pathway Signal Transduction Staging Streptozocin Techniques Testing Tight Junctions Up-Regulation Vascular Diseases Vascular Endothelial Growth Factors Vascular Endothelium Vascular Permeabilities Visual Acuity Wild Type Mouse Work adenoviral-mediated biological adaptation to stress cytokine diabetic diabetic patient endoplasmic reticulum stress in vivo inflammatory marker inhibitor/antagonist inorganic phosphate lipid metabolism macular edema new therapeutic target non-diabetic novel overexpression oxidation phosphatase inhibitor pigment epithelium-derived factor prevent public health relevance receptor research study response therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Features of diabetic retinopathy (DR) include leukocyte/endothelial interaction (leukostasis), breakdown of blood retinal barrier (BRB) and hyperpermeability. VEGF plays a crucial role in the development of hyperpermeability via activation of VEGF-R2 which subjected to negative control by oxidation of protein tyrosine phosphatases (PTPs). Despite the clinical evidence which shows that dyslipidemia may contribute to DR, its role has not been studied in detail. Diabetic dyslipidemia is characterized by an increase in arachidonic acid (AA) which is further metabolized by 12/15-lipoxygenase and other enzymatic pathways into proinflammatory lipid metabolites. Recently we demonstrated that upregulation of 12/15 lipoxygenase (12/15-LOX) and its lipid metabolites, 12-HETEs in DR contributes to retinal neovascularization via disrupting glial cells VEGF/PEDF balance. NADPH oxidase and endoplasmic reticulum (ER) are potential targets to the increased lipid metabolites of 12/15-LOX. The major goal of the current proposal is to investigate the hypothesis that activation of 12/15-LOX contributes to retinal inflammation during DR via NADPH oxidase-dependent mechanism which involves ER stress response, oxidation of PTPs and subsequent enhanced VEGF-R2 activity. Furthermore, VEGF-R2 activity is enhanced by VEGF produced by Muller cells which are activated by the excess lipid metabolites of 12/15-LOX in diabetic retina. Our hypothesis will be investigated via 3 specific aims 1) To determine whether 12/15-LOX pathway contributes to diabetes-induced retinal inflammation. This aim will be tested in vivo and in vitro by examining the effect of pharmacological or molecular modulation of 12/15-LOX expression and activity on diabetes or high glucose-induced increases in inflammatory cytokines, leukostasis, hyperpermeability and alterations in tight junction proteins (TJPs). To characterize the role of retinal versus the circulating leukocyte 12/15-LOX in diabetes-induced retinal inflammation we will utilize bone marrow transplantation (BMT) studies to determine the effect of wild type leukocytes with 12/15-LOX knockout retinal endothelial cells, and vise versa, on inflammatory cell infiltration and subsequent leukostasis and hyperpermeability. 2) To determine whether NADPH oxidase-mediated ER stress contributes to retinal inflammation induced by lipid metabolites of 12/15-LOX. For this aim, the effect of inhibiting NADPH oxidase or ER stress response on 12/15-LOX and HG-mediated inflammatory response will be tested. The impact of intraocular injection of HETEs in NADPH oxidase catalytic subunit NOX2-deficient mice will be compared to the effect of HETEs in wild type mice. 3) To test the hypothesis that enhanced VEGF-R2 signaling pathway plays a role in 12/15-LOX-mediated retinal inflammation, we will test whether PTP agonist or VEGF-R2 inhibitors prevent the pro-inflammatory effect of 12/15-LOX lipid metabolites in cultured retinal endothelial cells. We will also examine the impact of adenoviral-mediated sFlt1 overexpression on 12/15-LOX mediated retinal inflammation in vivo. Our experiments should establish 12/15-LOX inflammatory pathway as a potential therapeutic target to prevent the early inflammatory response during DR and in turn halts the progress of the disease to the late stage of retinal neovascularization and vision loss.

描述（由申请人提供）：糖尿病视网膜病变（DR）的特征包括白细胞/内皮相互作用（白细胞停滞）、血视网膜屏障（BRB）破坏和通透性过高。 VEGF 通过激活 VEGF-R2 在通透性过高的发展中发挥着至关重要的作用，而 VEGF-R2 受到蛋白酪氨酸磷酸酶 (PTP) 氧化的负控制。尽管临床证据表明血脂异常可能导致 DR，但其作用尚未得到详细研究。糖尿病血脂异常的特征是花生四烯酸 (AA) 增加，花生四烯酸 (AA) 进一步被 12/15-脂氧合酶和其他酶途径代谢为促炎性脂质代谢物。最近，我们证明 DR 中 12/15 脂氧合酶 (12/15-LOX) 及其脂质代谢物 12-HETE 的上调通过破坏神经胶质细胞 VEGF/PEDF 平衡而有助于视网膜新生血管形成。 NADPH 氧化酶和内质网 (ER) 是 12/15-LOX 脂质代谢物增加的潜在靶标。当前提案的主要目标是研究以下假设：12/15-LOX 的激活通过 NADPH 氧化酶依赖性机制导致 DR 期间的视网膜炎症，该机制涉及 ER 应激反应、PTP 氧化以及随后增强的 VEGF-R2 活性。此外，Muller 细胞产生的 VEGF 增强了 VEGF-R2 活性，而 Muller 细胞被糖尿病视网膜中过量的 12/15-LOX 脂质代谢物激活。我们的假设将通过 3 个具体目标进行研究：1) 确定 12/15-LOX 通路是否导致糖尿病引起的视网膜炎症。该目标将在体内和体外进行测试，通过检查 12/15-LOX 表达和活性的药理学或分子调节对糖尿病或高葡萄糖诱导的炎症细胞因子增加、白细胞停滞、通透性过高和紧密连接蛋白改变的影响。 TJP）。为了表征视网膜与循环白细胞 12/15-LOX 在糖尿病引起的视网膜炎症中的作用，我们将利用骨髓移植 (BMT) 研究来确定野生型白细胞与 12/15-LOX 敲除视网膜内皮细胞的作用，反之亦然，影响炎症细胞浸润以及随后的白细胞停滞和通透性过高。 2) 确定NADPH氧化酶介导的ER应激是否有助于12/15-LOX脂质代谢物诱导的视网膜炎症。为此，将测试抑制 NADPH 氧化酶或 ER 应激反应对 12/15-LOX 和 HG 介导的炎症反应的影响。将眼内注射 HETE 对 NADPH 氧化酶催化亚基 NOX2 缺陷小鼠的影响与 HETE 对野生型小鼠的影响进行比较。 3) 为了检验增强的 VEGF-R2 信号通路在 12/15-LOX 介导的视网膜炎症中发挥作用的假设，我们将测试 PTP 激动剂或 VEGF-R2 抑制剂是否会阻止 12/15-LOX 的促炎作用培养的视网膜内皮细胞中的脂质代谢物。我们还将研究腺病毒介导的 sFlt1 过表达对体内 12/15-LOX 介导的视网膜炎症的影响。我们的实验应建立12/15-LOX炎症通路作为潜在的治疗靶点，以预防DR期间的早期炎症反应，进而阻止疾病进展至视网膜新生血管和视力丧失的晚期阶段。