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

• 批准号: 8483282
• 负责人: Mohamed Al-Sayed Al-Shabrawey
• 金额: $ 31.95万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 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/8483282
• 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; Molecular; Muller' 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 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

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）：糖尿病性视网膜病（DR）的特征包括白细胞/内皮相互作用（Liukostasis），血液视网膜屏障（BRB）的分解和超透明度。 VEGF通过激活VEGF-R2的激活在高温性的发展中起着至关重要的作用，VEGF-R2通过氧化蛋白酪氨酸磷酸酶（PTPS）受到阴性对照。尽管临床证据表明血脂异常可能有助于DR，但尚未对其作用进行详细研究。糖尿病血脂异常的特征是蛛网膜酸（AA）的增加，这进一步由12/15-脂氧酶和其他酶促途径进一步代谢，以促进脂质脂质代谢物。最近，我们证明了12/15脂氧合酶（12/15-lox）及其脂质代谢产物的上调，DR中的12速率通过破坏神经胶质细胞VEGF/PEDF平衡有助于视网膜新生血管化。 NADPH氧化酶和内质网（ER）是对脂质代谢物增加的潜在靶标。当前建议的主要目的是研究以下假设：12/15-LOX的激活通过NADPH氧化酶依赖性机制有助于DR期间的视网膜炎症，该机制涉及ER应激反应，PTP的氧化以及随后增强的VEGF-R2活性。此外，Muller细胞产生的VEGF活性增强了VEGF-R2的活性，Muller细胞在糖尿病性视网膜中被12/15-lox的多余脂质代谢产物激活。我们的假设将通过3个特定目的进行研究1）确定12/15-LOX途径是否导致糖尿病引起的视网膜炎症。通过研究12/15-LOX表达的药理学或分子调节对糖尿病的影响，或在炎性细胞因子，白细胞，高渗透压和紧密连接蛋白（TJPS）改变的葡萄糖诱导的增加（TJP）中，将在体内和体外进行测试。为了表征视网膜和循环白细胞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具有12/15-Lox lox敲除型敲除视网膜内膜细胞和vise versa，以及对炎症的细胞的影响超重性。 2）确定NADPH氧化酶介导的ER应激是否有助于12/15-LOX脂质代谢产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。 HETE在NADPH氧化酶催化亚基NOX2缺陷小鼠中的眼内注射的影响将与HETE在野生型小鼠中的影响进行比较。 3）为了测试增强的VEGF-R2信号通路在12/15-Lox介导的视网膜炎症中起作用的假设，我们将测试PTP激动剂或VEGF-R2抑制剂是否可以防止培养的视网膜内皮细胞中12/15-Lox脂质脂肪的促炎作用。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炎症途径，作为潜在的治疗靶标，以防止DR期间的早期炎症反应，进而阻止疾病的进展到视网膜新血管形成和视力丧失的晚期。