Role of lipoxygenase pathway in early microvascular dysfunction during diabetic r

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），血液视网膜屏障（BRB）的破坏和高渗透性。VEGF通过激活VEGF- r2在高通透性的发展中起着至关重要的作用，而VEGF- r2受到蛋白酪氨酸磷酸酶（PTPs）氧化的负性控制。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。糖尿病性血脂异常的特征是花生四烯酸（AA）增加，经12/15-脂氧合酶等酶促途径代谢为促炎脂质代谢物。最近，我们证明了DR中12/15脂氧合酶（12/15- lox）及其脂质代谢产物12-HETEs的上调通过破坏胶质细胞VEGF/PEDF平衡有助于视网膜新生血管的形成。NADPH氧化酶和内质网（ER）是12/15-LOX脂质代谢产物增加的潜在靶点。本研究的主要目标是研究12/15-LOX的激活通过NADPH氧化酶依赖机制参与DR期间视网膜炎症的假设，该机制涉及内质网应激反应、ptp氧化和随后VEGF-R2活性增强。此外，糖尿病视网膜中过量脂质代谢物12/15-LOX激活Muller细胞产生的VEGF可增强VEGF- r2活性。我们的假设将通过三个具体目标进行研究：1)确定12/15-LOX通路是否参与糖尿病诱导的视网膜炎症。这一目标将在体内和体外通过检查12/15-LOX表达和活性的药理学或分子调节对糖尿病或高糖诱导的炎症细胞因子增加、白细胞停滞、高通透性和紧密连接蛋白（TJPs）改变的影响进行测试。为了描述视网膜与循环白细胞12/15-LOX在糖尿病诱导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敲除的视网膜内皮细胞，反之亦然，对炎症细胞浸润和随后的白细胞瘀滞和高通透性的影响。2)确定NADPH氧化酶介导的内质网应激是否与12/15-LOX脂质代谢物诱导的视网膜炎症有关。为此，我们将测试抑制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。将对NADPH氧化酶催化亚基nox2缺失小鼠眼内注射HETEs的影响与野生型小鼠进行比较。3)为了验证VEGF-R2信号通路增强在12/15-LOX介导的视网膜炎症中发挥作用的假设，我们将在培养的视网膜内皮细胞中检测PTP激动剂或VEGF-R2抑制剂是否能阻止12/15-LOX脂质代谢产物的促炎作用。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。我们的实验应该建立12/15-LOX炎症通路作为潜在的治疗靶点，以防止DR期间的早期炎症反应，从而阻止疾病进展到视网膜新生血管和视力丧失的晚期。

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.