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/内皮细胞相互作用（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在高渗透性的发展中起关键作用，VEGF-R2受到蛋白酪氨酸磷酸酶（PTPs）氧化的负控制。尽管临床证据表明血脂异常可能导致DR，但其作用尚未详细研究。糖尿病性血脂异常的特征在于花生四烯酸（AA）的增加，花生四烯酸（AA）通过12/15-脂氧合酶和其他酶途径进一步代谢成促炎性脂质代谢物。最近，我们证明，上调12/15脂氧合酶（12/15-LOX）及其脂质代谢产物，12-HETE在DR有助于通过破坏胶质细胞VEGF/PEDF平衡视网膜新生血管。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。目前的建议的主要目标是调查的假设，激活12/15-LOX有助于视网膜炎症在DR期间通过NADPH氧化酶依赖性机制，涉及ER应激反应，氧化的PTP和随后增强的VEGF-R2活性。此外，VEGF-R2活性通过由Muller细胞产生的VEGF增强，所述Muller细胞被糖尿病视网膜中12/15-LOX的过量脂质代谢物激活。我们的假设将通过3个具体目标进行研究1）确定12/15-LOX通路是否有助于糖尿病诱导的视网膜炎症。这一目标将通过检查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、高渗透性和紧密连接蛋白改变的影响来在体内和体外进行测试（TJP）。为了表征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敲除视网膜内皮细胞（反之亦然）对炎性细胞浸润和随后的白细胞停滞和高渗透性的影响。2)确定NADPH氧化酶介导的内质网应激是否有助于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。将在NADPH氧化酶催化亚基N 0X 2缺陷型小鼠中眼内注射HETE的影响与野生型小鼠中HETE的影响进行比较。3)为了测试增强的VEGF-R2信号传导途径在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抑制剂是否阻止12/15-LOX脂质代谢物在培养的视网膜内皮细胞中的促炎作用。我们还将研究腺病毒介导的sFlt 1过表达对体内12/15-LOX介导的视网膜炎症的影响。我们的实验将建立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阶段。

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.