Role of lipoxygenase pathway in early microvascular dysfunction during diabetic r

描述(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(白细胞淤积)、血视网膜屏障(BRB)破坏和高通透性。通过蛋白酪氨酸磷酸酶(PTPs)氧化负性调控的VEGF-R2的激活，在高通透性的形成中起着关键作用。尽管临床证据表明血脂异常可能与糖尿病视网膜病变有关，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)的增加，花生四烯酸通过12/15-脂氧合酶和其他酶途径进一步代谢为促炎性脂质代谢产物。最近，我们发现糖尿病视网膜病变中12/15脂氧合酶(12/15-LOX)及其脂代谢产物12-HETE的上调可能通过破坏胶质细胞的VEGF/PEDF平衡而促进视网膜新生血管的形成。NADPH氧化酶和内质网(ER)是12/15-LOX脂代谢产物增加的潜在靶点。本研究的主要目的是探讨12/15-LOX的激活通过NADPH氧化酶依赖的机制参与视网膜炎症的假说，该机制涉及内质网应激反应、PTPs的氧化以及随后增强的VEGF-R2活性。此外，糖尿病视网膜中过量脂代谢产物12/15-LOX激活的Muller细胞产生的血管内皮生长因子可增强血管内皮生长因子-R2的活性。我们的假说将通过3个特定的目标进行研究：1)确定12/15-LOX途径是否参与糖尿病引起的视网膜炎症。这一目标将在体内和体外通过检测药物或分子调节12/15-LOX的表达和活性在糖尿病或高糖引起的炎性细胞因子增加、白细胞停滞、高通透性和紧密连接蛋白(TJP)变化的影响来检验。为了确定视网膜和循环中的白细胞12/15-LOX在糖尿病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基因敲除的视网膜内皮细胞在炎症细胞浸润、随后的白细胞淤积和高通透性中的作用。2)探讨NADPH氧化酶介导的内质网应激在12/15-LOX脂代谢产物诱导的视网膜炎症中的作用。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。眼内注射HETES对NADPH氧化酶催化亚基NOX2缺陷小鼠的影响将与HETES对野生型小鼠的影响进行比较。3)为了验证增强的VEGF-R2信号通路在12/15-LOX介导的视网膜炎症中的作用，我们将检测PTP激动剂或VEGF-R2抑制剂是否能抑制12/15-LOX脂代谢产物对培养的视网膜内皮细胞的促炎作用。我们还将在体内检验腺病毒介导的sFlt1过表达对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，进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.