Role of Abnormal lysyl oxidase in the pathogenesis of diabetic retinopathy

赖氨酰氧化酶异常在糖尿病视网膜病变发病机制中的作用

• 批准号: 9248395
• 负责人: Sayon Roy
• 金额: $ 43.5万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9248395
• 关键词: AKT inhibition; Affinity; Apoptosis; Attenuated; Basement membrane; Binding; Biochemical; Biogenesis; Blindness; Blood Vessels; Blood capillaries; Cell Culture Techniques; Cell Death; Cell Proliferation; Cell Survival; Cells; Characteristics; Clinical; Clinical Trials; Data; Development; Diabetes Mellitus; Diabetic Retinopathy; Disease; Endothelial Cells; Enzyme Precursors; Enzymes; Extracellular Matrix; Extracellular Protein; Extravasation; FGF2 gene; Feedback; Fibronectins; Glucose; Goals; Human; In Vitro; Incidence; Inhibition of Apoptosis; Knockout Mice; LOXL2 gene; Lead; Lesion; Literature; MAPK1 gene; Mediating; Modality; Modeling; Molecular Genetics; Mus; Pathogenesis; Pathway interactions; Patients; Pericytes; Permeability; Prevalence; Process; Protein Isoforms; Protein-Lysine 6-Oxidase; Proteins; Public Health; Ras/Raf; Research; Retina; Retinal; Role; Signal Transduction; Testing; Up-Regulation; Vascular Permeabilities; aging population; animal facility; base; capillary; crosslink; diabetic; diabetic rat; extracellular; genetic approach; insight; monolayer; novel therapeutics; overexpression; prevent; public health relevance

 DESCRIPTION (provided by applicant): The overall goal of this project is to test the hypothesis that increased lysyl oxidase (LOX) expression and activation triggered by high glucose/diabetes compromises basement membrane (BM) function and thereby induces excess permeability and vascular cell death in diabetic retinopathy (DR). The hypothesis is based on findings that diabetes increases expression of LOX, which is required for the formation and maturation of the BM, and that HG-induced LOX upregulation and over-activation compromises endothelial barrier function. Importantly, inhibition of HG-induced LOX overexpression results in reduced cell monolayer permeability. While LOX is known for its cross-linking activity, LOX propeptide (LOX-PP), released during LOX biogenesis, is known for regulating cell proliferation and survival through modulation of Akt and Erk pathways. Our preliminary data indicates that HG-induced LOX overexpression promotes apoptosis in retinal endothelial cells, and that the process may be exacerbated by LOX-PP, which is upregulated by HG. Furthermore, preliminary data indicates that LOX binds with high affinity to extracellular fibronectin (FN) under HG condition, and interferes with LOX internalization into the cell, a process unique to LOX. Thus, the rationale for the proposed studies is that (i) HG-induced LOX overexpression contributes to capillary leakage, and together with increased LOX-PP promotes apoptosis by modulating Akt and Erk pathways, and (ii) HG-induced increased binding of LOX with ECM proteins, decreases LOX internalization, attenuates negative feedback signaling and thereby promotes LOX overexpression. The proposed studies will be performed using biochemical, molecular, and genetic approaches utilizing cell culture models, diabetic rats, and LOX heterozygous KO mice (LOX+/-). Towards this goal, we have generated LOX+/- mice colonies at our animal facility that are now available for the proposed studies. The specific aims of this proposal are to determine (i) whether inhibition of LOX overexpression and activation prevents apoptosis in retinal endothelial cells in vitro, and reduces vascular cell loss and permeability in diabetic rat retinas (ii) whether the decreased LOX level in LOX+/- mice is protective against diabetes-induced retinal vascular lesions, and (iii) mechanism(s) by which HG induces LOX overexpression and promotes retinal vascular cell loss. Findings from these studies are expected to identify a novel therapeutic strategy for preventing retinal vascular cell loss and capillary leakage, and provide mechanistic insights underlying HG-induced LOX overexpression in DR.

 描述(申请人提供)：本项目的总体目标是测试假设，即高糖/糖尿病引发的赖氨酰氧化酶(LOX)表达和激活增加会损害基底膜(BM)功能，从而导致糖尿病视网膜病变(DR)的过度通透性和血管细胞死亡。这一假说是基于以下发现：糖尿病增加LOX的表达，而LOX是骨髓形成和成熟所必需的，HG诱导的LOX上调和过度激活损害了内皮屏障功能。重要的是，抑制HG诱导的LOX过度表达导致细胞单层通透性降低。虽然LOX以其交联性而闻名，但在LOX生物发生过程中释放的LOX前肽(LOX-PP)通过调节Akt和Erk通路来调节细胞的增殖和生存。我们的初步数据表明，HG诱导的LOX过表达促进视网膜内皮细胞的凋亡，这一过程可能被HG上调的LOX-PP加剧。此外，初步数据表明，在HG条件下，LOX与细胞外纤维连接蛋白(FN)具有高亲和力，并干扰LOX内化到细胞内，这是LOX特有的过程。因此，这项研究的基本原理是：(I)HG诱导的LOX过度表达有助于毛细血管渗漏，并且LOX-PP的增加通过调节Akt和Erk途径促进细胞凋亡，以及(Ii)HG诱导LOX与ECM蛋白结合增加，减少LOX内化，减弱负反馈信号，从而促进LOX过度表达。拟议的研究将使用生化、分子和遗传学方法，利用细胞培养模型、糖尿病大鼠和LOX杂合子KO小鼠(LOX+/-)进行。为了实现这一目标，我们已经在我们的动物设施中产生了LOX+/-小鼠克隆，现在可以用于拟议的研究。这项建议的具体目的是确定(I)抑制LOX过表达和激活是否能防止体外培养的糖尿病大鼠视网膜内皮细胞的凋亡，并减少血管细胞的丢失和通透性；(Ii)LOX+/-小鼠LOX水平的降低是否对糖尿病引起的视网膜血管病变具有保护作用；以及(Iii)HG诱导LOX过表达并促进视网膜血管细胞丢失的机制(S)。这些研究结果有望确定一种新的治疗策略，以防止视网膜血管细胞丢失和毛细血管渗漏，并为HG诱导的DR LOX过度表达提供机制方面的见解。