Regulation of vascular leakage in age related macular degeneration

年龄相关性黄斑变性中血管渗漏的调节

• 批准号: 8385057
• 负责人: MEREDITH GREGORY-KSANDER
• 金额: $ 30.02万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8385057
• 关键词: Address; Age related macular degeneration; Age-Years; Agonist; American; Angiogenic Factor; Animal Model; Apoptosis; Apoptotic; Atrophic; Binding; Binding Proteins; Blindness; Blood; Blood Vessels; Cell surface; Cells; Choroidal Neovascularization; Cleaved cell; Data; Development; Doxycycline; Endothelial Cells; Extravasation; Eye; Failure; Fluorescein Angiography; Gene Targeting; Goals; Growth; Immune; Induction of Apoptosis; Inflammatory; Inflammatory Infiltrate; Integral Membrane Protein; Interleukin-1; Isolectin; Knock-in Mouse; Laboratories; Lasers; Lead; Lesion; Liquid substance; Maintenance; Mediating; Membrane; Metalloproteases; Modeling; Mouse Strains; Mus; Mutate; Optical Coherence Tomography; Pathogenesis; Pathway interactions; Patients; Phenotype; Play; Population; Protein Isoforms; Proteins; Recombinants; Regulation; Reporting; Retina; Retinal; Role; Site; Staining method; Stains; Structure of retinal pigment epithelium; System; TNF gene; Therapeutic; Time; Tissues; Transgenic Mice; Tumor Necrosis Factor Ligand Superfamily Member 6; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Vision; Wild Type Mouse; Work; bevacizumab; cell type; cytokine; cytotoxic; extracellular; in vivo; intravitreal injection; macrophage; mouse model; neovascular; new growth; new therapeutic target; novel; overexpression; photoreceptor degeneration; prevent; protein function; success

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is a leading cause of blindness in Americans 60 years of age and older. There are two forms of AMD: Dry or atrophic and Wet or exudative. However, the Wet form of AMD, which is characterized by choroidal neovascularization (CNV), is associated with more severe vision loss caused primarily by the leakage of fluid and blood from the abnormal vessels. Anti-VEGF therapy has emerged as the treatment of choice for patients with wet AMD. However, some patients never respond to anti-VEGF therapy, while still others stop responding after initial success, highlighting the fact that the pathobiology of wet AMD development and progression is still unknown. FasL is an important protein in maintaining immune privilege in the eye, where it is thought to induce apoptosis of infiltrating inflammatory cells. In fact, several reports indicate that FasL expressed on retinal pigment epithelial cells inhibits choroidal neovascularization by triggering apoptosis o vascular endothelial cells. However, FasL exists as both a membrane bound and soluble protein with potentially opposing functions and it is unclear how the different forms of FasL contribute to the development of CNV. We recently developed a unique knock-in mouse strain in which the FasL metalloproteinase cleavage sites were mutated to prevent cleavage of the membrane-bound protein. In these ¿CS mice, FasL is expressed by the physiologically relevant cell types, but these cells are unable to cleave FasL and therefore, can only express mFasL (termed ¿CS mice). For the first time, these mice have allowed us to study the in vivo function of membrane FasL in the absence of soluble FasL. To better understand the function of the different FasL isoforms in the development of choroidal neovascularization, we used a laser-induced murine model of CNV. Our preliminary data indicate that, in ¿CS mice, either the increased expression of mFasL (or the loss of sFasL) prevents vascular leakage following laser induced-CNV. Spectral domain optical coherence tomography (SD-OCT) and Isolectin-B staining demonstrated no difference in the size of the neovascular lesion between ¿CS mice and wild-type mice. However, fluorescein angiography revealed a significant decrease in vascular leakage in ¿CS mice as compared to wild-type mice. These results indicate that membrane FasL does not prevent the ingrowth of choroidal vessels, as previously documented, but rather plays a critical function in preventing vascular leakage, the primary cause of vision loss in patients with wet AMD. We hypothesize that membrane FasL inhibits vascular leakage in laser-induced CNV through Fas mediated pathways triggered in infiltrating macrophages and/or RPE cells. The goals of this proposal are as follows: (Aim 1) Determine whether the decreased vascular leakage in ¿CS mice is due to increased mFasL and/or a lack of sFasL and whether the decreased leakage coincides with a shift in the M1/M2 phenotype of infiltrating macrophages (Aim 2) Establish whether infiltrating macrophages and/or RPE are the critical Fas+ targets through which mFasL mediates inhibition of vascular leakage. PUBLIC HEALTH RELEVANCE: Age Related Macular Degeneration (AMD) is the primary cause of blindness in patients 60 years of age and older. The growth of abnormal blood vessels into the retina is the main cause of severe vision loss, in part due to vessel leakage of blood and fluid that causes damage to the surrounding retinal tissue. We have identified a novel protein that prevents vascular leakage and the goal of this study is to (i) determine whether the administration of this protein can be used to prevent vascular leakage in a mouse model, and (ii) elucidate the mechanism by which this protein functions.

描述（由申请人提供）：年龄相关性黄斑变性 (AMD) 是 60 岁及以上美国人失明的主要原因。 AMD 有两种形式：干性或萎缩性和湿性或渗出性。然而，湿性 AMD 的特点是脉络膜新生血管 (CNV)，它与更严重的视力丧失相关，这主要是由异常血管中的液体和血液渗漏引起的。抗 VEGF 疗法已成为湿性 AMD 患者的首选治疗方法。然而，一些患者对抗 VEGF 治疗从未产生反应，而还有一些患者在初步成功后停止反应，这凸显了湿性 AMD 发生和进展的病理学仍然未知的事实。 FasL 是维持眼睛免疫特权的重要蛋白质，被认为可以诱导浸润性炎症细胞凋亡。事实上，一些报告表明 FasL 表达 视网膜色素上皮细胞通过触发血管内皮细胞凋亡来抑制脉络膜新生血管形成。然而，FasL 以膜结合蛋白和可溶性蛋白的形式存在，具有潜在的相反功能，目前尚不清楚不同形式的 FasL 如何有助于 CNV的发展。我们最近开发了一种独特的敲入小鼠品系，其中 FasL 金属蛋白酶切割位点发生突变，以防止膜结合蛋白的切割。在这些 ¿CS 小鼠中，FasL 由生理相关细胞类型表达，但这些细胞无法裂解 FasL，因此只能表达 mFasL（称为 ¿CS 小鼠）。这些小鼠首次使我们能够在缺乏可溶性 FasL 的情况下研究膜 FasL 的体内功能。为了更好地了解不同 FasL 亚型在脉络膜新生血管形成中的功能，我们使用了激光诱导的 CNV 小鼠模型。我们的初步数据表明，在 CS 小鼠中，mFasL 表达的增加（或 sFasL 的丧失）可以防止激光诱导 CNV 后的血管渗漏。谱域光学相干断层扫描 (SD-OCT) 和异凝集素-B 染色表明 ¿CS 小鼠和野生型小鼠之间的新生血管病变大小没有差异。然而，荧光素血管造影显示，与野生型小鼠相比，CS 小鼠的血管渗漏显着减少。这些结果表明，膜 FasL 并不像之前所记录的那样阻止脉络膜血管向内生长，而是在防止血管渗漏方面发挥着关键作用，而血管渗漏是湿性 AMD 患者视力丧失的主要原因。我们假设膜 FasL 通过在浸润巨噬细胞和/或 RPE 细胞中触发的 Fas 介导途径抑制激光诱导的 CNV 中的血管渗漏。该提案的目标如下：（目标 1）确定 ¿CS 小鼠血管渗漏减少是否是由于 mFasL 增加和/或 sFasL 缺乏所致，以及渗漏减少是否与浸润巨噬细胞 M1/M2 表型的转变一致（目标 2）确定浸润巨噬细胞和/或 RPE 是否是 mFasL 的关键 Fas+ 靶点 介导抑制血管渗漏。 公共卫生相关性：年龄相关性黄斑变性 (AMD) 是 60 岁及以上患者失明的主要原因。异常血管生长到视网膜是严重视力丧失的主要原因，部分原因是血管渗漏和 对周围视网膜组织造成损害的液体。我们已经鉴定出一种可防止血管渗漏的新型蛋白质，本研究的目的是（i）确定该蛋白质的施用是否可用于预防小鼠模型中的血管渗漏，以及（ii）阐明该蛋白质的功能机制。