Investigating the function and mechanism of interleukin 7 receptor-expressing pro-angiogenic macrophages during experimental choroidal neovascularization

研究表达白细胞介素7受体的促血管生成巨噬细胞在实验性脉络膜新生血管形成过程中的功能和机制

• 批准号: 10563645
• 负责人: Jeremy A Lavine
• 金额: $ 40万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10563645
• 关键词: Ablation; Angiogenesis Pathway; Annexins; Area; Atrophic; Basement membrane; Blindness; Blood Vessels; Bone Marrow; Breeding; Cell Therapy; Cells; Choroid; Choroidal Neovascularization; Classification; Complement; Complement Receptor; Confocal Microscopy; Data; Dendritic Cells; Drusen; Endothelial Cells; Exudative age-related macular degeneration; Eye; Fibronectins; Flow Cytometry; Fluorescence-Activated Cell Sorting; Genes; Heterogeneity; Homeostasis; Human; Immune; Inflammatory; Interleukin 7 Receptor; Interleukin-1 beta; Knock-out; Laser injury; Lasers; Lesion; LoxP-flanked allele; Lymphocyte; Macrophage; Maps; Matrix Metalloproteinases; Measures; Modeling; Mus; Ontology; Operative Surgical Procedures; PTPRC gene; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Patients; Resistance; Role; System; T-Lymphocyte; Tamoxifen; Testing; Therapeutic; Vascular Endothelial Growth Factors; Vision; Wild Type Mouse; angiogenesis; choroidal angiogenesis; complement pathway; cytokine; diphtheria toxin receptor; experimental study; intravitreal injection; monocyte; novel; novel therapeutics; osteopontin; prevent; recruit; scaffold; single-cell RNA sequencing; targeted treatment; transcriptome

PROJECT SUMMARY: Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world, and is treated solely by inhibiting vascular endothelial growth factor (VEGF). Although highly effective, 15% of patients still lose vision despite maximal anti-VEGF therapy. Thus, a critical need exists for novel non-VEGF therapies. The complement pathway is genetically associated with AMD, macrophages express complement proteins and receptors, and macrophages are found in surgically excised choroidal neovascularization (CNV). Additionally, choroidal macrophage depletion results in choroidal atrophy, and both global macrophage depletion and inhibition of classical monocyte-derived macrophages reduce experimental CNV area. Therefore, macrophages are implicated in human CNV, steady state vascular homeostasis, and pathological choroidal angiogenesis. However, macrophages are known to be highly heterogeneous cells that can perform many diverse functions. Therefore, we performed single-cell RNA-sequencing on wildtype and classical monocyte- depleted mice to investigate macrophage origin, heterogeneity, and function during laser-induced CNV. We identified that interleukin-7 receptor (IL7R)-expressing macrophages are likely derived from classical monocytes and display a pro-angiogenic transcriptome. Based upon these data, our central hypothesis is that IL7R+ macrophages are critical pro-angiogenic cells during CNV pathogenesis. To test this hypothesis, we formulated the following specific aims: 1) Demonstrate that IL7R+ macrophages are necessary for CNV. In this aim, we will use Il7rCre x Rosa26GFP mice to fate map IL7R+ macrophages, Il7rCre x Cx3cr1iDTR mice to deplete IL7R+ macrophages, and Cx3cr1CrER x Il7rflox mice to knockout the IL7R. We will test the function of IL7R+ macrophages and the IL7R using the laser-induced CNV model. 2) Determine that IL7R+ macrophages are derived from classical monocytes and are sufficient to stimulate CNV. In this aim, we will use Ccr2CreER x Rosa26GFP mice to fate map classical monocytes and determine if classical monocytes can differentiate into IL7R+ macrophages. Furthermore, we will show that IL7R+ macrophages are sufficient to stimulate angiogenesis by isolating IL7R+ macrophages from mouse eyes after laser injury. We will then test the ability of IL7R+ vs IL7Rneg macrophages to rescue reduced CNV area in Ccr2-/- mice. Completion of these aims will determine that IL7R+ macrophages are necessary for CNV, derived from classical monocytes, and sufficient to stimulate angiogenesis. These data will set the stage for cell-based anti-CNV therapies. Cell-based therapies, targeting IL7R+ macrophages, have the potential to inhibit multiple pro- angiogenic pathways simultaneously, rather than inhibiting VEGF alone, and change the paradigm for nAMD therapy.

项目概要： 新生血管性年龄相关性黄斑变性（nAMD）是发达国家致盲的主要原因， 并且仅通过抑制血管内皮生长因子（VEGF）来治疗。虽然非常有效，但15%的 尽管进行了最大量的抗VEGF治疗，患者仍然丧失视力。因此，迫切需要新的非VEGF抑制剂。 治疗补体途径与AMD遗传相关，巨噬细胞表达补体 蛋白质和受体以及巨噬细胞在手术切除的脉络膜新血管形成（CNV）中被发现。 此外，脉络膜巨噬细胞耗竭导致脉络膜萎缩，并且总体巨噬细胞耗竭和全身性巨噬细胞耗竭均导致脉络膜萎缩。 和抑制经典的单核细胞衍生的巨噬细胞减少实验CNV面积。因此，我们认为， 巨噬细胞参与人CNV、稳态血管稳态和病理性脉络膜血管内皮细胞凋亡。 血管生成然而，已知巨噬细胞是高度异质性的细胞，其可以执行许多功能。 多种功能。因此，我们对野生型和经典单核细胞进行了单细胞RNA测序， 消耗小鼠研究巨噬细胞的起源，异质性，和功能在激光诱导的CNV。我们 发现表达白细胞介素-7受体（IL 7 R）的巨噬细胞可能来源于经典单核细胞， 并显示促血管生成转录组。基于这些数据，我们的中心假设是IL 7 R + 巨噬细胞是CNV发病过程中关键的促血管生成细胞。为了验证这一假设，我们制定了 具体目标如下： 1)证明IL 7 R+巨噬细胞是CNV所必需的。在这个目标中，我们将使用Il 7 rCre x Rosa 26 GFP IL 7 rCre x Cx 3cr 1 iDTR小鼠消耗IL 7 R+巨噬细胞， Cx 3cr 1CrER x Il 7 rflox小鼠以敲除IL 7 R。我们将检测IL 7 R+巨噬细胞的功能和IL 7 R 使用激光诱导的CNV模型。 2)确定IL 7 R+巨噬细胞来源于经典单核细胞，足以刺激CNV。 在这个目标中，我们将使用Ccr 2CreER x Rosa 26 GFP小鼠来对经典单核细胞进行命运图绘制，并确定经典单核细胞是否具有遗传毒性。 单核细胞可以分化成IL 7 R+巨噬细胞。此外，我们将表明IL 7 R+巨噬细胞 足以通过从激光损伤后的小鼠眼睛中分离IL 7 R+巨噬细胞来刺激血管生成。 然后，我们将测试IL 7 R+与IL 7 Rneg巨噬细胞拯救Ccr 2-/-小鼠中减少的CNV面积的能力。 这些目标的完成将确定IL 7 R+巨噬细胞是CNV所必需的，其来源于经典的 单核细胞，并足以刺激血管生成。这些数据将为基于细胞的抗CNV奠定基础 治疗靶向IL 7 R+巨噬细胞的基于细胞的疗法有可能抑制多种促凋亡因子， 同时抑制血管生成途径，而不是单独抑制VEGF，并改变nAMD的范式 疗法