Endothelial cell stability after pericyte loss through the Ang-1/Tie2 pathway

项目总结/摘要 内皮细胞是毛细血管的主要组成部分，几乎存在于身体的每个组织中 并且对于有效地将氧气和营养物输送到代谢活性组织是必需的。在毛细血管内， 内皮细胞（EC）的功能部分地由周细胞调节，周细胞是调节内皮细胞的特化血管周围细胞， 血管渗透性、稳定性和重塑。其中一个主要的信号通路负责周细胞- EC相互作用是血管生成素-1（Ang-1）/Tie 2途径。周细胞产生Ang-1，Ang-1与Tie 2结合， 酪氨酸激酶受体通过附近的内皮细胞表达，并促进EC重塑，抑制EC 细胞凋亡，降低EC层渗透性，并促进EC单层的完整性。 最近的研究已经确定周细胞损失或功能障碍是许多疾病进展的关键因素。 癌症、阿尔茨海默病和糖尿病视网膜病变等疾病。尽管许多研究已在体内使用 小鼠模型，以阐明周细胞损失在体内毛细血管功能障碍和疾病进展中的作用 模型通常缺乏所需的实验控制，并且严重依赖于非人类细胞。克服这些 挑战，我们建议分析的作用，周细胞EC相互作用，稳定EC功能，使用人类 细胞在高度受控的体外血管模型中。 使用3D纤维蛋白凝胶，其中包含EC内衬通道和贯穿凝胶主体的周细胞，我们将评估 周细胞如何在宏观和单细胞尺度上改变EC功能。为了模拟周细胞的损失 在糖尿病视网膜病变期间发生，我们将使用单纯疱疹病毒选择性诱导周细胞凋亡， 病毒-1胸苷激酶（HsvTK）系统。通过用“自杀基因”HsvTK转导周细胞， 通过加入药物更昔洛韦选择性诱导周细胞凋亡。我们将通过以下方式评估EC功能： 测量跨EC层的扩散、细胞-细胞连接的表达、基底膜形成，以及 EC存活、发芽和迁移。最后，我们将评估外源性Ang-1恢复稳定的能力， 周细胞丧失后的EC功能。我们假设，周细胞的损失将导致增加的渗透性， EC层，降低细胞-细胞连接的表达，并降低EC存活和增殖。 此外，我们预期外源性Ang-1在低水平表达后将促进稳定的EC表型， 周细胞的损失。 该项目将帮助我们了解周细胞损失在癌症等疾病进展中的作用， 老年痴呆症和糖尿病视网膜病变。使用高度受控的体外模型，我们可以更好地阐明 有助于疾病进展的分子事件，并测试这些疾病的潜在治疗靶点 在一个高度受控的环境中使用人类细胞。

PROJECT SUMMARY/ABSTRACT Endothelial cells are the primary components of capillaries, which are present in nearly every tissue of the body and are essential for efficient delivery of oxygen and nutrients to metabolically active tissues. Within capillaries, the function of endothelial cells (ECs) is regulated in part by pericytes, specialized perivascular cells that regulate vessel permeability, stability, and remodeling. One of the primary signaling pathways responsible for pericyte- EC interactions is the angiopoietin-1 (Ang-1)/ Tie2 pathway. Pericytes produce Ang-1, which binds to the Tie2 tyrosine kinase receptor expressed by nearby endothelial cells and promotes EC remodeling, inhibits EC apoptosis, decreases EC layer permeability, and promotes the integrity of EC monolayers. Recent research has identified pericyte loss or dysfunction as a key contributor to the progression of many diseases such as cancer, Alzheimer’s disease, and diabetic retinopathy. Though many studies have used in vivo mouse models to elucidate the role of pericyte loss in capillary dysfunction and disease progression, in vivo models often lack the desired experimental control and rely heavily on non-human cells. To overcome these challenges, we propose to analyze the role of pericyte-EC interactions in stabilizing EC function using human cells in a highly controlled in vitro vascular model. Using 3D fibrin gels containing EC-lined channels and pericytes throughout the bulk of the gel, we will assess how pericytes alter EC function on both a macroscopic and single-cell scale. Then, to mimic the pericyte loss that occurs during diabetic retinopathy, we will selectively induce apoptosis in pericytes using the herpes simplex virus-1 thymidine kinase (HsvTK) system. By transducing pericytes with the ‘suicide gene’ HsvTK we can selectively induce apoptosis in pericytes by adding the drug Ganciclovir. We will assess EC function by measuring diffusion across the EC layer, expression of cell-cell junctions, basement membrane formation, and EC survival, sprouting, and migration. Finally, we will assess the ability of exogenous Ang-1 to restore stable EC function following pericyte loss. We hypothesize that pericyte loss will induce increased permeability of the EC layer, decrease expression of cell-cell junctions, and decrease EC survival and proliferation. Additionally, we expect that exogenous Ang-1 will encourage a stable EC phenotype following low levels of pericyte loss. This project will help us understand the role of pericyte loss in the progression of diseases such as cancer, Alzheimer’s disease, and diabetic retinopathy. Using a highly controlled in vitro model we can better elucidate the molecular events contributing to disease progression and test potential therapeutic targets for these diseases in a highly controlled environment using human cells.