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

项目摘要/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一个组织中。 对于有效地将氧气和营养物质输送到新陈代谢活跃的组织是必不可少的。在毛细血管内， 内皮细胞(ECs)的功能部分由周细胞调节，周细胞是一种特殊的血管周围细胞，负责调节 血管通透性、稳定性和重塑。负责周细胞的主要信号通路之一- EC相互作用是血管生成素-1(Ang-1)/Tie2途径。周细胞产生Ang-1，与Tie2结合 邻近内皮细胞表达酪氨酸激酶受体促进内皮细胞重塑，抑制内皮细胞 细胞凋亡，降低EC层通透性，促进EC单层的完整性。 最近的研究发现，周细胞丢失或功能障碍是许多 癌症、阿尔茨海默病和糖尿病视网膜病变等疾病。尽管许多研究已经在体内使用了 小鼠模型阐明周细胞丢失在体内毛细血管功能障碍和疾病进展中的作用 模型往往缺乏所需的实验控制，严重依赖非人类细胞。要克服这些障碍 挑战，我们建议使用人类来分析周细胞-EC相互作用在稳定EC功能中的作用 细胞处于高度可控的体外血管模型中。 使用3D纤维蛋白凝胶，在整个凝胶中包含EC衬里的通道和周细胞，我们将评估 周细胞如何在宏观和单细胞尺度上改变EC的功能。然后，为了模拟周细胞的丢失 在糖尿病视网膜病变期间，我们将使用单纯疱疹病毒选择性地诱导周细胞凋亡。 病毒1型胸苷激酶(HSVtk)系统。通过转导带有自杀基因HSVtk的周细胞，我们可以 通过加入药物更昔洛韦选择性地诱导周细胞凋亡。我们将通过以下方式评估EC的功能 测量EC层的扩散，细胞-细胞连接的表达，基底膜的形成，以及 EC的生存、萌发和迁移。最后，我们将评估外源Ang-1恢复稳定的能力 周细胞丢失后内皮细胞功能恢复。我们假设周细胞丢失会导致血管通透性增加。 EC层，减少细胞间连接的表达，减少EC的存活和增殖。 此外，我们预计外源性Ang-1将在低水平后促进稳定的EC表型 周细胞丢失的症状。 这个项目将帮助我们了解周细胞丢失在癌症等疾病进展中的作用， 阿尔茨海默氏症和糖尿病视网膜病变。使用高度可控的体外模型，我们可以更好地阐明 疾病进展的分子事件和测试这些疾病的潜在治疗靶点 在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.