The Role of Age-Related Matrix Stiffening on Endothelial Cell Dysfunction and Res

描述(由申请人提供)：这个探索性项目的目标是调查新的假设，即随着衰老和动脉粥样硬化进展而增加的血管僵硬，有助于内皮细胞功能障碍。众所周知，在活体内，老年和动脉粥样硬化血管的内膜和中层往往比年轻的健康血管的顺应性差。此外，已有研究表明，动脉弹性是70岁以上、高血压和糖尿病患者心血管死亡率的独立预测因子。虽然血管的宏观力学特性已被很好地描述，但关于增加的基质硬度如何影响血管壁内的细胞，我们知之甚少。具体地说，目前尚不清楚血管壁僵硬是否能在细胞水平上积极促进疾病的进展。我们将制造经过调整的工程化基质，以模拟健康和动脉粥样硬化血管的硬度，以研究内皮细胞对基质硬度的特异性反应。该项目围绕三个具体目标进行组织。目的1：探讨基质弹性对血管内皮细胞形态改变、细胞骨架重排、整合素黏附和通透性的影响。为此，我们将研究不同硬度底物上内皮细胞的重排和形状、细胞骨架重排、整合素激活和单层通透性，以验证底物力学变化改变内皮细胞形态、局部黏附形成和连接完整性的假设。目的：研究正常和紊乱血流条件下基质弹性对ICAM-1和E-选择素表达、定位及单核细胞黏附的影响。为了达到这个目的，我们将检验这样一个假设，即更坚硬的基质，模拟动脉粥样硬化的血管，将增强内皮细胞对炎性细胞因子TNF-a的反应，导致ICAM-1和E-选择素的表达增加，单核细胞黏附增加，通透性增加。目的：研究小鼠体内内皮细胞连接结构和细胞定位与年龄和血管硬度的关系。在这个目标中，我们将使用双光子显微镜，这是一种最先进的成像方式，可以成像到组织深处几毫米处，以可视化完整动脉中内皮细胞连接蛋白和内皮细胞形态随年龄变化的3D定位。由于双光子显微镜将激发限制在飞升焦点体积内，并且能够在不解剖组织的情况下穿透到组织中，因此我们将能够精确地成像连接结构，而不会受到平面外荧光的干扰，并且只需对血管进行最少的操作。这一目标将首次提供完整动脉内皮随年龄变化的3D重建。此外，它还将在活体模型中验证我们在AIMS 1和2中获得的结果。总之，这项工作将阐明内皮细胞基底膜硬度的变化如何影响内皮细胞的功能，目的是确定新的治疗靶点，以防止细胞对血管僵硬的反应。 公共卫生相关性：在衰老和动脉粥样硬化进展过程中，血管僵硬。虽然这种僵硬在器官水平上引起了人们的强烈兴趣，因为它导致了心脏负荷的增加，但在细胞水平上增加的壁僵硬的影响在很大程度上被忽略了。这项建议将研究血管僵硬增加对血管内皮细胞的影响，内皮细胞是动脉粥样硬化发展的第一介质，以确定防止细胞行为异常和动脉粥样硬化进展的治疗方法。

DESCRIPTION (provided by applicant): The goal of this exploratory project is to investigate the novel hypothesis that increased blood vessel stiffness, which accompanies aging and atherosclerosis progression, contribute to endothelial cell dysfunction. It is now well-known that in vivo, the intimal and medial layers of aged and atherosclerotic vessels tend to be less compliant than younger, healthy vessels. Moreover, it has been shown that arterial elasticity is an independent predictor of cardiovascular mortality in patients over 70 years of age, and those with hypertension and diabetes mellitus. While the macro-mechanical properties of the vessel are well characterized, little is known about how increased matrix stiffness affects the cells within the vessel wall. Specifically, it is unknown whether vessel wall stiffness could actively promote disease progression at the cellular level. We will fabricate engineered substrates that are tuned to mimic the stiffness of healthy and atherosclerotic vessels to investigate endothelial cell-specific responses to matrix stiffness. This project is organized around three specific aims. Aim 1: Determine the effects of matrix elasticity on flow-mediated endothelial cell morphological changes, cytoskeletal rearrangement, integrin adhesion and permeability. In this aim, we will investigate endothelial cell realignment and shape, cytoskeletal rearrangement, integrin activation and monolayer permeability on varying stiffness substrates to test the hypothesis that changes in substrate mechanics alters endothelial cell morphology, focal adhesion formation, and junctional integrity. Aim 2: Investigate the effect of matrix elasticity on ICAM-1 and E-selectin expression and localization and monocyte adhesion in normal and disturbed flow conditions. In this aim, we will test the hypothesis that stiffer matrices, mimicking those of atherosclerotic vessels, will augment the response of endothelial cells to the inflammatory cytokine, TNF-a, causing increased expression of ICAM-1 and E-selectin, increased monocyte adhesion, and increased permeability. Aim 3: To investigate endothelial cell junctional structure and cell orientation in vivo in mice as a function of age and vessel stiffness. In this aim, we will use two-photon microscopy, a state-of-the-art imaging modality that can image several millimeters deep into tissue, to visualize the 3D localization of endothelial cell junctional proteins and endothelial cell morphology in intact arteries as a function of age. Because two-photon microscopy restricts excitation to a femtoliter focal volume and has the ability to penetrate into tissues without dissection of the tissue, we will be able to precisely image junctional structures without the confounding effects of out-of- plane fluorescence and with only minimal manipulation of the vessel. This aim will provide the first 3D reconstructions of the endothelium of intact arteries as a function of age. Moreover, it will validate the results we obtain in Aims 1 and 2 in an in vivo model. Together, this work will elucidate how changes in the stiffness of the basement membrane of the endothelium affects endothelial cell function, with the goal of identifying novel therapeutic targets to prevent the cells' response to vessel stiffness. PUBLIC HEALTH RELEVANCE: During aging and atherosclerosis progression, blood vessels stiffen. While this stiffening has been of intense interest at the organ level, because it results in increased load on the heart, the effect of increased wall stiffness at the cellular level has largely been ignored. This proposal will investigate the effects of increased vessel stiffness on the endothelium, the first mediator in the development of atherosclerosis, in order to identify treatments to prevent aberrant cell behavior and the progression of atherosclerosis.