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

描述（由申请人提供）：本探索性项目的目的是研究新的假设，即伴随衰老和动脉粥样硬化进展的血管硬度增加会导致内皮细胞功能障碍。现在众所周知，在体内，老化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顺应性差。此外，已经表明动脉弹性是70岁以上患者以及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性能得到了很好的表征，但关于增加的基质刚度如何影响血管壁内的细胞却知之甚少。具体而言，尚不清楚血管壁硬度是否可以在细胞水平上积极促进疾病进展。我们将制造工程基板，调整模拟健康和动脉粥样硬化血管的硬度，研究内皮细胞特异性反应基质硬度。该项目围绕三个具体目标展开。目标1：确定基质弹性对流动介导的内皮细胞形态学变化、细胞骨架重排、整合素粘附和通透性的影响。在这个目标中，我们将研究内皮细胞的重新排列和形状，细胞骨架重排，整合素激活和单层渗透性不同刚度的基板上测试的假设，即基板力学的变化改变内皮细胞形态，粘着斑的形成，和交界处的完整性。目标二：研究基质弹性对正常和干扰血流条件下ICAM-1和E-选择素表达和定位以及单核细胞粘附的影响。在这个目标中，我们将测试的假设，更硬的基质，模仿动脉粥样硬化血管，将增加内皮细胞的炎症细胞因子，TNF-α的反应，导致ICAM-1和E-选择素的表达增加，单核细胞粘附增加，并增加渗透性。目的3：研究小鼠体内内皮细胞连接结构和细胞取向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，一个国家的最先进的成像模式，可以成像几毫米深的组织，可视化的3D定位的内皮细胞连接蛋白和内皮细胞形态在完整的动脉作为年龄的函数。由于双光子显微镜限制激发到一个femtoliter的焦点体积，并有能力渗透到组织没有解剖的组织，我们将能够精确地成像交界处的结构，没有混淆的影响面外荧光和只有最小的操纵的血管。这一目标将提供完整动脉内皮的第一个3D重建作为年龄的函数。此外，它将在体内模型中验证我们在目标1和2中获得的结果。总之，这项工作将阐明内皮基底膜硬度的变化如何影响内皮细胞功能，目的是确定新的治疗靶点，以防止细胞对血管硬度的反应。 公共卫生相关性：在衰老和动脉粥样硬化进展过程中，血管收缩。虽然这种硬化在器官水平上引起了强烈的兴趣，因为它导致心脏负荷增加，但在细胞水平上增加的壁硬度的影响在很大程度上被忽略了。该提案将研究血管硬度增加对内皮细胞的影响，内皮细胞是动脉粥样硬化发展中的第一个介质，以确定预防异常细胞行为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.