Endothelial biomechanics in vascular aging

内皮生物力学在多种内皮功能中起着关键作用。我们早期的研究发现， 体外氧化脂质和体内血脂异常通过CD 36清除剂诱导显著的内皮硬化 受体和氧固醇的掺入。最近，我们发现在老化的动脉瘤中， 主要依赖于CD 36和小窝蛋白Caveolin-1（Cav 1）。我们的长期目标是阐明 负责年龄诱导的内皮生物力学变化的机制，并确定 这些机制对内皮功能障碍的贡献。在目前的提案中，我们提出了三个目标： 在目标1中，我们专注于阐明年龄诱导的EC硬化的机制。第一步（1A），确定 内皮特异性CD 36和/或Cav 1缺失是否可预防中度老年人的内皮硬化 （10-12月龄）和高龄（20-24月龄）小鼠以及是否表达/膜 这些蛋白质的定位随年龄而改变。然后（1B），我们将提供全面的脂质组学分析 随着年龄的增长，动脉组织中脂质成分的变化，并确定特定的脂质种类， 并诱导内皮硬化。在目标2中，我们专注于年龄相关的肌动蛋白重塑和破坏， 内皮屏障完整性。首先（目的2A），我们将确定内皮CD 36和Cav 1的作用， 在年龄相关的肌动蛋白重塑、连接形态学中，CD 36/Cav 1介导的氧化脂质摄取， 内皮对大分子的渗透性和单核细胞的浸润。在目标的第二部分（2B）中， 我们将研究氧化脂质，特别是氧化固醇，诱导肌动蛋白的分子机制。 重塑，侧重于一种新的假设，即氧固醇与RhoA竞争结合RhoA抑制剂。 蛋白质，GDI-1。在目标3中，这些研究被扩展以研究CD 36/Cav 1/氧化固醇- 依赖性内皮硬化诱导核形态畸变、DNA损伤和核 机械敏感性转录因子（3A）的易位，并探讨氧化甾醇诱导的 内皮硬化通过破坏称为肌动蛋白帽的核周结构而导致核变形 （3B）。然后，这些研究扩展到探索性子目标（3C），即比较分析 在体内老化内皮细胞和暴露于氧化脂质的内皮细胞中， 诱导内皮硬化。这些目标是通过内皮特异性功能丧失（CD 36和CD 38）来实现的。 Cav 1）和功能获得（Cav 1）遗传小鼠模型。结合原子力显微镜，脂质 质谱分析、通过机器学习算法分析的高分辨率共焦成像以及其他状态- 最先进的实验方法。总的来说，这些研究预计将提供重要的新的 深入了解衰老血管系统中内皮硬化的机制 以及脂质诱导的内皮硬化在年龄相关屏障破坏和细胞核异常中的作用。

Endothelial biomechanics plays a key role in multiple endothelial functions. Our earlier studies discovered that oxidized lipids in vitro and dyslipidemia in vivo induce significant endothelial stiffening via CD36 scavenger receptor and incorporation of oxysterols. Most recently, we found that endothelial stiffening in aging aortas critically depends on CD36 and the caveolar protein, Caveolin-1 (Cav1). Our long term goal is to elucidate the mechanisms responsible for age-induced changes in endothelial biomechanics and to determine the contribution of these mechanisms to endothelial dysfunction. In the current proposal, we address three goals: In Aim 1, we focus on elucidating the mechanism of age-induced EC stiffening. First (1A), we will determine whether endothelial-specific deletions of CD36 and/or Cav1 prevent endothelial stiffening in moderately aged (10-12 months old) and advanced aged (20-24 months old) mice and whether expression/membrane localization of these proteins is altered by age. Then (1B), we will provide a comprehensive lipidomics analysis of changes in lipid composition in arterial tissues with age and identify specific lipid species that accumulate with age and induce endothelial stiffening. In Aim 2, we focus on age-related actin remodeling and disruption of endothelial barrier integrity. First (aim 2A), we will determine the roles of endothelial CD36 and Cav1 and CD36/Cav1-mediated uptake of oxidized lipids in age-related actin remodeling, junctional morphology, endothelial permeability to macromolecules, and infiltration of monocytes. In the second part of the aim (2B), we will investigate the molecular mechanisms by which oxidized lipids, particularly oxysterols, induce actin remodeling, focusing on a novel hypothesis that oxysterols compete with RhoA for binding to a RhoA inhibitory protein, GDI-1. In Aim 3, these studies are extended to investigate the impact of CD36/Cav1/oxysterol- dependent endothelial stiffening in inducing distortion of nuclear morphology, DNA damage and nuclear translocation of mechanosensitive transcription factors (3A) and explore the hypothesis that oxysterol-induced endothelial stiffening results in nuclei distortion by disruption of a peri-nuclear structure called the actin cap (3B). These studies are then extended to an exploratory sub-aim (3C) of comparative analysis of transcriptomic changes in aged endothelium in vivo and in endothelial cells exposed to oxidized lipids that induce endothelial stiffening. These goals are achieved using endothelial-specific loss of function (CD36 and Cav1) and gain of function (Cav1) genetic mouse models. A combination of Atomic Force Microscopy, lipid mass-spectrometry, high resolution confocal imaging analyzed by machine-learning algorithms and other state- of-the-art experimental approaches. Taken together, these studies are expected to provide significant new insights into our understanding of the mechanisms responsible for endothelial stiffening in aging vasculature and the role of lipid-induced endothelial stiffening in age-related barrier disruption and nuclei abnormalities.