Network signature of low-flow endothelial dysfunction

项目摘要/摘要 内皮细胞是血管内稳态的重要调节因子，内皮功能障碍是血管内皮细胞损伤的标志。 心血管疾病。寻找新疗法的挑战是找到早期控制点，以防止 转移到广泛的病理信号传递谱并扰乱内皮网络。采用新的成像技术和 分析方法，我们已经确定了沿着血管内膜的动态钙信号的离散模式 这是血管功能的基础，并指导着普遍的血管反应的特异性、敏感性和强度。 这些模式由动态事件参数(频率、幅度、持续时间和空间)的配置文件定义 传播)，沿着内皮网络形成不同的特征。血管内皮细胞钙事件的复合谱 (从孤立的短暂瞬变到宽广的多细胞波)是由 质膜Trp通道(钙进入)和内质网IP3Rs(钙释放)。小的 电导钙激活的钾通道(Kca)通过发挥钙依赖作用，在这一信号转导过程中发挥关键作用 超极化和通过Trp通道(特别是流体切应力(FSS))放大钙内流 激活的TRPV4通道)。在外周动脉疾病患者的血流剥夺的远端动脉中， 内皮细胞表现出独特的截断的钙信号，其特征是空间受限的小幅度 转瞬即逝。这种异常的钙离子分布在低流量颈动脉结扎的小鼠模型中早期出现，导致 内皮功能障碍和血管重塑。这些低流量适应包括渐进性损失 内皮KCa2.3通道，提示早期失去KCA/TRPV4的协同作用。我们假设 低FSS条件下TRPV4-KCa2.3信号的中断会导致进行性的、高度的 限制性内皮细胞钙信号，促进内皮功能障碍和血管重塑。 目标1将表征TRPV4-KCa2.3信号在沿动脉的生理性钙信号中的作用 内皮细胞。我们将进行共聚焦成像(使用新的高含量分析)，并使用内皮- 特异性基因敲除小鼠(ecKCa2.3-/-和ecTRPV4-/-)以及人类外周动脉的合作 不同FSS下的信道影响。目标2将确定低/振荡FSS是否导致截断 TRPV4-KCa2.3依赖的内皮细胞钙信号导致内皮功能障碍和血管 改建。我们将使用部分结扎的小鼠模型来评估TRPV4的大小和时间过程。 KCa2.3-对钙信号、血管反应性和血管壁增厚的特定影响。目标3将决定 保存内皮细胞TRPV4-KCa2.3钙信号是否改善功能性和 慢性低流量引起的结构性血管改变。我们还将评估是否需要采取干预措施来保存 钙离子信号直接减轻低流量的病理影响。

PROJECT SUMMARY/ABSTRACT The endothelium is a crucial regulator of vascular homeostasis and endothelial dysfunction is a hallmark of cardiovascular disease. The challenge in searching for new therapies is finding early control points that prevent the shift to broad pathologic signaling profiles and disrupt the endothelial network. Employing novel imaging and analysis approaches, we have identified discrete patterns of dynamic Ca2+ signalling along the vascular intima that underlie vascular function and direct the specificity, sensitivity and intensity of prevailing vascular responses. These patterns, defined by profiles of dynamic event parameters (frequency, amplitude, duration and spatial spread), form distinct signatures along the endothelial network. The complex spectrum of endothelial Ca2+ events (from isolated brief transients to broad multicellular waves) result from positive feedback interaction between plasma membrane TRP channels (Ca2+ entry) and endoplasmic reticulum IP3Rs (Ca2+ release). Small conductance Ca2+-activated K+ channels (KCa) play a key role in this signaling by exerting Ca2+-dependent hyperpolarization and amplifying Ca2+ influx through TRP channels (particularly fluid shear stress (FSS)- activated TRPV4 channels). In flow-deprived distal arteries from patients with peripheral artery disease, the endothelium exhibits a distinctive truncated Ca2+ signature characterized by spatially restricted small amplitude transients. This anomalous Ca2+ profile appears early in a low-flow carotid ligation mouse model, giving rise to endothelial dysfunction and vascular remodelling. These low-flow adaptations involve progressive loss of endothelial KCa2.3 channels and suggest an early loss of cooperative KCa/TRPV4 action. We hypothesize that disruption of TRPV4-KCa2.3 signaling under conditions of low FSS causes a progressive, highly restricted endothelial Ca2+ signature that promotes endothelial dysfunction and vascular remodeling. Aim 1 will characterize the role of TRPV4-KCa2.3 signaling in physiologic Ca2+ signatures along the arterial endothelium. We will conduct confocal imaging (with novel high-content analysis) and employ endothelium- specific knockout mice (ecKCa2.3-/- and ecTRPV4-/-) as well as human peripheral arteries to elucidate cooperative channel impacts under differential FSS. Aim 2 will determine whether low/oscillatory FSS causes truncation of the TRPV4-KCa2.3-dependent endothelial Ca2+ signature that leads to endothelial dysfunction and vascular remodeling. We will employ a partial ligation mouse model to assess the magnitude and time course of TRPV4- KCa2.3-specific impacts on Ca2+ signaling, vasoreactivity and vascular wall thickening. Aim 3 will determine whether preservation of endothelial TRPV4-KCa2.3 Ca2+ signaling ameliorates development of functional and structural vascular changes resulting from chronic low flow. We will also assess whether interventions to preserve the Ca2+ signature directly abate pathologic impacts of low flow.