Network signature of low-flow endothelial dysfunction

项目概要/摘要 内皮是血管稳态的重要调节器，内皮功能障碍是血管内分泌失调的标志。 心血管疾病寻找新疗法的挑战是找到早期控制点， 转向广泛的病理信号特征并破坏内皮网络。采用新的成像技术， 通过分析方法，我们已经确定了动态Ca 2+信号沿着血管内膜的离散模式， 它是血管功能的基础，并指导主要血管反应的特异性、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）的轮廓定义 扩散），沿内皮网络形成不同的标记沿着。内皮细胞Ca2+事件的复杂谱 (from孤立的短暂瞬变到广泛的多细胞波）由正反馈相互作用引起， 质膜TRP通道（Ca2+进入）和内质网IP3Rs（Ca2+释放）。小 电导Ca2+激活的K+通道（KCa）通过产生Ca2+依赖性的信号传导在该信号传导中起关键作用。 超极化和通过TRP通道放大Ca2+内流（特别是流体剪切应力（FSS）- 激活TRPV4通道）。在外周动脉疾病患者的血流剥夺远端动脉中， 内皮细胞表现出独特的截断Ca2+特征，其特征在于空间限制的小振幅 临时工这种异常的Ca2+分布在低流量颈动脉结扎小鼠模型中出现较早，引起 内皮功能障碍和血管重塑。这些低流量的适应包括渐进的损失， 内皮KCa 2.3通道，并建议合作KCa/TRPV 4作用的早期损失。我们假设 在低FSS条件下TRPV4-KCa2.3信号传导的中断引起进行性的，高度的 限制内皮Ca2+信号，促进内皮功能障碍和血管重塑。 目的1将描述TRPV 4-KCa 2.3信号在动脉生理Ca 2+信号沿着中的作用 内皮细胞我们将进行共聚焦成像（新的高含量分析），并采用内皮细胞- 特异性敲除小鼠（ecKCa2.3-/-和ecTRPV4-/-）以及人外周动脉，以阐明协同作用 差分FSS下的信道影响。目标2将确定低/振荡FSS是否会导致 TRPV4-KCa2.3依赖性内皮Ca2+信号导致内皮功能障碍和血管 重塑我们将采用部分连接小鼠模型来评估TRPV4 - 1的幅度和时间过程。 KCa2.3特异性影响Ca2+信号传导、血管反应性和血管壁增厚。目标3将决定 内皮TRPV4-KCa2.3 Ca2+信号转导的保留是否改善了功能性和 慢性低流量导致的结构性血管变化。我们还将评估是否采取干预措施， Ca2+信号直接减轻低流量病理影响。

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.