Aging and neurovascular regulation of endothelial cell calcium signals, in vivo

描述(申请人提供)：交感神经和感觉血管周围神经(PVN)、平滑肌细胞(SMCs)和内皮细胞(ECs)之间的细胞间通讯对于调节血管运动张力和根据代谢需求将血液动员到身体组织的能力是必不可少的。肌内皮细胞连接(MEJ)对这一调节至关重要，它存在于内弹力板(IEL)的孔洞(Fenestrae)处，允许ECs和SMC之间的电信号和化学信号的交流。通过释放激活SMC的神经递质，PVN在通过MEJ启动细胞间通讯方面发挥着重要作用。增龄与内皮功能障碍和运动能力受损有关，但信号机制的潜在变化仍不清楚。大多数关于细胞间通讯的研究没有包括PVN的作用，而是集中在内皮细胞到SMC的通讯(例如，内皮依赖的超极化)以及起源于内皮细胞的信号如何影响SMC钙离子。因此，本研究的目的是研究室旁核激活的血管内皮细胞内钙信号的特征。在年轻(3-4个月)和老年(24-26个月)的小鼠中，本项目的重点是刺激交感神经和感觉神经如何通过MEJ影响局部的EC钙信号。我的初步研究表明，衰老与感觉神经抑制交感血管收缩的能力下降以及1-肾上腺素受体的脱敏有关。根据将肾上腺素受体激活与全球内皮细胞钙信号联系起来的报道，我的数据导致了总体假设，即来自SMC室旁核激活的局部内皮细胞钙信号随年龄而改变。为了在体内研究这些功能关系，我开发了一种新的活体制剂来研究肠系膜动脉 (MA)在麻醉转基因GCaMP2中。这些动物是在室内饲养的，并表达EC特定的钙指示剂(GCaMP2)，使得能够在体内对局部EC钙离子进行高速共聚焦成像，而不需要外部染料或担心其潜在的人工制品。在选择性抑制感觉或交感神经传递之前和期间，使用该模型刺激PVN将达到以下特定目的：(1)确定PVN激活如何在血流控制过程中调节EC钙信号；(2)确定衰老对在体EC Ca~(2+)信号和血管运动功能的影响。这项研究的完成将对EC钙信号在年轻和衰老血管系统血流控制中的作用提供有价值的新见解，可能作为治疗年龄相关性内皮功能障碍的新治疗策略的基础。

DESCRIPTION (provided by applicant): Intercellular communication between sympathetic and sensory perivascular nerves (PVNs), smooth muscle cells (SMCs) and endothelial cells (ECs) is essential to the regulation of vasomotor tone and the ability to mobilize blood to body tissues in accord with metabolic demand. Critical to this regulation are myoendothelial junctions (MEJs), which exist at holes (fenestrae) in the internal elastic lamina (IEL) and allow communication of electrical and chemical signals between ECs and SMCs. By releasing neurotransmitters that activate SMCs, PVNs play an important role in the initiating cell-cell communication through MEJs. Advancing age is linked to endothelial dysfunction and impaired exercise capacity, but the underlying changes in signaling mechanisms are still unclear. The majority of studies on intercellular communication do not incorporate the role of PVN, instead focusing on EC-to-SMC communication (e.g., endothelium-dependent hyperpolarization) and how signals originating in ECs affect SMC Ca2+. Therefore, the purpose of this proposal is to characterize EC Ca2+ signals arising from PVN activation of SMCs. In Young (3-4 month) and Old (24-26 month) mice, this project is focused on how stimulation of sympathetic and sensory nerves affects local EC Ca2+ signaling via MEJs. My preliminary studies show aging to be associated with a decrease in the ability of sensory nerves to inhibit sympathetic vasoconstriction along with desensitization of ¿1-adrenoreceptors. In light of reports linking adrenoreceptor activation to global EC Ca2+ signaling, my data led to the overall hypothesis that local EC Ca2+ signals arising from PVN activation of SMC are altered with aging. To investigate these functional relationships in vivo, I have developed a novel intravital preparation to study mesenteric arteries (MA) in anesthetized transgenic GCaMP2. These animals are bred in house and express an EC-specific Ca2+ indicator (GCaMP2), enabling high speed confocal imaging of local EC Ca2+ in vivo without the need for external dyes or concern for their potential artifacts. Using this model to stimulate PVNs before and during selective inhibition of sensory or sympathetic neurotransmission will address the following Specific Aims: (1) Determine how PVN activation modulates EC Ca2+ signals during blood flow control; and (2) Determine the impact of aging on EC Ca2+ signals and vasomotor function in vivo. Completion of the proposed studies will provide valuable new insight with respect to the role of EC Ca2+ signaling during blood flow control in the young and aging vasculature that may serve as the basis for novel therapeutic strategies for treating age-related endothelial dysfunction.