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

描述（由申请人提供）：交感神经和感觉血管周围神经（PVNs）、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舒缩张力和根据代谢需求动员血液到身体组织的能力至关重要。这种调节的关键是肌内皮连接（mej），它存在于内部弹性层（IEL）的孔（窗）中，允许ECs和SMCs之间的电信号和化学信号交流。通过释放激活SMCs的神经递质，PVNs在通过mej启动细胞间通讯中发挥重要作用。年龄增长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尚不清楚。大多数关于细胞间通讯的研究都没有纳入PVN的作用，而是关注ec到SMC的通讯（例如，内皮依赖性超极化）以及源自ec的信号如何影响SMC Ca2+。因此，本提案的目的是表征由PVN激活SMCs引起的EC Ca2+信号。在幼龄（3-4个月）和老年（24-26个月）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EC Ca2+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肾上腺素受体的脱敏有关。根据将肾上腺素受体激活与全局EC Ca2+信号联系起来的报道，我的数据导致了整体假设，即由SMC的PVN激活引起的局部EC Ca2+信号随着年龄的增长而改变。为了在体内研究这些功能关系，我开发了一种新的活体准备来研究肠系膜动脉

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.