Impact of aging on calcium and electrical signaling in microvascular endothelium

描述（由申请人提供）：在未来20年里，65岁及以上的美国人数量预计将从目前的约13%（八分之一）增加到约20%（五分之一）。研究衰老生理是了解、治疗和预防心血管疾病发展的有效途径；是美国公民的头号杀手，也是人类生活质量下降的罪魁祸首。衰老与重要组织和器官的灌注不足有关，在血管内皮功能障碍中起着不可或缺的作用。在控制血液流入微循环的阻力动脉中，Ca2+和内皮依赖性血管舒张背后的电信号通路的相互作用涉及“内皮源性超极化”（EDH）。功能关系支持启动[例如，激活小钙激活和中等钙激活的K+通道（SKCa/IKCa）]和超极化（通过间隙连接）沿网络分支的内皮和在内皮之间的传播，作为协调组织灌注（即氧输送）与组织实质细胞代谢需求的高效机制。虽然衰老与氧化应激（例如，线粒体产生过氧化氢）有关，但在组织灌注受损的内皮功能障碍背景下，缺乏与EDH相关的衰老研究。因此，该项目的目标是确定衰老过程中线粒体来源的氧化应激如何与内皮细胞Ca2+和控制血管舒张和功能性充血的电信号途径相互作用。我将检验线粒体来源的Ca2+和氧化应激的相互作用改变微血管阻力动脉内皮中的电信号的中心假设。为了研究这些关系，我将采用一种全新的制备完整的微血管内皮管，通过处理刚解剖的小鼠腹部骨骼肌的腹壁上动脉，去除平滑肌细胞、外膜、血管周围神经和血流。使用完整的内皮管(长度：~ 3mm，宽度：~60 ？m)从幼年（4-6个月），中期（12-14个月）和老年（24- 26个月）C57BL/6小鼠中分离，我将采用同步光学测量关键信号事件（例如，细胞内Ca2+和H2O2的产生）和细胞内膜电位（Vm）的记录。目的1将确定随着年龄的增长，氧化应激通过激活（SKCa/IKCa）改变内皮细胞Vm的机制。目的2将确定线粒体在Ca2+缓冲中的作用（SKCa/IKCa），以影响随着年龄的增长而产生的超极化。目的3将确定线粒体活性氧的产生，并评估其在老年Vm中的作用。该项目将独特地确定线粒体在原生完整微血管内皮中处理Ca2+和氧化应激信号的作用。这个项目的结果将

DESCRIPTION (provided by applicant): The number of Americans 65 years and older is expected to increase to ~20% (1 in 5) from the current ~13% (1 in 8) over the next two decades. Scientific research efforts for resolving aging physiology constitute an effective approach towards understanding, treating and preventing the development of cardiovascular disease; the number one killer of American citizens and culprit for diminishing quality of human life. Aging is associated with under-perfusion of vital tissues and organs with an integral role for vascular endothelial dysfunction. In resistance arteries that control blood flow into the microcirculation, the interaction of Ca2+ and electrical signaling pathways underlying endothelium-dependent vasodilation involves "endothelium-derived hyperpolarization" (EDH). Functional relationships support the initiation [e.g., activation of small- and intermediate-calcium-activated K+ channels (SKCa/IKCa)] and spread (via gap junctions) of hyperpolarization along and among the endothelium of network branches as a highly effective mechanism for coordinating tissue perfusion (i.e., oxygen delivery) with the metabolic demand of tissue parenchymal cells. Whereas aging is associated with oxidative stress (e.g., hydrogen peroxide production by mitochondria), there is a paucity of aging research concerned with EDH in the context of endothelial dysfunction underlying impaired tissue perfusion. Therefore, the goal of this project is to determine how mitochondrial-derived oxidative stress during aging interacts with endothelial cell Ca2+ and electrical signaling pathways that govern vasodilation and functional hyperemia. I will test the central hypothesis that the interaction of mitochondrial-derived Ca2+ and oxidative stress alter electrical signaling in the endothelium of microvascular resistance arteries. To investigate these relationships, I will employ a novel preparation of intact microvascular endothelial tubes, whereby freshly-dissected superior epigastric arteries of mouse abdominal skeletal muscle are treated to remove smooth muscle cells, adventitia, perivascular nerves and blood flow. Using intact endothelial tubes (length: ~3 mm, width: ~60 ?m) isolated from of Young (4-6 month), Intermediate (12-14 month), and Old (24- 26 month) C57BL/6 mice, I will employ simultaneous optical measurements of key signaling events (e.g., intracellular Ca2+ and H2O2 production) with intracellular recordings of membrane potential (Vm). Aim 1 will determine the mechanism by which oxidative stress alters endothelial Vm via activation of (SKCa/IKCa) with advancing age. Aim 2 will determine the role of mitochondria in Ca2+ buffering to impact (SKCa/IKCa) for ensuing hyperpolarization with advancing age. Aim 3 will determine mitochondrial production of reactive oxygen species and evaluate its role governing Vm in old age. This project will uniquely determine the role of mitochondrial handling of Ca2+ and oxidative stress signals in native intact microvascular endothelium. Results from this project will provide critical new insight towards developing therapeutic strategies for reversing endothelial dysfunction to promote tissue blood flow and sustain the quality of life during aging.