Impact of aging on calcium and electrical signaling in microvascular endothelium

描述(由申请人提供)：65岁及以上的美国人的数量预计将在未来20年内从目前的~13%(1/8)增加到~20%(1/5)。解决衰老生理学的科学研究努力是了解、治疗和预防心血管疾病发展的有效途径；心血管疾病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流不足有关，对血管内皮功能障碍起着不可或缺的作用。在控制血流进入微循环的阻力动脉中，钙离子与内皮依赖性血管扩张相关的电信号通路的相互作用涉及“内皮源性超极化”(EDH)。功能关系支持超极化的启动[例如，激活小的和中等的钙激活的K+通道(SKCa/IKCa)]和在网络分支的内皮之间传播(通过缝隙连接)，作为协调组织灌流(即，氧气输送)和组织实质细胞的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，但在组织灌注量受损的基础上，与EDH相关的衰老研究很少。因此，该项目的目标是确定衰老过程中线粒体衍生的氧化应激如何与管理血管扩张和功能性充血的内皮细胞钙离子和电信号通路相互作用。我将验证一个中心假设，即线粒体衍生的钙离子和氧化应激的相互作用会改变微血管阻力动脉内皮细胞的电信号。为了研究这些关系，我将使用一种新的完整微血管内皮管的制备方法，在这种方法中，新鲜解剖的小鼠腹部骨骼肌上腹上动脉被处理，以去除平滑肌肉细胞、外膜、血管周围神经和血流。使用从青年(4-6月)、中期(12-14个月)和老年(24-26个月)C57BL/6小鼠分离的完整内皮管(长~3 mm，宽~60？m)，我将采用同步光学测量关键信号事件(例如，细胞内钙和H_2O_2产生)和细胞内记录膜电位(Vm)。目的1将确定氧化应激通过激活(SKCa/IKCa)随年龄增长改变内皮细胞Vm的机制。目的2将确定线粒体在钙缓冲冲击(SKCa/IKCa)中的作用，以促进随年龄增长而发生的超极化。目的3将测定线粒体产生的活性氧物种，并评估其在老年VM中的调控作用。该项目将独一无二地确定线粒体处理钙离子和氧化应激信号在天然完整的微血管内皮细胞中的作用。这个项目的结果将是 为开发逆转内皮功能障碍的治疗策略提供关键的新见解，以促进组织血流并维持衰老期间的生活质量。

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.