Intercellular Coordination of Blood Flow Control

描述（由申请人提供）：我们的长期目标集中于定义信号传导事件，该信号传导事件协调微血管中内皮细胞（EC）和平滑肌细胞（SMC）的活性，微血管控制氧气和营养物质的输送，雅阁组织代谢需求。我们的工作假设是，局部控制血流反映了EC和SMC的供血动脉（FA）和小动脉，其中包括微血管阻力网络之间的活动协调。用乙酰胆碱（ACh）刺激启动互补信号，该互补信号沿内皮沿着传播以使连续的SMC沿着血管分支松弛：（1）通过激活Ca 2+敏感性K+通道（KCa）的超极化，称为“快速传导血管舒张”。（RCVD;速度>几mm/s），并通过机电耦合介导，从而细胞内Ca 2+（[Ca 2 +]i）和SMC（2）释放一氧化氮的Ca 2+波，称为“慢传导传导血管舒张”（SCVD;速度，~110 5 m/s），并通过药物力学偶联介导（即，Ca 2+敏化），由此SMC张力变化独立于[Ca 2 +]i。该项目的重点是了解SCVD如何启动，传播和与RCVD相互作用以控制组织血流。实验进行使用一个建立的模型，仓鼠FA与钙离子指示剂在体外补充体内研究，使用转基因小鼠表达的钙离子指示蛋白（GCaMP 2）在小动脉EC。内皮细胞Ca 2+波的Ca 2+来源尚不清楚。目的1将通过在钙库耗竭前后刺激兰尼碱和1，4，5-三磷酸肌醇（IP 3）受体来确定钙库的释放是否是SCVD的组成部分。为了测试细胞外Ca 2+（[Ca 2 +]o）是否是Ca 2+波的组成部分，沿着[Ca 2 +]o进入EC对其进行操纵。在无肌源性张力的情况下，Ca 2+波传播< 300 5 m，但当存在张力时传播约1 mm。目的2将确定为什么肌源性张力需要通过操纵跨壁压和SMC激活有效传播EC Ca 2+波。我们将评估SMC张力是否（如果是，如何）影响EC [Ca 2 +]i，以及EC传播Ca 2+波是否需要[Ca 2 +]i的阈值水平。当KCa拮抗剂抑制超极化和RCVD时，Ca ~（2+）波速度减慢至~ 205 m/s。目的3将通过评估Vm的变化（使用跨壁压、肾上腺素能激动剂或通过操纵[K+]o）如何影响Ca 2+波沿着EC的传播，确定RCVD如何与SCVD相互作用。此外，尽管KCa在ACh刺激的位点被激活，我们将探讨为什么Ca 2+波在远程位点不激活KCa。解决Ca 2+波的性质以及RCVD和SCVD之间的相互作用将为考虑在糖尿病，高血压和缺血等病理生理条件下各自的信号通路可能受到影响提供关键的新见解。我们打算利用这些知识来促进治疗血管疾病的新策略的发展，并促进氧气和营养物质向全身组织的输送。公共卫生相关性：该研究项目的目标是了解电信号和化学信号如何协调血管壁细胞以产生扩张，从而增加全身组织的血流和氧气输送。我们专注于最小的动脉（供应）血管，因为这些血管网络的分支是血流控制的部位。了解血管扩张信号如何产生并在血管网络中协调，为开发用于治疗与血管并发症和受损组织灌注相关的疾病（例如糖尿病和高血压）的新疗法提供了新的见解。

DESCRIPTION (provided by applicant): Our long-term goals center on defining the signaling events which coordinate the activity of endothelial cells (EC) and smooth muscle cells (SMC) in microvessels that control the delivery of oxygen and nutrients in accord with tissue metabolic demand. Our working hypothesis is that the local control of blood flow reflects the coordination of activity among EC and SMC of the feed arteries (FA) and arterioles which comprise microvascular resistance networks. Stimulating with acetylcholine (ACh) initiates complementary signals that propagate along the endothelium to relax consecutive SMC along vessel branches: (1) Hyperpolarization via activation of Ca2+-sensitive K+ channels (KCa), referred to as `rapid-conducted vasodilation' (RCVD; velocity > several mm/s) and mediated by electromechanical coupling, whereby intracellular Ca2+ ([Ca2+]i) and SMC (`myogenic') tone change with membrane potential (Vm); (2) A Ca2+ wave that releases nitric oxide, referred to as `slow-conducted conducted vasodilation' (SCVD; velocity, ~110 5m/s) and mediated through pharmacomechanical coupling (i.e., Ca2+ sensitization), whereby SMC tone changes independent of [Ca2+]i. This project is focused on understanding how SCVD is initiated, propagated and interacts with RCVD to control tissue blood flow. Experiments are performed using an established model of hamster FA with Ca2+ indicators in vitro complemented by in vivo studies using transgenic mice expressing a Ca2+ indicator protein (GCaMP2) in arteriolar EC. The source(s) of Ca2+ underlying endothelial Ca2+ waves is unknown. Aim 1 will determine whether release of Ca2+ from internal stores is integral to SCVD by stimulating ryanodine and inositol 1,4,5- trisphosphate (IP3) receptors before and after store depletion. To test whether extracellular Ca2+ ([Ca2+]o) is integral to Ca2+ waves, [Ca2+]o is manipulated along with its entry into EC. In the absence of myogenic tone, Ca2+ waves travel < 300 5m but propagate for ~1 mm when tone is present. Aim 2 will determine why myogenic tone is required for effective propagation of EC Ca2+ waves by manipulating transmural pressure and SMC activation. We will evaluate whether (and if so, how) SMC tone affects EC [Ca2+]i and whether a threshold level of [Ca2+]i is required for EC to propagate Ca2+ waves. When hyperpolarization and RCVD are inhibited by KCa antagonists, the velocity of Ca2+ waves slows to ~20 5m/s. Aim 3 will determine how RCVD interacts with SCVD by evaluating how changing Vm (with transmural pressure, adrenergic agonists, or by manipulating [K+]o) affects the propagation of Ca2+ waves along EC. Further, whereas KCa are activated at the site of ACh stimulation, we will explore why Ca2+ waves do not activate KCa at remote sites. Resolving the nature of Ca2+ waves and the interaction(s) between RCVD and SCVD will provide critical new insight for considering how respective signaling pathways may be affected during such pathophysiological conditions as diabetes, hypertension, and ischemia. We intend for this knowledge to facilitate the development of novel strategies for treating vascular disease and promoting the delivery of oxygen and nutrients to tissues throughout the body. PUBLIC HEALTH RELEVANCE: The goal of this research project is to understand how electrical and chemical signals coordinate cells of the blood vessel wall to produce dilation and thereby increase blood flow and oxygen delivery to tissues throughout the body. We focus on the smallest of arterial (supply) vessels because these branches of the vascular network are the site of blood flow control. Understanding how vasodilator signals originate and are coordinated in vascular networks provides new insight for developing novel therapies for treating diseases associated with vascular complications and impaired tissue perfusion, e.g. diabetes and hypertension.