Intercellular Coordination of Blood Flow Control

描述(申请人提供)：我们的长期目标集中在定义信号事件，这些信号事件协调微血管中内皮细胞(EC)和平滑肌细胞(SMC)的活动，根据组织代谢需求控制氧气和营养的输送。我们的工作假设是，血流的局部控制反映了构成微血管阻力网络的供血动脉(FA)和小动脉的EC和SMC之间的活动协调。用乙酰胆碱(ACh)刺激，启动沿内皮传播的互补信号，以松弛沿着血管分支的连续的SMC：(1)通过激活钙敏感的K+通道(KCA)的超极化，被称为“快速传导血管扩张”(RCVD；速度和GT；几mm/S)，并由机电耦合介导，由此细胞内的钙([Ca+]i)和SMC(‘肌源性’)的音调随膜电位(Vm)改变；(2)释放一氧化氮的钙波，称为‘慢传导血管扩张’(SCVD；SMC张力变化不依赖于[Ca~(2+)]i。本项目致力于了解SCVD是如何启动、传播并与RCVD相互作用以控制组织血流的。实验使用已建立的具有钙指示剂的仓鼠FA模型进行，并辅之以体内研究，使用在小动脉内皮细胞中表达钙指示剂蛋白(GCaMP2)的转基因小鼠。内皮细胞内钙波背后的钙离子来源(S)未知。目的1通过刺激Ryanodine和1，4，5-三磷酸肌醇(IP3)受体，确定细胞内钙释放是否参与SCVD的发生。为了测试细胞外钙([Ca~(2+)]_o)是否是钙波的组成部分，[Ca~(2+)]_o随着[Ca~(2+)]_o进入内皮细胞而被操纵。在没有肌源性张力的情况下，Ca~(2+)波传播约300~5米，当有肌源性张力时，Ca~(2+)波传播约1 mm。目的2将通过控制跨壁压力和SMC激活来确定为什么EC钙波的有效传播需要肌源性张力。我们将评估SMC音调是否(如果是)影响EC[Ca~(2+)]i，以及EC是否需要[Ca~(2+)]i的阈值水平来传播Ca~(2+)波。当超极化和RCVD被KCA拮抗剂抑制时，Ca~(2+)波的速度减慢到~20.5m/S。目标3将通过评估Vm的变化(使用跨壁压力、肾上腺素能激动剂或通过操纵[K+]o)影响Ca~(2+)波沿EC的传播来确定RCVD与SCVD的相互作用。此外，虽然Kca在ACh刺激的部位被激活，但我们将探索为什么钙波不激活远程位置的Kca。解决钙波的本质以及RCVD和SCVD之间的相互作用(S)将为研究各自的信号通路在糖尿病、高血压和缺血等病理生理条件下如何受到影响提供关键的新见解。我们打算利用这一知识来促进开发治疗血管疾病的新策略，并促进氧气和营养物质向全身组织的输送。与公共健康相关：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的细胞以产生扩张，从而增加血液流量和向全身组织的氧气输送。我们把重点放在最小的动脉(供应)血管上，因为这些血管网络的分支是血流控制的地方。了解血管扩张信号如何在血管网络中产生和协调，为开发治疗与血管并发症和组织灌注受损相关的疾病(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.