Mechanisms of Heterocellular Signaling at the Myoendothelial Junction

描述（由申请人提供）：我们最近发现血红蛋白在肌内皮连接处富集，这是内皮细胞和平滑肌细胞在阻力动脉中接触的解剖位置。这是一个重要的发现，因为它表明血红蛋白在红细胞外具有重要和活跃的作用。该蛋白是少数真正极化的蛋白之一，定位于内皮来源的肌内皮连接处，sirna诱导的蛋白量减少显著改变了动脉反应性，包括对苯肾上腺素的收缩和对乙酰胆碱的扩张。我们得出的机制是基于有证据表明，单体血红蛋白是一种有效的一氧化氮清除剂，内皮一氧化氮合酶（eNOS）和血红蛋白在一个大分子复合物中被发现。基于这项工作，以及大量强有力的初步数据，我们假设肌内皮连接处的血红蛋白是一氧化氮信号的一种新的调节剂，可以影响血压调节。我们将通过两个具体目标来验证这一假设：1.)研究内皮血红蛋白a基因消融/过表达对动脉功能的影响；2.)阐明AHSP和eNOS如何调节MEJ的血红蛋白表达和双加氧酶活性。这些目标将通过研究来阐明，研究首先集中在固定血红蛋白小鼠和血红蛋白过表达小鼠上，以确定内皮中该蛋白的缺失/过表达对动脉反应性、全组织血流量、外周阻力和血压的影响。此外，α地中海贫血的2个血红蛋白a等位基因缺失的人类模型将用于研究这种全基因组杂合缺失对脉管系统的影响。接下来，我们将研究血红蛋白伴侣血红蛋白稳定蛋白（AHSP）如何将血红蛋白运输到肌内皮连接处，或直接作为血红蛋白氧化还原状态的调节剂，改变一氧化氮结合的能力。该建议的总和统一并建立在我们之前的R01中获得的数据上，允许我们第一次提出关于完整动物中肌内皮连接功能的直接问题。事实上，我们相信部分答案可以为外周血管控制血压的全新理解提供基础，并衍生出未开发的控制高血压的药理学靶点。

DESCRIPTION (provided by applicant): We have recently discovered that hemoglobin ¿ is enriched in myoendothelial junctions, the anatomical location where endothelial cells and smooth muscle cells make contact in the resistance arteries. This was a significant finding because it demonstrated that hemoglobin ¿ had an important and active role outside of erythrocytes. This protein is one of only a few truly polarized proteins to be localized to endothelial-derived myoendothelial junctions, and the siRNA-induced decrease in the amount of the protein significantly altered arterial reactivity, including constriction to phenylephrine and dilation to acetylcholine. The mechanism we derived was based on evidence indicating that monomeric hemoglobin ¿ is a potent scavenger of nitric oxide, and that endothelial nitric oxide synthase (eNOS) and hemoglobin ¿ were found to be in a macromolecular complex. Based on this work, as well as a plethora of strong preliminary data, we hypothesize that hemoglobin ¿ at the myoendothelial junction is a novel regulator of nitric oxide signaling which can impact blood pressure regulation. We will test this hypothesis using two specific aims: 1.) investigate the effects of endothelial hemoglobin a gene ablation/over-expression on arterial function and 2.) elucidate how AHSP and eNOS regulate hemoglobin ¿ expression and dioxygenase activity at the MEJ. These aims will be elucidated using studies focused first on a floxed hemoglobin ¿ mouse as well as a hemoglobin a over-expressing mouse to determine the effects of deletion/over-expression of this protein in endothelium on arterial reactivity, whole tissue blood flow, peripheral resistance and blood pressure. In addition, a human model of the disease alpha thalassemia where 2 alleles of hemoglobin a are deleted will be used to study the effects of this genome-wide heterozygous deletion on the vasculature. Next we will investigate how the hemoglobin ¿ chaperone hemoglobin ¿ stabilizing protein (AHSP) may traffic hemoglobin ¿ to the myoendothelial junction or act directly as a regulator of the hemoglobin a redox state, altering the ability of nitric oxide to bind. The sum of this proposal unites and builds on the dat obtained from our previous R01 by allowing us for the first time to ask the direct question as to the function of the myoendothelial junction in intact animals. Indeed, we believe part of the answers could provide the basis for a completely new understanding of blood pressure control by the peripheral vasculature, as well as the derivation of unexplored pharmacological targets for control of hypertension.