Mechanisms of Heterocellular Signaling at the Myoendothelial Junction

描述(申请人提供)：我们最近发现，血红蛋白在肌内皮细胞连接中丰富，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。这是一项重要的发现，因为它证明了血红蛋白在红细胞外具有重要和活跃的作用。这种蛋白质是为数不多的几种真正极化的蛋白质之一，定位于内皮细胞来源的肌内皮连接，而siRNA诱导的蛋白质数量的减少显著改变了动脉的反应性，包括对苯肾上腺素的收缩和对乙酰胆碱的扩张。我们推导出的机制是基于证据表明，单体血红蛋白是一种强大的一氧化氮清除剂，内皮型一氧化氮合酶(ENOS)和血红蛋白被发现是在一个大分子复合体中。基于这项工作，以及大量强有力的初步数据，我们假设肌内皮细胞连接处的血红蛋白是一种新型的一氧化氮信号调节因子，可以影响血压调节。我们将使用两个具体目标来检验这一假设：1)研究血管内皮细胞血红蛋白a基因去除/过表达对动脉功能的影响。阐明AHSP和eNOS如何调节MEJ的血红蛋白表达和双加氧酶活性。为了阐明这些目的，我们将首先对一只小鼠和一只过表达的小鼠进行研究，以确定该蛋白在内皮细胞中的缺失/过表达对动脉反应性、全组织血流、外周阻力和血压的影响。此外，一个人类阿尔法地中海贫血模型，其中两个血红蛋白a等位基因缺失，将被用来研究这种全基因组杂合性缺失对血管系统的影响。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细胞连接，或者直接作为血红蛋白氧化还原状态的调节器，改变一氧化氮的结合能力。这项建议的总和统一并建立在我们从之前的R01获得的DAT上，允许我们第一次提出关于完整动物的肌内皮连接功能的直接问题。事实上，我们相信，部分答案可以为对外周血管系统控制血压的全新理解提供基础，以及推导出控制高血压的未知药理靶点。

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.