Endothelin and Angiotensin Receptor Signaling in Pulmonary Hypertension

描述(由申请人提供)：用于治疗肺动脉高压(PH)的首选药物清楚地表明，内皮素-1(ET-1)是这一致命疾病的主要参与者。血管紧张素(AngII)通过AT1受体刺激人肺血管内皮细胞(HLVEC)产生ET-1。ET-1通过其ETB受体刺激HLVEC产生血管扩张、抗增殖的一氧化氮(NO)和前列环素(PGI2)。在人肺血管平滑肌细胞(HLVSMC)中，ET-1刺激细胞增殖和血管收缩。在PH中，这种动态平衡调节被打破。AT1受体参与了PH的初始阶段，传递了许多参与PH病因学的信号。我们对受体信号基序的研究，结合突变受体的转染和信号级联检测，使我们能够控制AT1受体的信号传递。最近，我们现在可以利用构建的细胞穿透肽来模拟受体基序序列(S)，通过(野生型)内源性表达的受体来控制信号传递。我们打算在ETA和ETB受体中实现信号控制，然后在人肺血管内皮细胞和平滑肌细胞中调节这些信号转导。预计这种方法将导致对PH的新治疗。我们的工作假设是，涉及MAPK、Akt/PI3K/PTEN系统、小G蛋白RhoA和异源三聚体G蛋白的信号级联在不同的组合中发挥作用，通过AT1受体产生ET-1，通过ETB受体在人肺血管内皮细胞产生NO和PGI2，在人肺血管平滑肌细胞通过ETA/ETB受体产生收缩和增殖；这些信号相互作用可以通过使用改变的受体和细胞穿透肽来控制；通过改变这些信号，我们将能够减少ET-1，增加内皮细胞产生NO和前列环素，减少平滑肌细胞的生长和收缩。具体目标：1)a)我们将通过识别受体内的基序和构建突变结构来调节AT1、ETA和ETB受体信号转导，从而调节G1q、G1i、RhoA、Akt和MAPKs。B)然后，我们将利用膜通透性多肽调节内源性表达的人肺内皮细胞AT1和ETB受体以及血管内皮细胞ETA受体的信号。2)利用受体的改变和SA-1产生的多肽，调节血管紧张素Ⅱ对ET-1的表达和释放，以及ET-1对血管内皮细胞释放NO和前列环素的影响。这些结果将被用来控制从PH患者的肺移植中分离出的HLVEC中这些受体的功能。3)利用改变的受体和针对特定目的1开发的多肽，我们将调节人肺血管平滑肌细胞的收缩和增殖，包括从PH患者获得的细胞。4)对培养的平滑肌收缩的研究将以大鼠的生理实验为基础。公共卫生相关性：肺动脉高压是一种使人虚弱且大多致命的疾病。我们的实验，包括产生干扰血管紧张素和内皮素负面信号的多肽，旨在引导新的方法，这些方法应该被证明对治疗肺动脉高压有益。

DESCRIPTION (provided by applicant):Drugs of choice used in the treatment of pulmonary hypertension (PH) clearly show that endothelin-1 (ET-1) is a major participant in this fatal disease. Angiotensin (AngII) through its AT1 receptor stimulates the production of ET-1 in human lung vascular endothelial cells (HLVEC). ET-1 then stimulates the production of vasodilating, antiproliferative nitric oxide (NO) and prostacyclin (PGI2) through its ETB receptor in the HLVEC. In human lung vascular smooth muscle cells (HLVSMC) ET-1 stimulates cell proliferation and vasoconstriction. In PH this homeostatic regulation breaks down. The AT1 receptor, which is involved at the initial stage of PH, propagates many of the signals participating in the etiology of PH. Our studies on receptor signaling motifs combined with mutant receptor transfection and signal cascade determinations have enabled us to control the signaling of the AT1 receptor. More recently we can now control signaling by the (wild type) endogenously expressed receptors with the use of cell penetrating peptides constructed to mimic the receptor motifs sequence(s). We intend to achieve signal control in the ETA and ETB receptors and then regulate these signal transductions in human lung vascular endothelial and smooth muscle cells. It is anticipated that this approach will lead to new treatments of PH. Our working hypothesis is that signal cascades involving MAPKs, the Akt/PI3K/PTEN system, small G-protein RhoA and heterotrimeric G- proteins work in various combinations to produce ET-1 through the AT1 receptor and to produce NO and PGI2 through the ETB receptor in human lung vascular endothelial cells and to produce contraction and proliferation through the ETA/ETB receptors in human lung vascular smooth muscle cells; that these signal interactions can be controlled through the use of altered receptors and cell penetrating peptides; and that by altering these signals, we will be able to decrease ET-1 and increase NO and prostacyclin production by endothelial cells and decrease growth and contraction in smooth muscle cells. Specific aims, 1) a) We will regulate AT1, ETA and ETB receptor signaling through motif identification and mutation construction within the receptor to regulate G1q, G1i, RhoA, Akt and MAPKs. b) We will then regulate signaling in endogenously expressed AT1 and ETB receptors in human lung endothelial cells and ETA receptors in smooth muscle cells with use of membrane permeable peptides. 2) With use of the altered receptors and the peptides developed in SA 1, we will regulate the expression and release of ET-1 by AngII and the release of NO and prostacyclin in the HLVEC by ET-1. These results will then be used to control function of these receptors in HLVEC isolated from lung transplants of individuals with PH. 3) With use of the altered receptors and the peptides developed in Specific Aim 1, we will regulate contraction and proliferation in human lung vascular smooth muscle cells, including cells obtained from individuals with PH. 4) The studies on smooth muscle contraction in culture will be followed up with physiological experiments using rats. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension is a debilitating and mostly fatal disease. Our experiments, including the production of peptides which interfere with negative signals of angiotensin and endothelin, are designed to lead to new approaches which should prove beneficial in the treatment of pulmonary hypertension.