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）的药物明确表明内皮素-1 （ET-1）是这一致命疾病的主要参与者。血管紧张素（AngII）通过其AT1受体刺激人肺血管内皮细胞（HLVEC）中ET-1的产生。然后，ET-1通过其在HLVEC中的ETB受体刺激血管舒张、抗增殖的一氧化氮（NO）和前列环素（PGI2）的产生。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，这种自我平衡调节被打破。AT1受体参与PH的初始阶段，传播了许多参与PH病因学的信号。我们对受体信号传导基序的研究结合突变受体转染和信号级联测定，使我们能够控制AT1受体的信号传导。最近，我们可以利用细胞穿透肽来模拟受体基序序列，从而控制（野生型）内源性表达受体的信号传导。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。我们的工作假设是，涉及MAPKs、Akt/PI3K/PTEN系统、小G蛋白RhoA和异三聚体G蛋白的信号级联以不同的组合作用，通过AT1受体产生ET-1，通过人肺血管内皮细胞中的ETB受体产生NO和PGI2，并通过人肺血管平滑肌细胞中的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，我们将能够减少内皮细胞的ET-1，增加一氧化氮和前列环素的产生，减少平滑肌细胞的生长和收缩。具体目标：1)a)我们将通过受体内基序识别和突变构建调控AT1、ETA和ETB受体信号，调控G1q、G1i、RhoA、Akt和MAPKs。b)然后，我们将使用膜透性肽调节人肺内皮细胞内源性表达的AT1和ETB受体和平滑肌细胞中的ETA受体的信号传导。2)利用sa1中改变的受体和肽，我们将调节AngII对ET-1的表达和释放，以及ET-1对HLVEC中NO和前列环素的释放。这些结果将用于控制从ph个体肺移植分离的HLVEC中这些受体的功能。3)使用特异性靶1中开发的改变受体和肽，我们将调节人肺血管平滑肌细胞的收缩和增殖，包括从ph个体获得的细胞。4)平滑肌在培养中的收缩研究将在大鼠的生理实验中进行。公共卫生相关性：肺动脉高压是一种使人衰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信号的肽，旨在找到新的治疗肺动脉高压的方法。

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.