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）的药物选择清楚地表明，内皮素-1（ET-1）是这种致命疾病的主要参与者。血管紧张素Ⅱ（AngII）通过其AT 1受体刺激人肺血管内皮细胞（HLVEC）产生ET-1。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（PGI 2）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，这种稳态调节被打破。AT 1受体，这是参与PH的初始阶段，传播许多参与PH的病因学的信号。我们的研究结合突变体受体转染和信号级联测定受体信号基序使我们能够控制AT 1受体的信号。最近，我们现在可以使用构建的模拟受体基序序列的细胞穿透肽来控制（野生型）内源性表达受体的信号传导。我们的目的是在人肺血管内皮细胞和平滑肌细胞中实现ETA和ETB受体的信号调控，进而调控这些信号转导。我们的工作假设是，涉及MAPKs，Akt/PI 3 K/PTEN系统，小G蛋白RhoA和异源三聚体G蛋白以各种组合产生ET-1，1通过AT 1受体产生NO和PGI 2，并通过ETB受体产生收缩和增殖。人肺血管平滑肌细胞中的ETA/ETB受体;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;并且通过改变这些信号，我们将能够减少ET-1并增加内皮细胞产生的NO和前列环素，并减少平滑肌细胞的生长和收缩。具体目标：1）a）通过对AT 1、ETA和ETB受体的识别和突变，调控G1 q、G1 i、RhoA、Akt和MAPK等信号通路。B）然后我们将使用膜渗透性肽调节人肺内皮细胞中内源性表达的AT 1和ET B受体以及平滑肌细胞中的ETA受体中的信号传导。2)利用SA 1中的受体和肽，我们将通过AngII调节ET-1的表达和释放，并通过ET-1调节HLVEC中NO和前列环素的释放。然后，这些结果将用于控制从患有PH的个体的肺移植物分离的HLVEC中这些受体的功能。3）通过使用改变的受体和在特异性目的1中开发的肽，我们将调节人肺血管平滑肌细胞的收缩和增殖，包括得自PH个体的细胞。4）在培养物中对平滑肌收缩的研究之后将进行使用大鼠的生理学实验。

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.