ANP Modulates Cardiac Remodeling via Cardiac Fibroblasts

描述（由申请人提供）：心房利钠肽（ANP）是胎儿基因程序的一个众所周知的组成部分，在应激条件下在成人心脏中过表达。然而，很少有人关注ANP在应激/肥厚心脏中的功能。本申请将测试以下假设：ANP具有直接的抗生长和抗纤维化作用，其通过中断其信号级联而对抗转化生长因子（TGF）-β和血管紧张素II（ANGII）的促纤维化作用，已知转化生长因子（TGF）-β和血管紧张素II在应激条件下在心脏中过表达。我们已经表明，与非转基因（NTG）对照组相比，ANP基因纯合破坏（Nppa-/-）小鼠表现出基线心脏扩大和夸大的心脏重塑/纤维化，以及早期过渡到失败，响应于横向主动脉缩窄（TAC）诱导的压力超负荷应激。初步研究显示，在TAG处理的Nppa-/-小鼠心脏中，肌成纤维细胞转化和细胞外基质（ECM）分子表达显著增加。我们还证明了TAC诱导的心脏重塑和纤维化在表达TGF-β受体II型基因的诱导型显性负突变（DriTGF β RI 1）的小鼠中被消除，因此不响应TGF-β信号传导。最近，我们已经取得了令人兴奋的初步观察，ANP信号抑制TGF-β诱导的磷酸化SmadS在小鼠心脏成纤维细胞（CFs）的核转位，第一次定义了一个精确的分子机制，ANP信号可以防止心脏重塑/纤维化和失败的血流动力学应激。1）建立Nppa-/-、DnTGFbRII和对照NTG小鼠心脏在静息条件下和在TAG后压力超负荷应激期间的各个阶段（急性应激、纤维化和重构的早期代偿阶段和晚期失代偿/向衰竭的过渡阶段）的表型。这一目标将提供第一个严格的体内测试的作用，TGF-β信号转导介导压力超负荷诱导的LV纤维化/重塑和心钠素在调节这些过程。2)使用分离的CF作为体外模型，以确定导致促纤维化/生长因子刺激的CF增殖/转化和ECM表达的细胞内信号传导机制。这个目的将提供一个严格的测试的假设，ANP信号具有负面的，和TGF-β（和/或ANGII）信号具有积极的刺激作用，这些过程。ANP和/或其信号传导途径的组分将用于定义ANP级联阻断促纤维化/生长因子信号传导的特定位点，从而抑制CF的表型转化和ECM产生。这些机制研究将为心功能不全/衰竭患者的治疗干预提供更合理的依据，其中许多患者的ANP水平降低。

DESCRIPTION (provided by applicant): Atrial natriuretic peptide (ANP) is a well known component of the fetal gene program that is overexpressed in adult heart under stress conditions. Yet little attention has been devoted to the function of ANP in the stressed/hypertrophic heart. The current application will test the hypothesis that ANP has direct anti-growth and anti-fibrogenic effects that oppose the pro-fibrogenic actions of transforming growth factor (TGF)-beta and angiotensin II (ANGII), which are known to be overexpressed in heart under stress conditions, by interrupting their signaling cascades. We have shown that, compared to nontransgenic (NTG) controls, mice with homozygous disruption of the ANP gene (Nppa-/-) exhibit cardiac enlargement at baseline and exaggerated cardiac remodeling/fibrosis, as well as an early transition to failure, in response to transverse aortic constriction (TAC)-induced pressure overload stress. Preliminary studies revealed remarkable increases in myofibroblast transformation and expression of extracellular matrix (ECM) molecules in hearts of Nppa-/- mice subjected to TAG. We have also demonstrated that TAC-induced cardiac remodeling and fibrosis are abolished in mice that express an inducible dominant negative mutation of the TGF-beta receptor type II gene (DriTGFbRIl), and thus do not respond to TGF-beta signaling. Most recently, we have made the exciting preliminary observation that ANP signaling inhibits TGF-beta-induced nuclear translocation of phosphorylated-SmadS in mouse cardiac fibroblasts (CFs), defining for the first time a precise molecular mechanism by which ANP signaling may protect against cardiac remodeling/fibrosis and failure in response to hemodynamic stress. The Aims of this proposal are: 1) To establish the phenotypes of Nppa-/-, DnTGFbRII and control NTG mouse hearts under resting conditions and at various phases (acute stress, early compensatory stage of fibrosis and remodeling, and late decompensate/transition stage to failure) during pressure overload stress following TAG. This Aim will provide the first rigorous in vivo test of the role of TGF-beta signaling in mediating pressure overload-induced LV fibrosis/remodeling and of ANP in modulating these processes. 2) Using isolated CFs as an in vitro model, to define the intracellular signaling mechanisms leading to pro-fibrogenic/growth factor-stimulated CF proliferation/transformation and ECM expression. This Aim will provide a rigorous test of the hypothesis that ANP signaling has negative, and TGF-beta (and/or ANGII) signaling has positive stimulatory effects on these processes. ANP and/or components of its signaling pathway will be used to define the specific site(s) at which the ANP cascade intercepts pro-fibrogenic/growth factor signaling, thus inhibiting phenotypic transformation of CFs and ECM production. These mechanistic studies will provide a more rational basis for therapeutic intervention in patients with cardiac dysfunction/failure, many of whom have decreased levels of ANP.