Hedgehog Signaling in the Adventitia

描述(申请人提供)：我们的长期目标是确定控制外膜生长和功能的分子途径。Sonic Hedgehog(Shh)是胚胎中一种重要的形态因子和生长因子，在成人组织中是血管生成的，是组织特异性祖细胞形成和维持所必需的。我们的初步数据确定了一个新的Shh信号域，它局限于动脉外膜，支持常驻Sca1+壁细胞(SMC/周细胞)前体细胞(AdvSca1)。利用Patted(PTC)-LacZ报告基因小鼠，外膜Shh信号在E15.5-E16.5最早检测到，出生后第1天(P1)至P10最高，并与位于中膜和外膜之间的Shh蛋白共定位。在表达天然Shh基因的EGFP的小鼠中，AdvSca1细胞是强烈的EGFPpos，而PTC-LacZ是异质性的，这表明外膜内存在Shh旁分泌信号网络。在Shh-/-小鼠中，AdvSca1细胞的数量大大减少，这表明Shh在这些祖细胞的招募或生存中发挥了重要作用。AdvSca1细胞在体内不表达SMC标志物，但表达SMC分化所需的转录因子，如SRF和myocardin，并在体外容易分化为SMC样细胞。然而，与SMC不同的是，AdvSca1细胞在体内表达强大的SRF依赖转录的协同抑制因子，包括Msx1和KLF4，它们可能在维持SMC前体细胞在血管壁中发挥关键作用。我们的总体假设是，外膜Shh信号是一个新的信号网络的一部分，该网络在血管发育、生长和重塑中发挥重要作用，部分是通过控制常驻的AdvSca1前体细胞。我们提出了一个工作模型，其中Shh信号通过控制强大的SRF依赖的转录共抑制物来维持AdvSca1前体细胞处于未分化状态。为了验证我们的假设和工作模式：特定目标1将定位动脉外膜形成和出生后生长过程中Shh信号的活动，并确定动脉壁中哪些细胞在体内产生Shh并对Shh做出反应。特殊目的2将使用血管壁重建的动物模型以及Shh信号的获得和丢失的遗传方法来研究外膜Shh信号在体内的功能作用。特异靶3将确定AdvSca1细胞是否通过SRF依赖的转录共抑制因子的表达而维持作为SMC前体细胞，并检测KLF4缺乏对体内中膜、外膜和AdvSca1细胞发育的影响。公共卫生相关性：Hedgehog Signals in Exentitia Majesky-NIH RO1应用项目叙述已知的冠心病(CHD)、动脉瘤和中风的风险因素仅占美国这些疾病发病率的一半左右。本申请中提出的研究旨在通过研究动脉壁生长和修复的基本机制来确定冠心病、动脉瘤和中风的新的候选危险因素。这项工作是开发新的CDH风险基因测试的第一步，并可能导致识别新的药物靶点，以更有效地治疗这些疾病。

DESCRIPTION (provided by applicant): Our long-term goal is to identify molecular pathways that control growth and function of the tunica adventitia. Sonic hedgehog (Shh) is an essential morphogen and growth factor in embryos, is angiogenic in adult tissues, and is required for the formation and maintenance of tissue-specific progenitor cells. Our preliminary data identify a novel Shh signaling domain restricted to the arterial adventitia that supports resident Sca1+ mural (SMC/pericyte) progenitor cells (AdvSca1). Using patched (ptc)-lacZ reporter mice, adventitial Shh signaling was first detected at E15.5-E16.5, was highest from postnatal day 1 (P1) to P10, and colocalized with a sleeve of Shh protein deposited between the media and adventitia. In mice expressing EGFP from the native Shh locus, AdvSca1 cells were strongly EGFPpos while heterogeneous for ptc-lacZ revealing a Shh paracrine signaling network within the adventitia. In Shh-/- mice, AdvSca1 cells were greatly reduced in number suggesting important roles for Shh in recruitment or survival of these progenitor cells. AdvSca1 cells do not express SMC markers in vivo, yet they do express transcription factors required for SMC differentiation such as SRF and myocardin, and readily differentiate to SMC-like cells in vitro. Unlike SMCs, however, AdvSca1 cells express potent co-repressors of SRF-dependent transcription in vivo including Msx1 and KLF4 that may play key roles in maintaining SMC progenitors in the vessel wall. Our overall hypothesis is that Shh signaling in the adventitia is part of a novel signaling network that plays essential roles in vascular development, growth and remodeling, in part, via control of resident AdvSca1 progenitor cells. We propose a working model in which Shh signaling maintains AdvSca1 progenitors in an undifferentiated state via control of potent SRF- dependent transcriptional co-repressors. To test our hypothesis and working model: Specific Aim 1 will localize Shh signaling activity during formation and postnatal growth of the arterial adventitia, and determine which cells in the artery wall produce and respond to Shh in vivo. Specific Aim 2 will employ animal models for vascular wall remodeling together with genetic approaches for gain and loss of Shh signaling to examine functional roles of adventitial Shh signaling in vivo. Specific Aim 3 will determine if AdvSca1 cells are maintained as SMC progenitors by expression of SRF-dependent transcriptional co-repressors, and examine effects of KLF4-deficiency on development of the tunica media, the tunica adventitia and AdvSca1 cells in vivo. PUBLIC HEALTH RELEVANCE: Hedgehog Signaling in the Adventitia Majesky- NIH RO1 application Project Narrative The known risk factors for coronary heart disease (CHD), aneurysm and stroke can account for only about half of the incidence of these diseases in the United States. The research proposed in this application seeks to identify new candidate risk factors for CHD, aneurysm and stroke by studying basic mechanisms for artery wall growth and repair. This work is the first step toward development of new genetic tests for CDH risk, and may lead to identification of new drug targets for more effective treatment of these diseases.