Hedgehog Signaling in the Adventitia

描述（由申请人提供）：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）是胚胎中必不可少的形态因子和生长因子，在成人组织中具有血管生成作用，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，该域局限于动脉外膜，支持常驻Sca1+壁细胞（SMC/周细胞）祖细胞（AdvSca1）。使用补丁(ptc)-lacZ报告小鼠，首先在E15.5-E16.5处检测到外膜Shh信号，从出生后第1天（P1）到P10天最高，并且与沉积在介质和外膜之间的Shh蛋白套筒共定位。在表达来自天然Shh位点的EGFP的小鼠中，AdvSca1细胞是强烈的EGFPpos，而ptc-lacZ则是异质的，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在这些祖细胞的募集或存活中起重要作用。AdvSca1细胞在体内不表达SMC标记物，但却表达SMC分化所需的转录因子如SRF和心肌素，并且在体外很容易分化为SMC样细胞。然而，与SMCs不同的是，AdvSca1细胞在体内表达srf依赖性转录的有效共抑制因子，包括Msx1和KLF4，可能在维持SMC祖细胞在血管壁中的作用中发挥关键作用。我们的总体假设是外膜中的Shh信号是一个新的信号网络的一部分，该信号网络在血管发育、生长和重塑中起着至关重要的作用，部分通过控制常驻的AdvSca1祖细胞。我们提出了一个工作模型，其中Shh信号通过控制有效的SRF依赖性转录共阻遏子来维持AdvSca1祖细胞处于未分化状态。为了验证我们的假设和工作模型：特异性Aim 1将在动脉外膜形成和出生后生长过程中定位Shh信号活动，并确定体内动脉壁中哪些细胞产生Shh并对其做出反应。特异性目标2将采用血管壁重塑的动物模型以及Shh信号的获得和丢失的遗传方法来研究体内外源性Shh信号的功能作用。特异性目的3将确定是否通过srf依赖性转录共抑制因子的表达来维持AdvSca1细胞作为SMC祖细胞，并检查klf4缺乏对体内中膜、外膜和AdvSca1细胞发育的影响。在美国，已知的冠心病（CHD）、动脉瘤和中风的危险因素仅占这些疾病发病率的一半左右。本申请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，并可能导致确定新的药物靶点，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.