Hedgehog Signaling in the Adventitia

描述（由申请方提供）：我们的长期目标是确定控制外膜图尼卡生长和功能的分子途径。Sonic hedgehog（Shh）是胚胎中必需的形态发生和生长因子，在成体组织中是血管生成的，并且是组织特异性祖细胞的形成和维持所需的。我们的初步数据确定了一种新的Shh信号结构域限制在动脉外膜，支持居民Sca 1+壁（SMC/周细胞）祖细胞（AdvSca 1）。使用patched（ptc）-lacZ报告小鼠，外膜Shh信号首先在E15.5-E16.5检测到，从出生后第1天（P1）到P10最高，并与沉积在中膜和外膜之间的Shh蛋白套管共定位。在从天然Shh基因座表达EGFP的小鼠中，AdvSca 1细胞是强烈的EGFPpos，而ptc-lacZ的异质性揭示了外膜内的Shh旁分泌信号网络。在Shh-/-小鼠中，AdvSca 1细胞的数量大大减少，这表明Shh在这些祖细胞的招募或存活中起着重要作用。AdvSca 1细胞在体内不表达SMC标志物，但它们确实表达SMC分化所需的转录因子，如SRF和myocardin，并且在体外容易分化为SMC样细胞。然而，与SMC不同，AdvSca 1细胞在体内表达SRF依赖性转录的有效共阻遏物，包括Msx 1和KLF 4，其可能在维持血管壁中的SMC祖细胞中发挥关键作用。我们的总体假设是，外膜中的Shh信号传导是一种新型信号传导网络的一部分，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AdvSca 1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依赖性转录辅阻遏物来维持AdvSca 1祖细胞处于未分化状态。为了测试我们的假设和工作模型：特异性目标1将在动脉外膜的形成和出生后生长期间定位Shh信号传导活性，并确定动脉壁中的哪些细胞在体内产生和响应Shh。具体目标2将采用血管壁重塑的动物模型以及Shh信号的获得和丧失的遗传方法来检查外膜Shh信号在体内的功能作用。具体目标3将确定AdvSca 1细胞是否通过表达SRF依赖性转录辅阻遏物维持为SMC祖细胞，并检查KLF 4缺乏对体内图尼卡中膜、图尼卡外膜和AdvSca 1细胞发育的影响。公共卫生相关性：刺猬信号在外膜Majesky- NIH RO 1应用项目叙述已知的危险因素冠心病（CHD），动脉瘤和中风可以只占约一半的发病率，这些疾病在美国。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识别CHD、动脉瘤和中风的新候选风险因素。这项工作是开发新的CDH风险基因检测的第一步，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.