Microenvironmental Endogenous Reprogramming of Differentiated Smooth Muscle Cells

描述(申请人提供)：血管平滑肌细胞(SMCs)是高度专业化的细胞，收缩以维持血管张力，处于静止状态，表达高水平的SMC特异性蛋白，如平滑肌肌球蛋白重链(SMMHC)。然而，在病理条件下，SMC能够经历深刻的表型和功能变化，使它们成为病理性新生内膜形成的主要贡献者。近年来的一个重要的范式转变表明，具有分化为SMC和内皮细胞的能力的血管前体细胞(VPC)位于出生后血管外膜内的一个专门的生态位中。虽然新的数据支持外膜VPC的存在，但重要的未解答的问题仍然存在，包括它们的来源及其对血管维护、修复和病理损伤形成的贡献。利用一种创新的体内命运定位方法，我们意外地观察到成熟的SMC迁移到外膜，下调SM标志物的表达，并获得祖细胞标志物的表达。我们的初步发现表明，SMC来源的Sca1(+)细胞群可以从表达功能性前体细胞表型并具有分化为SMC的能力的成年血管中分离出来。有一小部分Sca1保留表达，表明有自我更新的潜能。我们的发现表明，从重新编程的成熟SMC中出现了常驻VPC。我们的总体假设是，成熟的SMC在血管发育的后期迁移到外膜，并根据仅在外膜表达的特定信号(例如微环境重新编程)重新编程为VPC；重新编程依赖于转录因子KLF4的诱导。血管损伤后，这些细胞的募集有助于新生内膜的形成。这一发现可能对确定VPC形成和维持的分子机制产生深远影响，并可能导致我们对体细胞重新编程的理解取得进展，以改善临床治疗应用。将成熟的中小企业作为居民VPC的来源是一种反教条主义的理论，它将挑战现有的概念。我们提出了两个目标来确定SMC来源的AdvSca1细胞在正常血管和血管损伤中的重要性和作用。在第一个目标中，我们将鉴定和定义成熟的SMC对外膜VPC池的贡献，我们将定义SMC来源的VPC的体外功能特性，我们将确定KLF4在体外和体内对SMC重编程的作用。对于第二个目标，我们将确定SMC来源的VPC在钢丝诱导的血管损伤反应中的命运。

DESCRIPTION (provided by applicant): Vascular smooth muscle cells (SMCs) are highly specialized cells that contract to maintain vascular tone, are quiescent and express high levels of SMC-specific proteins, such as smooth muscle myosin heavy chain (SMMHC). Under pathological conditions, however SMCs are capable of undergoing profound phenotypic and functional changes making them major contributors to pathological neointima formation. An important paradigm shift in recent years suggests that vascular progenitor cells (VPCs) with the capacity to differentiate into SMCs and endothelial cells reside in a specialized niche within the adventitia of postnatal vessels. While emerging data support the existence of adventitial VPCs, important unanswered questions remain, including their origin and their contribution to vessel maintenance, repair, and pathological lesion formation. Using an innovative in vivo fate-mapping approach, we made the unanticipated observation that mature SMCs migrate into the adventitia, downregulate SM marker expression, and gain expression of progenitor cell markers. Our preliminary findings demonstrate that a population of SMC-derived Sca1(+) cells can be isolated from adult vessels that express a functional progenitor cell phenotype and exhibit the capacity to differentiate into SMCs. A small percentage retained expression of Sca1, indicating the potential for self-renewal. Our findings suggest the emergence of resident VPCs from reprogrammed mature SMCs. Our overall hypothesis is that mature SMCs migrate to the adventitia during the later stages of vascular development and are reprogrammed to VPCs in response to specific signals expressed exclusively in the adventitia (e.g. microenvironmental reprogramming); reprogramming is dependent on induction on the transcription factor, Klf4. After vascular injury, recruitment of these cells contributes to neointima formation. Such a finding could have a profound impact on defining the molecular mechanisms underlying the formation and maintenance of VPCs and could lead to advances in our understanding of somatic cell reprogramming to improve clinical therapeutic applications. Mature SMCs as the source of resident VPCs is an anti-dogmatic theory that would challenge existing concepts. Two Aims are proposed to establish the importance and the role of SMC-derived AdvSca1 cells in normal vessels and in the setting of vascular injury. In Aim One, we will characterize and define the contribution of mature SMC to the adventitial VPC pool, we will define the in vitro functional characteristics of SMC-derived VPCs, and we will define the in vitro and in vivo role of Klf4 on SMC reprogramming. For Aim Two, we will determine the fate of SMC-derived VPCs in response to wire-induced vascular injury.