Reprogramming of mature smooth muscle cells to vascular progenitor cells

描述（由申请人提供）：血管平滑肌细胞（SMCs）是高度特化的细胞，表达高水平的SMCs特异性蛋白，如平滑肌肌球蛋白重链（SMMHC/Myh11）和平滑肌-肌动蛋白（A/Acta2）1。然而，在病理条件下，SMCs能够经历深刻的表型和功能变化，导致增殖性炎症表型。近年来一个重要的范式转变表明，血管祖细胞（VPCs）存在于出生后血管外膜内的一个特殊的生态位中。内皮细胞表达多种干细胞标记物，包括Sca1和CD34，它们有能力根据特定信号在体外分化成多个谱系，并可能导致体内内膜病变。然而，重要的未解之谜仍然存在，包括它们的起源、多能性和/或异质性的程度，以及它们对血管维持、修复和病理病变形成的贡献。使用一种创新的体内命运定位方法对表达Myh11的成熟SMC进行遗传标记，我们明确地确定成熟的SMCs在电线诱导的损伤中产生了大多数内膜SMCs。出乎意料的是，我们观察到一些成熟的SMCs反向迁移到外膜，没有检测到SM标记的表达，但获得了Sca1和CD34的表达。我们确定了AdvSca1祖细胞的一个独特亚群是通过一个我们称之为“内源性外源性重编程”的过程从成熟的SMCs中产生的，并表明这些细胞在动脉内稳态和疾病中发挥重要作用。我们的初步研究结果表明，smc衍生的AdvSca1细胞存在于外膜中，并有助于形成具有不同生物学特性和命运决定的居住vpc的异质群体。我们的总体假设是，成熟的SMCs在血管发育的后期阶段被重新编程为功能不同的VPCs亚群；重编程至少部分依赖于转录因子Klf4的诱导和肿瘤抑制因子PTEN的缺失。血管损伤后，这些细胞的动员和募集有助于内膜病变的形成和血管修复。我们的数据可能对确定常驻VPCs形成和维持的内源性分子机制产生深远的影响，这可能导致在原位操纵这些细胞以改善在动脉粥样硬化/再狭窄、动脉瘤形成、缺血组织和肿瘤血管生成等情况下的治疗应用。成熟的大血管SMCs，作为常驻VPCs的来源，不仅涉及SMC去分化，但重编程为功能祖表型，尚未被描述。本文提出了三个目标：确定SMC衍生的VPCs对血管损伤的分化潜力和反应（Aim 1）；确定Klf4和PTEN在SMC重编程中的作用并确定SMC重编程的基本内源性途径（Aim 2）；确定SMC衍生的VPCs在血管损伤反应中的体内命运和功能（Aim 3）。

DESCRIPTION (provided by applicant): Vascular smooth muscle cells (SMCs) are highly specialized cells that express high levels of SMC-specific proteins, such as smooth muscle myosin heavy chain (SMMHC/Myh11) and smooth muscle-�ctin (�A/Acta2)1. Under pathological conditions, however, SMCs are capable of undergoing profound phenotypic and functional changes resulting in a proliferative, inflammatory phenotype. An important paradigm shift in recent years suggests that vascular progenitor cells (VPCs) reside in a specialized niche within the adventitia of postnatal vessels. Adventitial VPCs express several stem cell markers, including Sca1 and CD34, have the capacity to differentiate in vitro into multiple lineages in response to specific signals, and potentially contribute to intimal lesions in vivo. However, important unanswered questions remain, including their origin, their degree of pluripotency and/or heterogeneity, and their contribution to vessel maintenance, repair, and pathological lesion formation. Using an innovative in vivo fate-mapping approach to genetically label mature Myh11- expressing SMC, we definitively established that mature SMCs give rise to the majority of intimal SMCs in response to wire-induced injury. Quite unexpectedly, we made the observation that some mature SMCs reverse migrate into the adventitia, exhibit no detectable expression of SM markers, but gain expression of Sca1 and CD34. We established that a distinct subpopulation of AdvSca1 progenitors arise from mature SMCs through a process we are calling "endogenous adventitial reprogramming" and suggest these cells play important roles in arterial homeostasis and disease. Our preliminary findings suggest SMC-derived AdvSca1 cells reside in the adventitia and contribute to a heterogeneic population of resident VPCs that possess distinct biological properties and fate decisions. Our overall hypothesis is that mature SMCs are reprogrammed during the later stages of vascular development to a subpopulation of functionally distinct VPCs; reprogramming is dependent at least in part on induction of the transcription factor, Klf4 and loss of the tumor suppressor, PTEN. After vascular injury, mobilization and recruitment of these cells contributes to intimal lesion formation and vessel repair. Our data could have a profound impact on defining the endogenous molecular mechanisms underlying the formation and maintenance of resident VPCs, which could lead to the potential to manipulate these cells in situ to improve therapeutic applications in settings suc as atherosclerosis/restenosis, aneurysm formation, ischemic tissues, and tumor angiogenesis. Mature macrovessel SMCs, as the source of resident VPCs, involving more than SMC dedifferentiation, but reprogramming to a functional progenitor phenotype, has not been described. Three Aims are proposed to define the differentiation potential and response of SMC- derived VPCs to vascular injury (Aim One), to define the role of Klf4 and PTEN on SMC reprogramming and identify essential endogenous pathways involved in SMC reprogramming (Aim Two), and define the in vivo fate and function of SMC-derived VPCs in response to vascular injury (Aim Three).