Reprogramming of mature smooth muscle cells to vascular progenitor cells

描述(申请人提供)：血管平滑肌细胞(SMC)是高度特化的细胞，表达高水平的SMC特异性蛋白，如平滑肌肌球蛋白重链(SMMHC/MYH11)和Smooth Muscle-�ctin(�A/Acta2)1。然而，在病理条件下，SMC能够经历深刻的表型和功能变化，导致增殖和炎症表型。近年来一个重要的范式转变表明，血管前体细胞(VPC)位于出生后血管外膜内的一个专门的小生境中。外膜VPC表达多种干细胞标记物，包括Sca1和CD34，具有在体外根据特定信号分化为多个谱系的能力，并可能在体内导致内膜损伤。然而，重要的问题仍未得到解答，包括它们的起源，它们的多能性和/或异质性的程度，以及它们在血管维持、修复和病理损伤形成中的作用。使用一种创新的体内命运图谱方法对表达MYH11的成熟SMC进行基因标记，我们明确地证实，成熟的SMC在线状损伤后能够产生大多数的内膜SMC。出乎意料的是，我们观察到一些成熟的SMC反向迁移到外膜，没有检测到SM标记的表达，但有Sca1和CD34的表达。我们发现，一组不同的AdvSca1前体细胞通过我们称之为“内源性外膜重编程”的过程从成熟的SMC分化而来，并提示这些细胞在动脉内稳态和疾病中发挥重要作用。我们的初步发现表明，SMC来源的AdvSca1细胞存在于外膜，并有助于形成具有不同生物学特性和命运决定的异质性常驻VPC群体。我们的总体假设是，成熟的SMC在血管发育的后期被重新编程为功能不同的VPC亚群；重新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的丢失。血管损伤后，这些细胞的动员和募集有助于内膜损伤的形成和血管修复。我们的数据可能对确定驻留VPC形成和维持的内源性分子机制产生深远影响，这可能导致在原位操纵这些细胞的潜力，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环境中的治疗应用。成熟的大血管SMC作为常驻VPC的来源，不仅涉及SMC的去分化，而且重新编程为功能性的祖细胞表型，尚未被描述。我们提出了三个目标，以确定SMC来源的VPC对血管损伤的分化潜能和反应(目标一)，确定KLF4和PTEN在SMC重编程中的作用，并确定参与SMC重编程的重要内源性途径(目标二)，以及确定SMC来源的VPC在血管损伤反应中的体内命运和功能(目标三)。

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).