Hypoxia and cardiac stem cell homing

 描述（由申请人提供）：干细胞治疗正在成为治疗晚期心脏病患者的有益策略。然而，移植的干细胞在心肌中的有限归巢和存活代表了治疗功效的障碍。在最初的资助期内，我们专注于低氧预处理（HP），通过以HIF- 1α依赖的方式激活CXC趋化因子受体4（CXCR 4）来增强心脏干细胞的归巢和存活，从而改善年轻小鼠细胞移植后的结果。不幸的是，老化的C-MSC对HP的反应很差，这表明需要研究在老化细胞中调节HP的分子机制来优化这种新的治疗策略。HIF-1 α相关的组织修复损伤与新血管形成减少有关，这是一个受HIF-1α依赖性信号调节的过程。我们的初步数据表明，老化的sca-1+心脏间充质干细胞（C-MSC）的染色质修饰可能是受损的HIF-1α反应和损害其功能的基础。具体而言，衰老细胞在HIF-1α靶基因CXCR 4和IL-10的启动子处表现出显著更高的组蛋白3赖氨酸27三甲基化（H3 K27 me 3，一种抑制性染色质修饰），这是干/祖细胞归巢和存活的关键调节因子。此外，衰老细胞表现出组蛋白甲基转移酶增强子zeste同源物2（EZH 2）的表达增加，EZH 2是多梳阻遏复合物2（PRC 2）的催化组分。EZH 2是唯一已知的能够在哺乳动物细胞中诱导H3 K27 me 3的酶。EZH 2还可以与组蛋白脱乙酰酶（HDAC）合作，其通过从组蛋白去除特定乙酰基来抑制转录。我们假设在老化的C-MSC中，EZH 2的异常表达通过H3 k27 me 3诱导的基因“沉默”并通过与HDAC 11相互作用导致关键HIF-1α依赖性基因的抑制，从而损害HP反应并损害治疗效果。我们提出了三个目标来检验我们的假设。在目标1中，我们将确定EZH 2在C-MSC中受转录因子E2 F1调节的机制，并且我们将靶向老年C-MSC中的E2 F1，以确定它是否与EZH 2表达和功能的变化机制相关。在目的2中，我们将进行遗传和生化实验，以确定HDAC 11是否作为多蛋白EZH 2-PRC 2复合物的一部分，与IL-10启动子相关，并抑制HIF-1α诱导的IL-10基因在老年C-MSC中的表达。在目的3中，我们将消融老化C-MSC中的EZH 2以研究对细胞移植的功能应答（即，细胞归巢、血管生成、心脏修复）。这将是第一个深入研究表观遗传重塑在调节衰老干细胞功能中的作用的研究。我们的研究结果可能会提高心脏干细胞治疗的疗效，并确定新的分子靶点，可以改善老年人的心脏修复。

 DESCRIPTION (provided by applicant): Stem cell therapy is emerging as a beneficial strategy to treat patients with advanced heart disease. However, limited homing and survival of transplanted stem cells in the myocardium represents a barrier to therapeutic efficacy. During the initial term of funding, we focused on hypoxia preconditioning (HP) as a means to enhance homing and survival of cardiac stem cells by activating CXC chemokine receptor-4 (CXCR4) in HIF- 1α-dependent manner, which led to improved outcomes following cell transplantation in young mice. Unfortunately, aged C-MSC respond poorly to HP, suggesting that research into the molecular mechanisms that regulate HP in aged cells is needed to optimize this novel therapeutic strategy. Age-related impairments in tissue repair are associated with decreased neovascularization, a process that is regulated by HIF-1α- dependent signaling. Our preliminary data suggest that chromatin modifications in aged sca-1+ cardiac mesenchymal stem cells (C-MSC) may underlie impaired HIF-1α responses and compromise their function. Specifically, aged cells exhibit significantly greater histone 3 lysine 27 trimethylation (H3K27me3, a repressive chromatin modification) at the promoters of the HIF-1α target genes CXCR4 and IL-10, which are crucial regulators of stem/progenitor cell homing and survival. Moreover, aged cells exhibit increased expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb Repressor Complex 2 (PRC2). EZH2 is the only known enzyme capable of inducing H3K27me3 in mammalian cells. EZH2 may also cooperate with histone deacetylases (HDAC), which repress transcription by removing specific acetyl groups from histones. We hypothesize that in aged C-MSC, aberrant expression of EZH2 leads to repression of key HIF-1α-dependent genes through H3k27me3-induced gene "silencing" and by interacting with HDAC11, thus impairing HP responses and compromising therapeutic efficacy. We propose three aims to test our hypothesis. In Aim 1, we will determine the mechanisms whereby EZH2 is regulated by the transcription factor E2F1 in C-MSC, and we will target E2F1 in aged C-MSC to determine whether it is mechanistically linked to changes in EZH2 expression and function. In Aim 2, we will carry out genetic and biochemical experiments to determine whether HDAC11 functions as a part of the multi-protein EZH2-PRC2 complex that associates with the IL-10 promoter and represses HIF-1α-induced IL-10 gene expression in aged C-MSC. In Aim 3, we will ablate EZH2 in aged C-MSC to investigate functional responses to cell transplantation (i.e., cell homing, angiogenesis, cardiac repair). This will be the first i-depth study to investigate the role of epigenetic remodeling in regulating the function of aged stem cells. Our findings may lead to enhanced efficacy of cardiac stem cell therapy and identify new molecular targets that could improve cardiac repair in the elderly.