Hypoxia and cardiac stem cell homing

 描述(由申请人提供)：干细胞疗法正在成为治疗晚期心脏病患者的一种有益的策略。然而，移植干细胞在心肌中有限的归巢和存活是治疗效果的障碍。在最初的资助期间，我们专注于低氧预适应(HP)作为一种手段，通过以HIF-1α依赖的方式激活CXC趋化因子受体-4(CXCR4)来增强心脏干细胞的归巢和存活，从而改善小鼠细胞移植后的结果。不幸的是，老年C-MSC对Hp的反应很差，这表明需要研究调节老年细胞Hp的分子机制来优化这一新的治疗策略。组织修复中与年龄相关的损伤与新生血管减少有关，这一过程受HIF-1α依赖的信号调节。我们的初步数据表明，老年SCA-1+心脏间充质干细胞中染色质的修饰可能是导致HIF-1α反应受损并损害其功能的基础。具体地说，老年细胞在HIF-1α靶基因CXCR4和IL-10的启动子上表现出显著更高的组蛋白3赖氨酸27三甲基化(H3K27me3，一种抑制性染色质修饰)，这两个基因是干/祖细胞归巢和存活的关键调节因素。此外，衰老的细胞表现出组蛋白甲基转移酶增强子2(EZH2)的表达增加，组蛋白甲基转移酶增强子2是多梳抑制物复合体2(PRC2)的催化成分。EZH2是唯一已知的能够在哺乳动物细胞中诱导H3K27me3的酶。EZH2还可能与组蛋白脱乙酰酶(HDAC)合作，组蛋白脱乙酰酶通过从组蛋白中去除特定的乙酰基来抑制转录。我们推测，在老年C-MSC中，EZH2的异常表达通过H3K27me3诱导的基因沉默和与HDAC11的相互作用，导致关键的HIF-1α依赖基因的抑制，从而削弱Hp的反应，影响治疗效果。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EZH2在C-MSC中受转录因子E2F1调控的机制，并以老年C-MSC中的E2F1为靶点，以确定它是否与EZH2表达和功能的变化有关。在目标2中，我们将进行遗传学和生化实验，以确定HDAC11是否作为与IL-10启动子相关的多蛋白EZH2-PrC2的一部分，并抑制HIF-1α诱导的老年C-MSC的IL-10基因表达。在目标3中，我们将去除老年C-MSC中的EZH2，以研究细胞移植的功能反应(即细胞归巢、血管生成、心脏修复)。这将是第一次深入研究表观遗传重塑在调节衰老干细胞功能中的作用。我们的发现可能会提高心脏干细胞治疗的疗效，并发现新的分子靶点，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.