Combining Targeted Demethylation with Noncoding RNA-mediated mRNA Stabilization as a Strategy for Therapeutic Arteriogenesis in the Aged

摘要： 老龄化和与年龄相关的疾病，如外周动脉疾病(PAD)，导致相当大的发病率和 死亡率。衰老与损伤引起的炎性动脉生成反应受损有关。我们定义了一个 巨噬细胞信号轴激活mRNA稳定蛋白HUR促进血管内皮生长因子-A的表达 动脉形成所必需的。我们试图了解衰老对这一途径的影响。中年(52- 周龄小鼠的血流恢复和动脉生成较年轻小鼠减少(12- 周龄)小鼠建立股动脉结扎缺血模型。在老龄小鼠中，缺血肌肉组织和 巨噬细胞显示VEGF-A表达降低。衰老的巨噬细胞表现为全球DNA增加 甲基化，尽管巨噬细胞HUR表达正常，但HUR与VEGF-A的结合减少 信使VEGF-A信使核糖核酸半衰期缩短。有点令人惊讶的是，Dicer1之前 在衰老的巨噬细胞中表达下调，被认为是血管内皮生长因子-A的失稳基因。DNMT 抑制剂RG108导致Dicer1和VEGF-A表达增加，并增加Hur与VEGF-A的结合 MRNA.MIR-29作为一种3p的miRNA，似乎对Dicer1表达的变化特别敏感。老态 巨噬细胞的miR-29表达降低，其种植部位位于VEGF-A的3‘端非编码区附近 到HUR结合部位。将miR-29基因导入巨噬细胞可模拟血管内皮细胞生长因子-A的表达。 髓系Dicer1缺失的小鼠与老龄小鼠的表型相似，血流恢复较慢， 降低血管内皮生长因子-A的表达，减少HUR与血管内皮生长因子-A mRNA的结合，从而缩短 信使核糖核酸半衰期。我们的假设是，衰老获得了Dicer1的甲基化，从而减少了 DICER1剂量敏感的microRNAs(即miR-29-3p)导致HUR与VEGF-A mRNA的结合减少 血管内皮生长因子-A的表达减少以及由此导致的血管/动脉生成依赖。我们的目标 寻求1)确定Dicer1启动子甲基化是老年小鼠血管内皮生长因子-A受损的主要机制。 以及2)确定Dicer1参与的分子机制。 敏感的microRNA，miR-29-3p，促进与VEGF-A mRNA的Hur结合，从而传递信息 稳定状态。我们的研究将导致范式的转变，从Dicer1作为VEGF-A的负调节因子，到Dicer1 一个积极的监管机构。Dicer1去甲基化或非编码对巨噬细胞血管内皮生长因子-A表达的挽救作用 RNA(即miR-29)模拟，可能在开发治疗策略方面具有深远的影响， 在严重的年龄相关性血管病变的背景下，促进动脉生成和相关组织的保存。

ABSTRACT: Aging and age-related diseases like peripheral artery disease (PAD) lead to considerable morbidity and mortality. Aging is associated with impaired inflammatory arteriogenesis responses to injury. We defined a macrophage signaling axis that activates the mRNA stabilizing protein, HuR, to promote VEGF-A expression required for arteriogenesis. We seek to understand the effects of aging on this pathway. Moderately aged (52- week-old) mice demonstrated reduced blood flow recovery and decreased arteriogenesis relative to young (12- week-old) mice in a femoral artery ligation model of ischemia. In aged mice, ischemic muscle tissue and macrophages revealed reduced VEGF-A expression. Aged macrophages demonstrated increased global DNA methylation, and though macrophage HuR expression was normal, there was reduced HuR binding to VEGF-A mRNA with consequent shortened VEGF-A mRNA half-life. Somewhat surprisingly, Dicer1, previously established as destabilizing for VEGF-A mRNA, was downregulated in aged macrophages. The DNMT inhibitor, RG108, led to increased Dicer1 and VEGF-A expression and increased HuR binding to VEGF-A mRNA. miR-29, as a 3p miRNA, appears to be particularly sensitive to changes in Dicer1 expression. Aged macrophages had decreased expression of miR-29, whose seeding site in the 3′-UTR of VEGF-A is adjacent to the HuR binding site. Transfection of macrophages with miR-29 mimic increased VEGF-A expression. Myeloid Dicer1-deleted mice were phenotypically similar to aged mice, having decreased blood flow recovery, decreased VEGF-A expression, and decreased HuR binding to VEGF-A mRNA with consequent shortened mRNA half-life. Our hypothesis is that aging acquired methylation of Dicer1 with consequent reductions in Dicer1 dose-sensitive microRNAs (i.e. miR-29-3p) results in reduced binding of HuR to VEGF-A mRNA and reductions in both VEGF-A expression and consequent VEGF-A dependent angio/ arteriogenesis. Our aims seek to 1) define Dicer1 promoter methylation in aged mice to be a major mechanism of impaired VEGF-A- mediated arteriogenesis with aging; and 2) define the molecular mechanisms whereby the Dicer1 dose- sensitive microRNA, miR-29-3p, promotes HuR-binding to VEGF-A mRNA with consequent message stabilization. Our studies will lead to a paradigm shift from Dicer1 as a negative regulator of VEGF-A to that of a positive regulator. The rescue of macrophage VEGF-A expression by demethylation of Dicer1 or noncoding RNA (i.e. miR-29) mimics, may have profound implications in the development of treatment strategies that can promote arteriogenesis and associated tissue preservation in the setting of severe age-related vasculopathies.