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

摘要： 衰老和年龄相关疾病，如外周动脉疾病（PAD），导致相当大的发病率， mortality.衰老与对损伤的炎性动脉生成反应受损有关。我们定义了一个 激活mRNA稳定蛋白HuR以促进VEGF-A表达的巨噬细胞信号传导轴 动脉生成所必需的。我们试图了解衰老对这一途径的影响。中等年龄（52- 周龄）小鼠表现出相对于年轻小鼠（12- 周龄）小鼠在股动脉结扎缺血模型中。在老年小鼠中，缺血性肌肉组织和 巨噬细胞显示VEGF-A表达减少。老年巨噬细胞表现出增加的整体DNA 甲基化，尽管巨噬细胞HuR表达正常，但HuR与VEGF-A的结合减少， mRNA，随之缩短VEGF-A mRNA半衰期。令人惊讶的是，Dicer 1，以前 在衰老的巨噬细胞中，被认为是VEGF-A mRNA的不稳定因素的蛋白质表达下调。关于DNMT 抑制剂RG 108导致Dicer 1和VEGF-A表达增加，HuR与VEGF-A结合增加 mRNA。miR-29作为一种3 p miRNA，似乎对Dicer 1表达的变化特别敏感。岁 巨噬细胞中miR-29的表达降低，其接种位点位于VEGF-A的3′-UTR附近， 到HuR结合位点。用miR-29模拟物转染巨噬细胞增加VEGF-A表达。 髓样Dicer 1缺失的小鼠在表型上与老年小鼠相似，具有降低的血流恢复， 降低VEGF-A的表达，并降低HuR与VEGF-A mRNA的结合， mRNA半衰期。我们的假设是，衰老获得了Dicer 1的甲基化，随之而来的是Dicer 1的减少。 Dicer 1剂量敏感性microRNA（即miR-29- 3 p）导致HuR与VEGF-A mRNA的结合减少， VEGF-A表达和随后的VEGF-A依赖性血管/动脉生成的减少。我们的目标 试图1）确定老年小鼠中Dicer 1启动子甲基化是VEGF-A受损的主要机制， 介导的动脉生成与衰老;和2）定义分子机制，其中Dicer 1剂量- 敏感的microRNA，miR-29- 3 p，促进HuR与VEGF-A mRNA的结合， 稳定化我们的研究将导致从Dicer 1作为VEGF-A的负调节剂到Dicer 1作为VEGF-A的负调节剂的范式转变。 积极的调节器。Dicer 1或非编码区去甲基化对巨噬细胞VEGF-A表达的拯救作用 RNA（即miR-29）模拟物，可能对开发治疗策略具有深远的意义， 在严重的年龄相关性血管病的情况下促进动脉生成和相关组织保存。

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.