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

将靶向去甲基化与非编码 RNA 介导的 mRNA 稳定相结合作为治疗老年人动脉生成的策略

• 批准号: 10631563
• 负责人: Alan Ross Morrison
• 金额: $ 4.92万
• 依托单位: OCEAN STATE RESEARCH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10631563
• 关键词: Age; Aging; Binding; Binding Sites; Blood Cells; Blood Vessels; Blood flow; Cholesterol; DNA Methylation; Disease; Dose; Grant; Growth; Half-Life; Impairment; Inflammatory; Ischemia; Lead; Ligation; Mediating; Medicine; Messenger RNA; Methylation; MicroRNAs; Modeling; Molecular; Morbidity - disease rate; Mus; Muscle; Myelogenous; Pathway interactions; Peripheral arterial disease; Persons; Phenotype; Proteins; Recovery; Signal Transduction; Site; Therapeutic; Tissue Preservation; Transfection; Untranslated RNA; Untranslated Regions; Vascular Diseases; Vascular Endothelial Growth Factors; age effect; age related; aged; demethylation; femoral artery; healing; improved; inhibitor; macrophage; mortality; new growth; older patient; promoter; response to injury; therapy development; treatment strategy

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.

抽象的： 衰老和与年龄相关的疾病，如外周动脉疾病 (PAD) 会导致相当大的发病率和 衰老与损伤后炎症动脉生成反应受损有关。 巨噬细胞信号轴激活 mRNA 稳定蛋白 HuR，促进 VEGF-A 表达 我们试图了解衰老对该途径的影响（52-）。 与年轻小鼠（12-周龄）相比，小鼠表现出血流恢复减少和动脉生成减少 周龄）小鼠股动脉结扎缺血模型。 巨噬细胞显示 VEGF-A 表达减少。 衰老的巨噬细胞显示整体 DNA 增加。 甲基化，虽然巨噬细胞 HuR 表达正常，但 HuR 与 VEGF-A 的结合减少 令人惊讶的是，Dicer1 之前的 mRNA 导致 VEGF-A mRNA 半衰期缩短。 DNMT 被认为会破坏 VEGF-A mRNA 的稳定性，并在衰老的巨噬细胞中下调。 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 miRNA，似乎对 Dicer1 表达的变化特别敏感。 巨噬细胞的 miR-29 表达降低，其播种位点位于 VEGF-A 的 3′-UTR 附近 用 miR-29 模拟物转染巨噬细胞可增加 VEGF-A 的表达。 髓样 Dicer1 缺失的小鼠在表型上与老年小鼠相似，血流恢复减少， VEGF-A 表达，并减少 HuR 与 VEGF-A mRNA 的结合，从而缩短 我们的假设是衰老导致 Dicer1 甲基化，从而导致 Dicer1 的甲基化减少。 Dicer1 剂量敏感的 microRNA（即 miR-29-3p）会导致 HuR 与 VEGF-A mRNA 的结合减少， 减少 VEGF-A 表达和随后的 VEGF-A 依赖性血管/动脉生成。 寻求 1) 确定老年小鼠 Dicer1 启动子甲基化是 VEGF-A- 受损的主要机制 介导衰老过程中的动脉生成；2) 确定 Dicer1 剂量的分子机制 敏感的 microRNA，miR-29-3p，促进 HuR 与 VEGF-A mRNA 结合并产生后续信息 我们的研究将导致从 Dicer1 作为 VEGF-A 负调节因子到作为 VEGF-A 负调节因子的范式转变。 通过 Dicer1 或非编码去甲基化来拯救巨噬细胞 VEGF-A 表达。 RNA（即 miR-29）模拟物可能对治疗策略的开发具有深远的影响，这些策略可以 在严重的与年龄相关的血管病变的情况下促进动脉生成和相关的组织保存。