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: 10826740
• 负责人: Alan Ross Morrison
• 金额: $ 6.79万
• 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: 2022-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10826740
• 关键词: 3' Untranslated Regions; Acute; Age; Aging; Angioplasty; Atherosclerosis; Binding; Binding Sites; Biogenesis; Blood Cells; Blood Vessels; Blood flow; Bypass; Cholesterol; Chronic; Clinical; CpG Islands; DNA; DNA Methylation; Data; Diabetes Mellitus; Disease; Dose; Genes; Grant; Growth; Half-Life; Homeostasis; Impairment; Infiltration; Inflammatory; Injury; Integrins; Interleukin-1 beta; Ischemia; Lead; Lesion; Ligation; Macrophage; Mechanics; Mediating; Medicine; Messenger RNA; Methylation; MicroRNAs; Modeling; Molecular; Morbidity - disease rate; Mus; Muscle; Myelogenous; Operative Surgical Procedures; Outcome; Pathway interactions; Perception; Peripheral arterial disease; Persons; Phenotype; Play; Protein Isoforms; Proteins; Recovery; Role; Signal Pathway; Signal Transduction; Site; Source; Therapeutic; Tissue Preservation; Tissues; Transcript; Transcription Coactivator; Transfection; Translations; Untranslated RNA; VEGFA gene; Vascular Diseases; Vascular Endothelial Growth Factors; Vascularization; adhesion receptor; age effect; age related; aged; angiogenesis; chemokine receptor; cytokine; demethylation; falls; femoral artery; healing; improved; inhibitor; limb ischemia; mRNA Instability; mRNA Stability; mortality; new growth; novel; older patient; posttranscriptional; promoter; response to injury; synergism; therapy development; translational potential; 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（如周围动脉疾病（PAD））导致相当大的发病率和 死亡。衰老与炎症动脉生成对损伤的反应受损有关。我们定义了一个 激活mRNA稳定蛋白HUR的巨噬细胞信号轴促进VEGF-A表达 动脉生成所必需的。我们试图了解衰老对这一途径的影响。适中年龄（52- 一周大的小鼠表现出相对于年轻人的血流恢复降低和动脉生成的改善（12-- 一周大的）小鼠缺血的股动脉连接模型。在老年小鼠中，缺血性肌肉组织和 巨噬细胞显示VEGF-A表达降低。老化的巨噬细胞表明全局DNA增加 甲基化，尽管巨噬细胞HUR表达正常，但与VEGF-A结合减少了 mRNA缩短了VEGF-A mRNA半衰期。有些令人惊讶的是，dicer1，以前 在老化的巨噬细胞中被下调为VEGF-A mRNA的不稳定。 DNMT 抑制剂RG108导致DICER1和VEGF-A表达增加，并增加了与VEGF-A的HUR结合 mRNA。 MiR-29作为3p miRNA，似乎对DICER1表达的变化特别敏感。老化 巨噬细胞改善了miR-29的表达，其在VEGF-A的3'-UTR中的播种位点相邻 到HUR绑定站点。用miR-29模拟巨噬细胞转染巨噬细胞增加了VEGF-A表达。 髓样丁香1骨的小鼠在表型上与老年小鼠相似，血流恢复降低， VEGF-A表达降低，并降低了与VEGF-A mRNA结合，因此缩短了 mRNA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microRNA（即miR-29-3p）导致HUR与VEGF-A mRNA的结合降低 VEGF-A表达和随之而来的VEGF-A依赖性血管生成的降低。我们的目标 寻求1）在老年小鼠中定义DICER1启动子甲基化是VEGF-A-的主要机制 随着衰老的介导的动脉生成； 2）定义分子机制，dicer1剂量 - 敏感的microRNA，miR-29-3p，将hur绑定促进到vegf-a mRNA，并导致消息 稳定。我们的研究将导致从DICER1作为VEGF-A的负调节器的范式转变为 一个积极的调节器。通过DICER1脱甲基或非编码来拯救巨噬细胞VEGF-A表达 RNA（即miR-29）模仿，可能对可以发展的治疗策略具有深远的影响 在严重的年龄相关血管病的情况下，促进动脉生成和相关的组织制备。