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

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.