MiR-144-5p limits experimental abdominal aortic aneurysm formation by mitigating M1 macrophage-associated inflammation: Suppression of TLR2 and OLR1

Background: It has been noted that dysregulation of MicroRNAs (miRNAs) contributes to the formation of abdominal aortic aneurysm (AAA), a vascular disease associated with progressive aortic dilatation and degradation, and pathological infiltration and activation of inflammatory cells, such as macrophages. Our microarray data revealing that miR-144-5p was the top 1 downregulated miRNA in mouse AAA tissues as compared to normal aortas motivated us to explore its role in AAA development.

Methods: We profiled miRNA and mRNA expression in Angiotensin II (Ang II)- (n = 3) and saline-infused abdominal aortas (n = 4) via Agilent microarrays, and further validated the data with real-time QPCR. In vivo, miR-144-5p or control agomirs were given to apoE^-/- mice with Ang II infusion-induced AAA. In vitro, mouse RAW 264.7 macrophages and human THP-1 macrophage-like cells were transfected with miR-144-5p or control agomirs/antagomirs, and oxidized Low Density Lipoprotein (ox-LDL) was used to stimulate M1 macrophage polarization.

Results: Based on the microarray and real-time QPCR validation data, we identified miR-144-5p as a novel downregulated miRNA in AAA tissues. Overexpression of miR-144-5p by utilizing its specific agomirs in vivo significantly attenuated Ang II-induced aortic dilatation and elastic degradation in apoE^-/- mice and improved their survival. AAA incidence was reduced by miR-144-5p as well. MiR-144-5p polarized macrophages to M2 type in Ang II-infused aortas. Further, the expression levels of two predictive targets for miR-144-5p, Toll Like Receptor 2 (TLR2) and ox-LDL Receptor 1 (OLR1), were higher in AAA specimens, and negatively correlated to miR-144-5p (Pearson correlation coefficient r < -0.9, P < .01). These two molecules were then confirmed as novel miR-144-5p targets via dual-luciferase assay. MiR-144-5p agomirs suppressed ox-LDL-induced upregulation of M1 macrophage markers, including interleukin 1β (IL1β), tumor necrosis factor α (TNFα), prostaglandin-endoperoxide synthase 2 (PTGS2) and nitric oxide synthase 2 (NOS2), in macrophages probably by targeting TLR2. MiR-144-5p also inhibited the signaling transduction of pathways downstream to TLR2 and OLR1, including NF-κB and ERK1/2 pathways, whose abnormal activation contributed AAA formation.

Conclusion: Our work suggests miR-144-5p as a novel regulator for AAA pathology. Management of miR-144-5p and its targets TLR2 and OLR1 provides therapeutic potential for limiting AAA formation.