ULTRASOUND-TARGETED MIRNA-26A THERAPY FOR ABDOMINAL AORTIC ANEURYSM

Abdominal aortic aneurysm (AAA) affects ∼5% of older adults. AAA is characterized by marked vessel inflammation, human aortic smooth muscle cell (haSMC) depletion and extracellular matrix (ECM) degradation. MicroRNA-26a (miR-26a) is reduced in human AAA and murine models and may contribute to haSMC apoptosis. We hypothesize that targeted miR-26a therapy via ultrasound-targeted microbubble destruction (UTMD) will reverse the AAA inflammatory response, replenish haSMC content and prevent AAA progression. In-vivo we used a combination of intraluminal elastase infusion and extraluminal calcium-chloride to create a clinically relevant rat AAA model. At day 3, rats undergo UTMD 2.5μg scrambled-control miR or miR-26a charge-coupled to 1×109 cationic bubbles intravenously). High power intermittent ultrasound is applied over the infrarenal aorta for targeted transfection of the aortic wall. Ultrasound imaging, ex-vivo aortic wall staining and expression of biomarkers (matrix metalloproteinases (MMPs)), targets (Mothers against decapentaplegic homologs (SMADs)) and inflammatory cytokines). In-vitro, haSMCs were stimulated with the inflammatory interleukin-1β (IL-1β; 20 ng/ml) for 12 hours and transfected with miR-26a, antimiR-26a and scrambled-control miR to elucidate effects of miR-26a on AAA pathogenic pathways. The in-vivo AAA model increased infrarenal aortic diameter by 92±1%, with UTMD of miR-26a then decreasing diameter by 24%, compared to UTMD of scrambled-control miR by day 28. AAA model downregulated miR-26a expression (0.7±0.1-fold; p < 0.05), increased inflammatory responses (IL-6;p < 0.05)and TGF-β; < 0.05) expression) and expression of MMPs (MMP-2; p < 0.01) and MMP-9; p < 0.01)) similar to human AAA. At 24h after UTMD of miR26a, there was MMP-9 (p < 0.05) and MMP-2 (p < 0.05) mRNA downregulation, with concomitant reduction in IL-6 (p < 0.05) and TGF-β (p < 0.05) expression. Histologically, miR-26a decreased apoptosis and increased regeneration of haSMCs in the aortic wall at day 28 post-surgery. In-vitro, IL-1β stimulated haSMCs showed significant downregulation of miR-26a (0.3±0.046-fold; p < 0.05). Then, qRT-PCR (71.5±9.46-fold; p < 0.001) and FACS assay (75.3±1.7%; p < 0.001) confirmed successful transfection of treated cells. Following miR-26a transfection, direct miR-26a targets, SMAD1 (0.6±0.07-fold;p < 0.05) and SMAD4 (0.5± 0.05-fold; p < 0.001), were significantly downregulated. Expression of differentiation marker, alpha-smooth muscle actin (0.5±0.06-fold; p < 0.01), decreased, which was associated with an increase in proliferation following miR-26a transfection. In-vivo, UTMD of miR-26a resulted in downregulation of MMPs and inflammatory cytokines, suggesting an anti-inflammatory effect, with reduced progression of AAA. In-vitro, downregulation of SMADs and haSMC differentiation markers following transfection suggests a phenotypic switch of haSMCs to the de-differentiated synthetic state with greater survival and ECM production. This study demonstrates the potential for the development of effective, safe and clinically-applicable miRNA-based UTMD therapies for AAA.