ELUCIDATING THE MOLECULAR MECHANISMS OF MIR-26A ON THE PATHOGENESIS OF ABDOMINAL AORTIC ANEURYSM - IN VITRO STUDIES

Abstract

Despite guideline recommendations for repair of Abdominal Aortic Aneurysm (AAA) when aortic diameter reaches 5.5 cm, the cumulative rates of aneurysm-related death remain high. Unfortunately, no therapies exist to fully prevent aneurysm growth. AAA is characterized by abnormal angiogenesis, impaired extracellular matrix synthesis and marked vessel inflammation -as such these are potential targets for preventative therapies. MicroRNA-26a (miR-26a) is a novel anti-angiogenic regulator of vascular smooth muscle cell (VSMC) function that is downregulated in AAA, and is a viable therapeutic target against AAA. We hypothesized that miR-26a can reverse the active molecular pathways in AAA. Cultured isolated rat aortic VSMCs and rat aortic endothelial cells (RAECs) were stimulated with the inflammatory interleukin-1β (IL-1β; 10 ng/ml) and then transfected with miR-26a, antimiR-26a and scrambled miR (siPORT™ NeoFX™ Transfection) to elucidate AAA cellular and molecular pathogenic pathways and the effects of miR-26a. Real-time qPCR (n=6 per group) and western blotting (n=6 per group) were performed to detect the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). Direct targets of miR-26a -phosphatase and tensin homolog (PTEN) and connective tissue growth factor (CTGF)- were quantified to assess miR-26a’s mechanism of action. Lastly, the angiogenic capacity of RAECs transfected with miR-26a or antimiR-26a were compared to control using the Matrigel™ Assay (n=6 per group). Upon IL-1β stimulation, miR-26a transfection reduced MMP-2 and MMP-9 mRNA levels back to baseline (p < 0.05) whereas antimiR-26a transfection showed no reduction. Stimulating rat VSMCs with IL-1β increased protein levels of active MMP-2 and MMP-9 in control and scramble miR-transfected groups. Transfection with miR-26a showed a reduction in levels of active and total MMP-2 and MMP-9 protein levels compared to control groups. Conversely, upon IL-1β stimulation, miR-26a transfection upregulated TIMP 1-4 levels (p < 0.05) whereas antimiR-26a transfection showed reduction back to baseline. TGFβ mRNA levels were downregulated after miR-26a transfection (p < 0.05) and upregulated after antimiR-26a transfection in VSMCs (p < 0.05). Lastly, overexpression of miR-26a led to reduced angiogenesis, with lower number of tubes and nodes, as compared to the control non-transfected RAECs (p < 0.0001) and antimiR-26a-transfected cells (p < 0.05). MiR-26a attenuates the inflammatory state of VSMCs through the downregulation of MMPs and upregulation of TIMPs and TGF-β, inhibits angiogenesis of RAECs and directly targets CTGF and PTEN, which are key molecules involved in AAA pathogenesis. This suggests that miR-26a is a viable therapeutic option for AAA.