Abstract 15322: mIR-376c is Decreased in Marfan Syndrome and Results in Increased Smad4/TGF-beta Signaling in vitro

IntroductionMarfan syndrome (MFS) is a phenotypically heterogeneous multisystem disease caused by pathogenic variants in FBN1. The predominant cardiovascular manifestation is thoracic aortic aneurysm (TAA), which is characterized by dysregulation of TGF-β signaling. MicroRNA contribute to tissue-specific responses that modify phenotype via post-transcriptional gene regulation, and represent potential therapeutic targets. We have previously demonstrated that miR-376c, a known regulator of Smad4/TGF-β signaling, is decreased in MFS-TAA tissue.HypothesisThat miR-376c contributes to MFS-TAA by influencing vascular smooth muscle cell (VSMC) phenotype and Smad4/TGF-β signaling.MethodsmiR-376c was knocked down (KD) in a human aortic VSMC line using an si-anti-miR-376c with HiPerFect (Qiagen®) transfection reagent, and compared with a siRNA negative control (NC) (n=12). VSMC contractile phenotype marker, SM22-α was assessed with western Blot (WB). Smad4 subcellular localization was assessed with immunofluorescence (IF) and total fraction with WB. TGF-β and MMP2 release both during and 24-hours post-stimulation with 20%FCS was compared to basal release using ELISA.ResultsSM22-α was increased 1.6-fold (p=0.05) in miR-376c-KD compared to NC, indicating a predominantly contractile phenotype. Smad4 was increased 1.4-fold (p=0.01) in miR-376c-KD, with increased localization to the nucleus. There was no difference in basal secretion of TGF-β or MMP2 between mir-376c-KD and NC. During stimulation, miR-376c-KD had a 3-fold increase in TGF-β release (p=0.0001), with a 1.4- fold increase in MMP2 release (p=0.008) 24-hours post-stimulation, which was not observed in NC.ConclusionsThis work demonstrates that when the miR-376c down-regulation seen in MFS-TAA is replicated in an in vitro VSMC model, despite a predominantly contractile phenotype, there is a significant increase in TGF-β release associated with increased Smad4. This adaptive response may be deleterious due to the resultant sustained increase in MMP2 release, which is known to degrade extracellular matrix and thereby propagate TAA formation. This work furthers our understanding of the pathogenesis of MFS-TAA and suggests that miR-376c may be a target for future therapy.