Abstract 82: TGF-β Regulates Matrix Production in Aortic Fibroblasts Through MicroRNA-29b

Abstract

Abdominal aortic aneurysms (AAA) cause ∼15,000 annual deaths in the United States. The mechanisms of AAA expansion remain incompletely explored. MicroRNAs (miRs) regulate gene expression post-transcriptionally, and play crucial roles in vascular integrity. TGF-β can act as a profibrotic stimulus, is a known regulator of miR-29b expression in some cell types, and may play a protective role in AAA development. Numerous extracellular matrix genes are predicted targets of miR-29b. We have previously shown that miR-29b is decreased in both human AAA, and in aneurysmal aortic segments in murine AAA models. Further inhibition of miR-29b in vivo reduces abdominal aortic aneurysm progression in two murine AAA models: the porcine-pancreatic-elastase (PPE) model in C57Bl/6 mice, and the angiotensin II-infusion (AngII) model in ApoE -/- mice, and is associated with a pro-fibrotic response within the vessel wall. We performed vascular cell culture studies using human aortic adventitial fibroblasts (AAFs) and aortic smooth muscle cells (ASMCs) to evaluate the expression and regulation of miR-29b. Treatment with recombinant human TGFβ1 significantly decreased miR-29b levels in aortic adventitial fibroblasts, but not smooth muscle cells (vs. untreated cells). TGFβ1 treatment increased soluble collagen production by AAFs nearly 4-fold, and increased gene expression of COL1A1 and COL3A1 . In ASMCs, TGFβ1 primarily increased ELN expression, and COL1A1 and COL3A1 to a lesser extent. We altered miR-29b expression in vitro by transfecting AAFs and ASMCs with either an antagomir (anti-29b) to inhibit activity or a pre-miR (pre-29b) to enhance activity (vs. scrambled miR control). Successful transfection (>50% of all cells) was confirmed by visual fluorescent microscopic analysis and FACS for labeled tag. Anti-29b significantly augmented the effects of TGFβ treatment upon soluble collagen protein levels and collagen gene expression in AAFs, while pre-29b inhibited this response nearly to untreated-control levels. A similar gene expression response was seen in ASMCs. These data suggest that TGFβ regulation of miR-29b in aortic fibroblasts leads to a profibrotic response, partly explaining its protective effects in AAA development.