Abstract 9648: Aldosterone Increases Transforming Growth Factor-β Expression to Activate Autophagy and Increase Aortic Fibroblast Collagen Synthesis and Vascular Stiffness

Abstract

Elevated levels of aldosterone (ALDO) are associated with aortic remodeling and increased arterial stiffness; however, the role of fibroblasts in these processes remains unknown. Accordingly, human aortic fibroblasts (HAoF) were treated with ALDO (10 -9 -10 -7 mol/L) or a vehicle control (V) for up to 72 h. Compared to V, ALDO increased HAoF expression of transforming growth factor-β (TGFβ) in a dose- and time-dependent manner with a maximal increase of 2.9 ± 0.3-fold (p<0.01) at 72 h. This change was associated with a 252.6 ± 28.8 % (p<0.01) increase in collagen III protein levels in ALDO-treated HAoF; these findings were confirmed by immunohistochemistry. Increased TGFβ expression was also associated with an increase in reactive oxygen species generation (126.4 ± 15.6 vs. 435.5 ± 38.6 fluorescent units, p<0.01) and activation of autophagy as demonstrated by increased beclin1 expression (211.3 ± 23.2% vs. V, p<0.01), lipidation of LC3BI to LC3BII (p<0.01) and autophagic flux assay. To demonstrate that autophagy was necessary to increase collagen III expression, HAoF were transfected with beclin1 siRNA to decrease expression by 69%. Inhibition of autophagy led to a 78% decrease (p<0.01) in collagen III expression without influencing TGFβ levels. To confirm these findings in vivo , C57Bl/6 mice received ALDO (50 μg/kg/d) or V for 21 days. In ALDO-treated mice, aortic TGFβ and LC3BII expression were increased with a concomitant 3.5 ± 0.5-fold (p<0.01) increase in collagen deposition. Pulse wave velocity (PWV), a marker of arterial stiffness, was increased in ALDO-treated mice (288.4 ± 22.4 vs. 389.6 ± 19.8 cm/sec, p<0.01). Treatment with imatinib (40 mg/kg/d) for 14 days downregulated vascular TGFβ expression, diminished collagen deposition, and decreased PWV by 41.1 ± 9.8% (p<0.01). To determine the clinical relevance of these findings, human aorta segments from patients with atherosclerotic vascular disease had higher levels of TGFβ, LC3BII, and fibrosis compared to segments from subjects without disease. These data indicate that ALDO activates fibroblasts to modulate vascular collagen deposition and increase vascular stiffness through a mechanism that involves, in part, TGFβ-mediated activation of autophagy in fibroblasts.