Abstract 13511: Extracellular Soluble Biglycan Induces Osteogenic Phenotypic Changes in Human Aortic Valve Interstitial Cells via Smad-Dependent and -Independent Pathways
Stimulation of Toll-like receptor (TLR) 2 and TLR4 in human aortic valve interstitial cells (AVICs) induces the expression of pro-osteogenic proteins. Identification of endogenous agents that activate TLR2 or TLR4 could provide insights into the mechanism of aortic valve inflammation and osteogenesis that play important roles in the progression of calcific aortic valve disease. Accumulation of biglycan (BGN) in calcific, stenotic areas of aortic valves has been reported, and soluble BGN can up-regulate the expression of a phospholipid-transfer protein in AVICs through TLR2-dependent mechanism. We hypothesized that soluble BGN functions as an endogenous ligand to TLR2/4 to up-regulate the expression of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1), and induces osteogenic phenotypic changes in human AVICs. Methods and results: Stimulation of human AVICs with soluble BGN (expressed by Murine myeloma cell, 0.05, 0.10 and 0.20 µg/ml) increased the expression and release of BMP-2 and TGF-β1. TLR2 silencing reduced BMP-2 while TLR4 silencing reduced TGF-β1 following BGN stimulation. Prolonged BGN stimulation led to osteogenic phenotypic changes with elevated cellular alkaline phosphatase (ALP) levels and activity, and accumulation of calcium deposits. These changes were reduced by neutralizing BMP-2 or TGF-β1. Further, BMP-2 (100 ng/ml) and TGF-β1 (5.0 ng/ml) each up-regulated the expression of ALP, and stimulation of human AVICs with a combination of these two pro-osteogenic mediators induced osteogenic changes comparable to BGN stimulation. Interestingly, both BMP-2 and TGF-β1 induced ERK1/2 phosphorylation. Inhibition of ERK1/2 suppressed the effect of BMP-2 and TGF-β1 in the induction of osteogenic changes while silencing Smad1 and Smad3 attenuated the effect of BMP-2 and TGF-β1, respectively.
Conclusions: Extracellular soluble BGN induces the expression of BMP-2 and TGF-β1 by human AVICs via TLR2/4. BMP-2 and TGF-β1 contribute to the mechanism underlying the BGN-induced osteogenic changes via Smad-dependent and -independent pathways. These findings highlight the potential role of soluble BGN, as an endogenous TLR ligand, in promoting the progression of calcific aortic valve disease.
- © 2012 by American Heart Association, Inc.