Background While biglycan and oxidized low-density lipoprotein (oxLDL) accumulation has been observed in calcific, stenotic aortic valves, their role in the pathogenesis of calcific aortic valve disease is poorly understood. We hypothesized that soluble biglycan induces the osteogenic response in human aortic valve interstitial cells (AVICs) via Toll-like receptor (TLR) 2 and TLR4, and mediates the pro-osteogenic effect of oxLDL. Methods and Results AVICs of stenotic valves express higher levels of biglycan. Stimulation of cells from normal valves with biglycan increased the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) among the chondrogenic/osterogenic markers examined, and caused accumulation of calcium deposits. TLR2 silencing, but not TLR4 silencing, reduced BMP-2 and ALP levels following biglycan stimulation although co-immunoprecipitation revealed that biglycan intercts with both TLR2 and TLR4. Biglycan induced the phosphorylation of ERK1/2, p38 MAPK and NF-κB. Inhibition of ERK1/2 markedly reduced the up-regulation of BMP-2 and ALP expression by biglycan while inhibition of p38 MAPK or NF-κB had a moderate effect. Stimulation of AVICs with oxLDL up-regulated biglycan expression and release. Knockdown neutralization of biglycan reduced the effect of oxLDL on BMP-2 and ALP expression. Conclusion Extracellular soluble biglycan induces the expression of BMP-2 and ALP in human AVICs primarily via TLR2 and contributes to the the pro-osteogenic effect of oxLDL. These findings highlight the potential role of soluble biglycan and oxLDL in the development of calcific aortic valve disease.

Objective Calcific aortic valve disease is a leading cardiovascular disease in the elderly, and progressive calcification results in the failure of valvular function. Aortic valve interstitial cells (AVICs) from stenotic valves express higher levels of bone morphogenetic protein-2 (BMP-2) in response to Toll-like receptor 4 (TLR4) stimulation. We recently found that TLR4 interacts with Notch1 in human AVICs. This study tests the hypothesis that Notch1 promotes the pro-osteogenic response of human AVICs. Approach and Results AVICs isolated from diseased human valves expressed higher levels of BMP-2 and alkaline phosphatase (ALP) following lipopolysaccharide (LPS) stimulation. The augmented pro-osteogenic response is associated with elevated cellular levels of Notch1, and enhanced Notch1 cleavage in response to LPS stimulation. Inhibition or silencing of Notch1 suppressed the pro-osteogenic response in diseased cells, and the Notch 1 ligand Jagged1 enhanced the response in AVICs isolated from normal human valves. Interestingly, ERK1/2 and NF-κB phosphorylation induced by LPS was markedly reduced by inhibition or silencing of Notch1 and enhanced by Jagged1. Inhibition of ERK1/2 or NF-κB also reduced BMP-2 and ALP expression induced by LPS. Conclusions Notch1 mediates the pro-osteogenic response to TLR4 stimulation in human AVICs. Elevated Notch1 levels and enhanced Notch1 activation play a major role in augmentation of the pro-osteogenic response of AVICs of stenotic valves through modulation of ERK1/2 and NF-κB activation. These pathways could be potential therapeutic targets for prevention of the progression of calcific aortic valve disease.