Background-Calcific aortic stenosis is the third most common cardiovascular disease in the United States. We hypothesized that the mechanism for aortic valve calcification is similar to skeletal bone formation and that this process is mediated by an osteoblast-like phenotype. Methods and Results-To test this hypothesis, we examined calcified human aortic valves replaced at surgery (nϭ22) and normal human valves (nϭ20) removed at time of cardiac transplantation. Contact microradiography and microcomputerized tomography were used to assess the 2-dimensional and 3-dimensional extent of mineralization. Mineralization borders were identified with von Kossa and Goldner's stains. Electron microscopy and energy-dispersive spectroscopy were performed for identification of bone ultrastructure and CaPO 4 composition. To analyze for the osteoblast and bone markers, reverse transcriptase-polymerase chain reaction was performed on calcified versus normal human valves for osteopontin, bone sialoprotein, osteocalcin, alkaline phosphatase, and the osteoblast-specific transcription factor Cbfa1. Microradiography and micro-computerized tomography confirmed the presence of calcification in the valve. Special stains for hydroxyapatite and CaPO 4 were positive in calcification margins. Electron microscopy identified mineralization, whereas energy-dispersive spectroscopy confirmed the presence of elemental CaPO 4 . Reverse transcriptase-polymerase chain reaction revealed increased mRNA levels of osteopontin, bone sialoprotein, osteocalcin, and Cbfa1 in the calcified valves. There was no change in alkaline phosphatase mRNA level but an increase in the protein expression in the diseased valves. Conclusions-These findings support the concept that aortic valve calcification is not a random degenerative process but an active regulated process associated with an osteoblast-like phenotype.

aortic valve stenosis is the most common indication for surgical valve replacement. Inflammation appears to be one of the mechanisms involved in aortic valve calcification, and valve interstitial cells seem to contribute to that process. Although Toll-like receptors (TLRs) play an important role in the cellular inflammatory response, it is unknown whether human aortic valve interstitial cells (HAVICs) express functional TLRs. We examined the expression of TLR2 and TLR4 in human aortic valve leaflets and in isolated HAVICs and analyzed the response of cultured HAVICs to the TLR2 and TLR4 agonists peptidoglycan (PGN) and LPS. Abundant TLR2 and TLR4 proteins were found in human aortic valve leaflets and in isolated HAVICs, and both receptors were detected in the membrane and cytoplasm of cultured HAVICs. Stimulation by either PGN or LPS resulted in the activation of the NF-B signaling pathway and the production of multiple proinflammatory mediators, including IL-6, IL-8, and ICAM-1. In addition, stimulation by either PGN or LPS upregulated the expression of bone morphogenetic protein-2 (BMP-2) and Runx2, factors associated with osteogenesis. This study demonstrates for the first time that HAVICs express TLR2 and TLR4 and that stimulation of HAVICs by PGN or LPS induces the expression of proinflammatory mediators and the upregulation of osteogenesis-associated factors. These results suggest that TLR2 and TLR4 may play a role in aortic valve inflammation and stenosis. cytokines; intracellular cell adhesion molecule-1; bone morphogenetic protein-2; Runx2

Greater expression of TLR2 and TLR4 and greater pro-inflammatory and pro-osteogenic responses to TLR2 and TLR4 agonists in AVICs than PVICs are associated with osteogenic phenotypic changes. These innate immune receptors may play a critical role in aortic valve calcification and stenosis.

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.