Objective Mononuclear cell infiltration in valvular tissue is one of the characteristics in calcific aortic valve disease. The inflammatory responses of aortic valve interstitial cells (AVICs) play an important role in valvular inflammation. However, it remains unclear what may evoke AVIC inflammatory responses. Accumulation of biglycan has been found in diseased aortic valve leaflets. Soluble biglycan can function as a danger-associated molecular pattern to induce the production of pro-inflammatory mediators in cultured macrophages. We tested the hypothesis that soluble biglycan induces AVIC production of pro-inflammatory mediators involved in mononuclear cell infiltration through Toll-like receptor (TLR)-dependent signaling pathways. Methods Human AVICs isolated from normal aortic valve leaflets were treated with specific siRNA and neutralizing antibody against TLR2 or TLR4 before biglycan stimulation. The production of ICAM-1 and MCP-1 were assessed. To determine the signaling pathway involved, phosphorylation of ERK1/2 and p38 MAPK was analyzed, and specific inhibitors of ERK1/2 and p38 MAPK were applied. Results Soluble biglycan induced ICAM-1 expression and MCP-1 release in human AVICs, but had no effect on IL-6 release. TLR4 blockade and knockdown reduced ICAM-1 and MCP-1 production induced by biglycan, while knockdown and neutralization of TLR2 resulted in greater suppression of the inflammatory responses. Biglycan induced the phosphorylation of ERK1/2 and p38 MAPK, but ICAM-1 and MCP-1 production was reduced only by inhibition of the ERK1/2 pathway. Further, inhibition of ERK1/2 attenuated NF-κB activation following biglycan treatment. Conclusions Soluble biglycan induces the expression of ICAM-1 and MCP-1 in human AVICs through TLR2 and TLR4, and requires activation of the ERK1/2 pathway. AVIC inflammatory responses induced by soluble biglycan may contribute to the mechanism of chronic inflammation associated with calcific aortic valve disease.

Aortic valve calcification causes the progression of calcific aortic valve disease (CAVD). Stimulation of aortic valve interstitial cells (AVICs) with lipopolysaccharide (LPS) up-regulates the expression of osteogenic mediators, and NF-κB plays a central role in mediating AVIC osteogenic responses to Toll-like receptor 4 (TLR4) stimulation. Diseased aortic valves exhibit greater levels of oxidized low-density lipoprotein (oxLDL). This study tested the hypothesis that oxLDL augments the osteogenic responses in human AVICs through modulation of NF-κB and Notch1 activation. AVICs isolated from normal human aortic valves were treated with LPS (0.1 µg/ml), oxLDL (20 µg/ml) or LPS plus oxLDL for 48 h. OxLDL alone increased cellular bone morphogenetic protein-2 (BMP-2) levels while it had no effect on alkaline phosphatase (ALP) levels. Cells exposed to LPS plus oxLDL produced higher levels of BMP-2 and ALP than cells exposed to LPS alone. Further, LPS plus oxLDL induced greater NF-κB activation, and inhibition of NF-κB markedly reduced the expression of BMP-2 and ALP in cells treated with LPS plus oxLDL. OxLDL also induced Notch1 activation and resulted in augmented Notch1 activation when it was combined with LPS. Inhibition of Notch1 cleavage attenuated NF-κB activation induced by LPS plus oxLDL, and inhibition of NF-κB suppressed the expression of BMP-2 and ALP induced by the synergistic effect of Jagged1 and LPS. These findings demonstrate that oxLDL up-regulates BMP-2 expression in human AVICs and synergizes with LPS to elicit augmented AVIC osteogenic responses. OxLDL exerts its effect through modulation of the Notch1-NF-κB signaling cascade. Thus, oxLDL may play a role in the mechanism underlying CAVD progression.

Background-While calcific aortic stenosis is common, calcification of the other three heart valves is not. The aortic valve interstitial cell (VIC) has been implicated in the pathogenesis of aortic stenosis. Pro-inflammatory stimulation of aortic VICs induces an osteogenic and inflammatory phenotypic change. We hypothesized that the VICs of the other heart valves do not undergo these changes. Using isolated human VICs from normal aortic, mitral, pulmonary and tricuspid valves, our purpose was to compare the osteogenic response to pro-inflammatory stimulation via TLR-4. Materials And Methods-Aortic, pulmonic, mitral, and tricuspid (n=4 for each valve type) VICs were isolated from hearts valves explanted from patients undergoing cardiac transplantation. Cells were cultured and grown to confluence in passage 2-6 before treatment with LPS (100-200ng/mL) for 24 or 48 hours. Cells were characterized by immunofluorescent staining. TLR-4 expression was analyzed (immunoblotting, flow cytometry). BMP-2 and intercellular adhesion molecule-1 (ICAM-1) production were determined (immunoblotting). Monocyte chemoattractant protein-1 (MCP-1) levels were determined by ELISA. Statistics were by Mann-Whitney U test. Results-TLR-4 stimulation induced BMP-2 production only in aortic VICs (p<0.05). ICAM-1 production and MCP-1 secretion increased in a similar fashion among TLR4-stimulated VICs from all four valves.

Background While calcific aortic stenosis is common, calcification of the other three heart valves is not. The aortic valve interstitial cell (VIC) has been implicated in the pathogenesis of aortic stenosis. Pro-inflammatory stimulation of aortic VICs induces an osteogenic and inflammatory phenotypic change. We hypothesized that the VICs of the other heart valves do not undergo these changes. Using isolated human VICs from normal aortic, mitral, pulmonary and tricuspid valves, our purpose was to compare the osteogenic response to pro-inflammatory stimulation via TLR-4. Materials And Methods Aortic, pulmonic, mitral, and tricuspid (n=4 for each valve type) VICs were isolated from hearts valves explanted from patients undergoing cardiac transplantation. Cells were cultured and grown to confluence in passage 2-6 before treatment with LPS (100-200ng/mL) for 24 or 48 hours. Cells were characterized by immunofluorescent staining. TLR-4 expression was analyzed (immunoblotting, flow cytometry). BMP-2 and intercellular adhesion molecule-1 (ICAM-1) production were determined (immunoblotting). Monocyte chemoattractant protein-1 (MCP-1) levels were determined by ELISA. Statistics were by Mann-Whitney U test. Results TLR-4 stimulation induced BMP-2 production only in aortic VICs (p<0.05). ICAM-1 production and MCP-1 secretion increased in a similar fashion among TLR4-stimulated VICs from all four valves. Conclusions Pro-inflammatory stimulation induces an osteogenic phenotype in aortic VICs but not mitral, pulmonic, or tricuspid VICs. We conclude that this differential osteogenic response of aortic VICs contributes to the pathogenesis of calcific aortic stenosis.

Background: Autophagy is a physiological process that plays an important role in maintaining cellular functions. When aortic valve interstitial cells (AVICs) are stimulated with inflammatory or mechanical stress, one response is elevated pro-osteogenic activity. We hypothesized that autophagy is important in the prevention or regulation of this proosteogenic activity in AVICs. Materials and methods: AVICs were isolated. Autophagy activity was examined and its role in AVIC’s pro-osteogenic activity was determined using chemical inhibitors and genetic techniques. The pro-osteogenic biomarker bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase (ALP) were analyzed by immunoblotting and calcium deposition assay. Results: Human AVICs from normal aortic valve donors displayed significantly higher autophagic activity than those from calcified aortic valve donors as indicated by lower protein levels of light chain 3-II. Suppression of autophagy by 3-methyladenine, bafilomycin, or knockdown of Atg7 gene induced the expression of BMP-2 and ALP, increased ALP activity, and calcium deposit formation in normal AVICs. Conversely, upregulation of autophagy with rapamycin or overexpression of Atg7 gene decreased the levels of BMP-2 and ALP in diseased AVICs. Conclusions: Our data showed that autophagy negatively regulates the pro-osteogenic activity in human AVICs, suggesting that upregulation of autophagy may prevent the progression of calcific aortic valve disease.