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. Author manuscript; available in PMC: 2013 Nov 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2012 Sep 13;32(11):2711–2720. doi: 10.1161/ATVBAHA.112.300116

Biglycan induces the expression of osteogenic factors in human aortic valve interstitial cells via Toll-like receptor 2

Rui Song 1,2, Qingchun Zeng 1,3, Lihua Ao 1, Jessica A Yu 1, Joseph C Cleveland 1, Ke-seng Zhao 2, David A Fullerton 1, Xianzhong Meng 1,*
PMCID: PMC3665416  NIHMSID: NIHMS411792  PMID: 22982459

Abstract

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.

Keywords: Biglycan, oxLDL, AVIC, BMP-2, TLR2

Calcific aortic valve disease is a chronic inflammatory disease. This disease affects a large number of people 65 years or older, and is the second most common indication for cardiac surgery. Due to a limited understanding of the mechanisms of valvular calcification and stenosis, pharmacological intervention is currently unavailable.

Calcific aortic valve diasease was originally considered to be the result of the natural degenerative aging of inert matrix-based tissue. In recent years, the active and cell-based nature of the disease process has been recognized and thus the potential for pharmacological intervention has arisen 1, 2. While the pathogenic mechanism of calcific aortic valve disease remains elusive, the inflammatory and pro-osteogenic changes in valvular tissue are implicated in the development and progression of this disease. Aortic valve calcification and stenosis occur within the valve tissue, and aortic valve interstitial cells (AVICs) have been demonstrated to play an important role in the inflammatory and osteogenic responses 3, 4. Our recent studies found that stimulation of Toll-like receptor (TLR) 2 or TLR4 in human AVICs with bacterial ligands not only elicits the production of inflammatory mediators, but also induces the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP), two important biomarkers of calcific aortic valve disease 5, 6. Further, AVICs isolated from stenotic aortic valves exhibit elevated levels of BMP-2 and ALP in the absence of stimulation 6, 7. However, the mechanism underlying the pro-osteogenic phenotype of AVICs of diseased valves is unclear, and the role of endogenous activators of TLR2 and TLR4 in the inflammatory and osteogenic responses in human AVICs remains to be determined.

Biglycan, a member of the family of small proteoglycans, is a stationary component of the extracellular matrix and is present in most tissues 8. However, when biglycan is secreted by cells or released from the extracellular matrix, it becomes available in a soluble form. Soluble biglycan has been found to induce cytokine production in macrophages through TLR2 and TLR4 9, 10. Interestingly, biglycan accumulates in calcific, stenotic areas of human aortic valves, and soluble biglycan is capable of inducing AVIC expression of a phospholipid transfer protein through TLR2 11. It is unknown, however, whether AVICs of diseased valve express higher levels of biglycan and whether soluble biglycan induces an osteogenic response in human AVICs. Therefore, the present study is sought to examine biglycan expression in AVICs from diseased human aortic valves and to determine the effect of biglycan on the expression of chondrogenic/osterogenic biomarkers in human AVICs.

Oxidized low-density lipoprotein (oxLDL) is implicated in vascular calcification associated with atherosclerosis 12, 13. Elevated levels of oxLDL in blood correlate with aortic valve calcification and fibrosis 14, and oxLDL accumulation in calcific, stenotic aortic valves is well described 11, 15-19. Our recent study found that oxLDL is capable of up-regulating the expression of BMP-2 in human coronary artery endothelial cells while native LDL is not 20. OxLDL has been shown to modulate biglycan expression in vascular smooth muscle cells 21. However, it remains unknown whether biglycan contributes to the mechanism underlying the pro-osteogenic effect of oxLDL. Further, it is unclear whether oxLDL is pro-osteogenic to human AVICs. We aimed to determine the effect of oxLDL on the expression of BMP-2 and ALP, as well as the role of biglycan in the effect of oxLDL on human AVICs.

We hypothesized that soluble biglycan induces an osteogenic response in human AVICs via TLR2 and TLR4, and mediates the pro-osteogenic effect of oxLDL on human AVICs. The purpose of this study was to determine: 1) whether biglycan expression is up-regulated in AVICs of stenotic human aortic valves, 2) the effect of soluble biglycan on the expression of chondrogenic/osterogenic biomarkers by human AVICs, 3) the mechanism by which soluble biglycan exerts its effect, 4) whether oxLDL induces biglycan expression and release in AVICs, and 5) the role of biglycan in oxLDL effect on AVICs.

Materials and Methods

Materials

Antibodies against BMP-2, TLR2, TLR4, Runx2, Osx, Msx2, Pit-1 and Sox9, as well as specific siRNA for human TLR2, TLR4 and biglycan and scrambled siRNA, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phosphorylated ERK1/2, total ERK1/2, phosphorylated p38 MAPK, total p38 MAPK, phosphorylated NF-κB and total NF-κB were purchased from Cell Signaling, Inc. (Beverly, MA). Medium 199 was purchased from Lonza (Walkersville, MD). Recombinant human biglycan (expressed by a murine myeloma cell line; endotoxin-free), antibodies against biglycan and ALP were purchased from R&D System (Minneapolis, MN). Biglycan ELISA kit was obtained from Uscn Life Science Inc. (Germany). OxLDL was purchased from Biomedical Technologies Inc. (Stoughton, MA). HiPerFect® transfection reagent and other transfection-related reagents were purchased from Dharmacon (Lafayette, CO). Pierce Cell Lysis Buffer for immunoprecipitation was purchased from Thermo Fisher Scientific (Rockford, IL). All other chemicals and reagents, including Bay11-7082, were from Sigma-Aldrich Chemical Co. (St Louis, MO).

Cell isolation and treatment

Stenotic aortic valve leaflets were obtained from 5 patients (4 males and 1 female, age 55.8±13.2 years) undergoing valve replacement at the University of Colorado Hospital. Normal aortic valve leaflets were collected from the explanted hearts of 6 patients (4 males and 2 females, age 59.0±8.1 years) with cardiomyopathy and undergoing heart transplantation at the University of Colorado Hospital. These valve leaflets were thin and did not exhibit histological abnormality. All patients gave informed consent for the use of their valves for this study approved by the University of Colorado Denver Institutional Review Board.

AVICs were isolated and cultured using a previously described method 22 with modification 5. Briefly, valve leaflets were subjected to sequential digestions with collagenase, and cells were collected by centrifugation. Cells were cultured in M199 growth medium containing penicillin G, streptomycin, amphotericin B and 10% fetal bovine serum. Cells from passages 4 to 6 were used for this study. Cells were subcultured on plates, and treated when they reached 80 to 90% confluence.

To determine the effect of soluble biglycan on the expression of BMP-2, ALP, Runx2, Osx, Msx2, Pit-1 and Sox9, cells from normal valves were treated with recombinant biglycan (0.05, 0.10 and 0.20 μg/ml) for 48 h. Levels of these chondrogenic/osterogenic biomarkers in cell lysates were assessed by immunoblotting.

The effect of biglycan on phosphorylation of ERK1/2, p38 MAPK and NF-κB was determined following stimulation of cells with biglycan for 60 to 240 min. To determine the role of ERK1/2, p38 MAPK and NF-κB in the effect of biglycan on human AVICs, ERK1/2 inhibitor (PD98059; 10-40 μM), p38 MAPK inhibitor (SB203580; 5-20 μM) and IKK inhibitor (Bay11-7082; 5-10 μM) were added to culture medium 30 min before the addition of biglycan.

To determine the effects of oxLDL on biglycan expression and release, cells were stimulated with oxLDL (20 and 40 μg/ml) for 2 to 24 h. Levels of biglycan mRNA and protein in cells, and levels of biglycan protein in culture medium were analyzed.

To determine the role of biglycan in oxLDL-induced osteogenic response, cells were treated with oxLDL for 48 h in the presence of a polyclonal antibody against human biglycan (10 μg/ml) or following pretreatment with human biglycan siRNA. Levels of BMP-2 and ALP in cell lysate were assessed.

Immunofluorescent staining

Immunofluorescent staining was applied to character cell cultures as described previously 23. After permeabilization with a methanol/acetone mixture, cells on chamber slides were fixed in 4% paraformaldehyde, incubated with a rabbit polyclonal antibody against human α-smooth muscle actin overnight at 4°C. After washing with PBS, cells were incubated with Cy3-tagged secondary antibody against the primary antibody (imaged on the red channel). Nuclei were stained with bis-benzimide (DAPI, imaged on the blue channel), and glycoproteins on cell surfaces with Alexa 488-tagged wheat germ agglutinin (WGA, imaged on the green channel). Microscopy was performed with a Leica DMRXA digital microscope (Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany) equipped with Slidebook software (I. I. I. Inc., Denver, CO).

Gene knockdown

To knockdown TLR2, TLR4 and biglycan, cells (60–70% confluent) in 24-well plates were incubated with a mixture of siRNA (60 nM) and transfection reagent (6 μl per ml of medium) for 48 h. Control cells were treated with scrambled siRNA and transfection reagent.

Real-time RT-PCR analysis

Total RNA was extracted using a Qiagen RNeasy Mini Kit (Valencia, CA, USA). Reverse transcription (RT) and PCR were performed in triplicate using iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and iQ™ SYBR® Green Supermix according to the manufacturer’s instructions. Amplification was carried out for 40 cycles including denaturation at 94°C for 10 seconds and annealing/extension at 59°C for 30 seconds. The following primers were used to amplify specific cDNA fragments: biglycan (forward: 5’- TGCAGA ACA ACG ACA TCT CC -3’; reverse:5’- GCC TTC TCA TGG ATC TTG GA -3’) (GenBank™ accession number NM_001711); GAPDH (forward: 5’- CAT GGC CTC CAA GGA GTA AG -3’; reverse: 5’- AGG GGT CTA CAT GGC AAC TG -3’).

Biglycan mRNA levels were quantified by real-time PCR using the iQ™ 5 Multicolor Real-time PCR Detection system (Bio-Rad, Hercules, CA, USA). Biglycan mRNA levels normalized to GAPDH mRNA were calculated using the 2-ΔΔT method 24.

Immunoblotting

Western blotting was applied to analyze biglycan, BMP-2, ALP, Runx2, Osx, Msx2, Pit-1, Sox9, TLR2, TLR4, phosphorylated and total ERK1/2, phosphorylated and total p38 MAPK, and phosphorylated and total NF-κB, with β-actin as a loading control. After stimulation, human AVICs were lysed in a sample buffer (100 mM Tris-HCl, pH 6.8, 2% SDS, 0.02% bromophenol blue and 10% glycerol). Cell lysates were resolved on 4-20% SDS-PAGE gels and the proteins were transferred onto PVDF membranes. After being blocked with 5% skim milk solution, membranes were incubated with primary antibodies, followed by peroxidase-linked secondary antibodies specific to the primary antibodies. Protein bands were revealed using the ECL system. Band density was analyzed using the National Institutes of Health ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD).

ELISA

Cell culture supernatants were collected. Biglycan levels were analyzed using an ELISA kit following the manufacturer’s protocol.

Alizarin red S staining

Alizarin red S staining for calcium deposits was performed as described previously 6. Briefly, cell monolayers were washed twice with phosphate-buffered saline (PBS) and fixed for 15 min in 4% paraformaldehyde, followed by incubation with 0.2% alizarin red solution (pH 4.2) for 30 min. Excessive dye was removed by washing with distilled water. Alizarin red staining was examined and photographed with a Nikon Eclipse TS100 microscope (Tokyo, Japan). To quantitatively analyze Alizarin red stain, wells were rinsed with distilled water, and alizarin red stains were bleached with 10% acetic acid at 85 °C. Supernatant was spectrophotometrically analyzed at 450 nm 25.

Co-immunoprecipitation

Human AVICs (80–90% confluent) in 6-well plates were gently rinsed 3 times with PBS. Cells were lysed in Pierce Cell Lysis Buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol, pH 7.4), and the lysates centrifuged at 112 xg for 10 min at 4°C. Clarified lysates were incubated with a mouse monoclonal antibody against human TLR2 (4 μg/sample), human TLR4 (4 μg/sample) or non-immune mouse IgG (4 μg/sample) with G-coupled Sepharose beads overnight at 4°C with rocking. Precipitation was performed at 4°C for 2 h using 20 μL of protein G-agarose per sample. The beads were gently washed 5 times with 0.5 mL of lysis buffer and resuspended in 100 μL of 2× SDS sample buffer. Biglycan, TLR2 and TLR4 were analyzed by immunoblotting.

Statistical analysis

Data are presented as mean ± standard error (SE). Statistical analysis was performed using StatView software (Abacus Concepts, Calabasas, CA). Analysis of variance (ANOVA) with the post hoc Bonferroni/Dunn test was used to analyze differences between experimental groups, and a paired ‘t’ test was applied to analyze data comparisons between normal and diseased AVICs. Statistical significance was defined as P≤0.05. Non-parametric Mann-Whitney U test was performed to confirm the difference of 2 group comparison. For multiple group comparisons, non-parametric Kruskal-Wallis test was performed to confirm the differences.

Results

AVICs of stenotic valves express high levels of biglycan

We analyzed biglycan mRNA and protein levels in AVICs isolated from normal and stenotic valves. As shown in Figures 1A and 1B, cells from diseased valves had markedly higher levels of biglycan mRNA (P<0.05) and protein.

Figure 1. AVICs of stenotic valves express higher levels of biglycan than cells of normal valves. AVICs were isolated from normal and stenotic human aortic valves and cultured in M199 medium.

Figure 1

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A. A representative immunoblot shows elevated cellular levels of biglycan in AVICs of 2 diseased valves in comparison to AVICs of 2 normal valves. B. Real-time RT-PCR revealed higher levels of biglycan mRNA in diseased AVICs. n=4 in each group; *P<0.05. C. AVICs from stenotic aortic valves grow to a lower density in comparison to AVICs from normal aortic valves although cells from these two sources exhibit similar morphology. In addition, AVICs from stenotic aortic valves and normal valves exhibit characteristics of myofibroblasts.

To examine whether cultures of AVICs from normal and stenotic aortic valves are phenotypically comparable, we characterized cell cultures. Phase contrast images showed comparable morphology between AVICs from normal valves and AVICs from stenotic valves (Figure 1C). However, AVICs of stenotic aortic valves grew slower. When equal numbers of cells were seeded, cultures of cells from stenotic valves had lower density after growth (49653±2675 cells/field using a 20x objective vs. 77903±3819 cells/field in normal cell cultures; P<0.05). Immunofluorescent staining with a specific antibody against human α-smooth muscle actin revealed that greater than 80% of cells are positive for α-smooth muscle actin in AVICs from either normal valves or stenotic valves. However, AVICs from stenotic valves exhibited abundant stress fibers (Figure 1C).

Soluble biglycan up-regulates the expression of BMP-2 and ALP in human AVICs

To determine the effect of soluble biglycan on the expression of chondrogenic/osterogenic biomarkers by human AVICs, we analyzed the levels of BMP-2, ALP, Runx2, Osx, Msx2, Pit-1 and Sox9 following biglycan treatment. As shown in Figures 2A and 2B, stimulation of cells with recombinant human biglycan resulted in a dose-dependent increase in protein levels of BMP-2 and ALP, and ALP activity. More importantly, prolonged stimulation with biglycan promoted the accumulation of calcium deposits and the formation of calcification nodules in human AVIC culture (Figure 2C). However, biglycan had no effect on the levels of Runx2, Msx2, Pit-1 and Sox9 although it slightly up-regulated the levels of Osx (supplemental data). These results demonstrate that soluble biglycan is potent to up-regulate the expression of BMP-2 and ALP, and that prolonged exposure to soluble biglycan induces pro-osteogenic changes in human AVICs.

Figure 2. Soluble biglycan induces BMP-2 and ALP expression, and calcification nodule formation in human AVICs.

Figure 2

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A. Human AVICs from normal valves were treated with biglycan (BGN; 0.05, 0.10 and 0.20 μg/ml) for 48 h. BMP-2 protein levels were analyzed with immunoblotting. Representative blots and densitometric data show dose-dependent induction of BMP-2 by biglycan. B. AVICs were treated with biglycan (BGN; 0.05, 0.10 and 0.20 μg/ml) for 48 h. ALP protein levels were analyzed with immunoblotting, and ALP activity was assessed by activity staining. Representative blots, densitometric data and images show dose-dependent induction of ALP by biglycan. C. AVICs were cultured in conditioning medium and incubated with biglycan (BGN) for 21 days. Images of Alizarin Red S staining and quantitative analysis of Alizarin Red S stain show a dose-dependent increase in calcium deposits (arrows). n = 3 or 4 in each group; *P<0.05 vs. untreated control. Scale bar = 200 μm.

Soluble biglycan up-regulates BMP-2 and ALP expression by human AVICs primarily through TLR2

Soluble biglycan has been reported to activate TLR2 and TLR4 in macrophages 9. We have found that stimulation of TLR2 or TLR4 up-regulates the expression of BMP-2 and ALP in human AVICs 6. To determine whether these two innate immune receptors play a role in mediating biglycan-induced BMP-2 and ALP expression in human AVICs, we applied specific siRNA to knockdown TLR2 and TLR4. As shown in Figure 3A, silencing of TLR2 markedly reduced protein levels of BMP-2 and ALP following biglycan stimulation. However, silencing of TLR4 had a minimal effect (Figure 3A). The results suggest that TLR2, but not TLR4, is involved in the up-regulation of BMP-2 and ALP expression in human AVICs by soluble biglycan.

Figure 3. Soluble biglycan interacts with TLR2 to induce the osteogenic response in human AVICs.

Figure 3

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A. Representative immunoblots show that treatment with specific siRNA reduced cellular TLR2 and TLR4 levels. Representative immunoblots and densitometric data show that silencing TLR2 markedly reduced cellular BMP-2 and ALP levels following treatment with biglycan (BGN, 0.10 μg/ml for 48 h) while silencing TLR4 had a minor effect on biglycan-induced BMP-2 and ALP expression. n = 3 in each group; *P<0.05 vs. untreated control; #P<0.05 vs. biglycan alone; †P<0.05 vs. biglycan + Scrambled siRNA. B. A higher level of TLR2 and a lower level of TLR4 are co-immunoprecipitated with biglycan in cells incubated with biglycan (0.10 μg/ml for 2 h).

To examine whether biglycan interacts with TLR2 and TLR4, we performed co-immunoprecipitation. Interestingly, both TLR2 and TLR4 were co-immunoprecipitated with biglycan in cells exposed to recombinant biglycan (Figure 3B). However, higher levels of TLR2 was co-immunoprecipitated with biglycan, indicating a preferential or stronger interaction of biglycan with TLR2.

ERK1/2 plays a major role in the mechanism underlying biglycan-induced BMP-2 and ALP expression

TLR2 activates multiple pro-inflammatory signaling pathways, including ERK1/2, p38 MAPK and NF-κB pathways 26. ERK1/2, p38 MAPK and NF-κB pathways have previously been shown to play important roles in regulating osteoblast activity 27, 28. We determined the effect of biglycan on phosphorylation of these signaling molecules. We found that biglycan induced the phosphorylation of ERK1/2, p38 MAPK and NF-κB (Figure 4A). To determine the role of ERK1/2, p38 MAPK and NF-κB in biglycan-induced expression of BMP-2 and ALP, we applied specific inhibitors prior to stimulation of cells with biglycan. Inhibition of ERK1/2 with PD98059 dose-dependently reduced the levels of BMP-2 and ALP following biglycan stimulation (Figure 4B). Inhibition of p38 MAPK with SB203580 and inhibition of NF-κB with Bay11-7082 also attenuated BMP-2 and ALP expression (Figures 4C and 4D). However, the effect of inhibition of p38 MAPK or NF-κB was moderate in comparison to that of ERK1/2 inhibition. Therefore, ERK1/2, p38 MAPK and NF-κB are all involved in mediating the up-regulation of BMP-2 and ALP expression in human AVICs by biglycan, and ERK1/2 plays a major role.

Figure 4. The ERK1/2, p38 MAPK and NF-κB pathways are involved in mediating biglycan-induced osteogenic response in human AVICs.

Figure 4

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A. Biglycan (BGN, 0.10 μg/ml) induced the phosphorylation of ERK1/2, p38 MAPK and NF-κB. B. Representative immunoblots and densitometric data show that inhibition of ERK1/2 with PD98059 markedly reduced BMP-2 and ALP levels following biglycan (BGN) stimulation. n=3 in each group; *P<0.05 vs. untreated control; #P<0.05 vs. biglycan alone. C and D. Representative immunoblots and densitometric data show that inhibition of p38 MAPK with SB203580 and inhibition of NF-κB with Bay11-7082 also attenuate BMP-2 and ALP expression following biglycan stimulation. n=3 in each group; *P<0.05 vs. untreated control; #P<0.05 vs. biglycan alone.

TLR2 mediates ERK1/2 phosphorylation and calcium deposit formation induced by biglycan

To determine whether TLR2 mediates biglycan-induced ERK1/2 phosphorylation, we stimulated cells with biglycan (BGN, 0.10 μg/ml) for 60 to 240 min in the presence of a TLR2-neutralizing antibody (10 μg/ml). The results in Figure 5A show that TLR2-neutralizing antibody suppressed ERK1/2 phosphorylation at all time points examined. A maximal reduction in ERK1/2 phosphorylation was observed at 240 min following biglycan stimulation.

Figure 5. TLR2 mediates biglycan-induced ERK1/2 phosphorylation and calcium deposit formation.

Figure 5

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A. Cells were treated with biglycan (BGN, 0.10 μg/ml) in the presence of TLR2-neutralizing antibody (10 μg/ml) for 60 to 240 minutes. Representative immunoblots and densitometric data show that TLR2-neutralizing antibody suppresses ERK1/2 phosphorylation at all time points examined following biglycan stimulation. B. Cells cultured in conditioning medium were stimulated with biglycan (BGN, 0.10 μg/ml) for 21 days in the presence of TLR2-neutralizing antibody (10 μg/ml). Images of Alizarin Red S staining and quantitative analysis of Alizarin Red S stain show that TLR2-neutralizing antibody reduces the formation of calcium deposits (arrows). n = 3 in each group; *P<0.05 vs. untreated control; #P<0.05 vs. biglycan alone; ‡P<0.05 vs. biglycan + non-immune IgG. Scale bar = 200 μm.

To confirm the role of TLR2 in biglycan-induced calcium deposit formation, we stimulated cells cultured in conditioning medium with biglycan (BGN, 0.10 μg/ml) for 21 days in the presence of the TLR2-neutralizing antibody (10 μg/ml). Alizarin Red S staining revealed that TLR2-neutralizing antibody significantly reduced the formation of calcium deposits (Figure 5B). Together, these results demonstrate that TLR2 mediates biglycan-induced ERK1/2 phosphorylation and is involved in the mechanism underling calcium deposit formation induced by biglycan.

OxLDL promotes biglycan expression and release in human AVICs

We have reported that oxLDL induces the expression of BMP-2 in human coronary artery endothelial cells while native LDL does not have an effect 20. In the present study, we observed that stimulation of human AVICs with oxLDL increased cellular levels of biglycan protein (Figure 6A). To examine whether the up-regulation of biglycan protein by oxLDL involves gene transcription, we employed real-time RT-PCR to determine the effect of oxLDL on biglycan mRNA levels. As shown in Figure 6B, biglycan mRNA levels increased in a time-dependent fashion following oxLDL stimulation. Elevated levels of biglycan mRNA were detected at 4 h of exposure to oxLDL, and the maximal increase (3.19 fold, P<0.05) in biglycan mRNA was observed at 8 h. Thus, oxLDL induces biglycan gene expression to increase biglycan protein levels in human AVICs.

Figure 6. OxLDL promotes biglycan expression and release in human AVICs.

Figure 6

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A. Human AVICs from normal valves were stimulated with oxLDL (20 and 40 μg/ml) for 48 h. Biglycan levels in cell lysates were analyzed with immunoblotting. A representative blot and densitometric data show increased biglycan levels in cells treated with oxLDL. Values are means ± SE; n=3; *P<0.05 vs. untreated controls. B. Cells were harvested at 2 to 12 h of oxLDL (20 μg/ml) stimulation for evaluation of biglycan mRNA levels (normalized by GAPDH levels). OxLDL up-regulated biglycan mRNA. n=3 in each group; *P<0.05 vs. untreated control. C. Culture supernatants were collected at 4 to 24 h of oxLDL (20 μg/ml) stimulation for evaluation of biglycan release by ELISA. OxLDL stimulation increased extracellular levels of biglycan. n=4 in each group; *P<0.05 vs. untreated control.

To determine whether oxLDL induces the secretion of biglycan, we analyzed biglycan levels in culture supernatants using ELISA. Biglycan levels in culture supernatants increased over time following oxLDL stimulation (Figure 6C). Extracellular biglycan levels increased by 4.5 fold (P<0.05) at 24 h of stimulation with 20 μg/ml of oxLDL. Together, the results demonstrate that oxLDL up-regulates biglycan expression and promotes biglycan release in human AVICs.

Biglycan contributes to the mechanism by which oxLDL induces the expression of BMP-2 and ALP in human AVICs

To determine whether biglycan is involved in the mechanism underlying the up-regulation of BMP-2 and ALP expression by oxLDL, we added a polyclonal antibody against human biglycan to culture medium prior to oxLDL stimulation. As shown in Figure 7A, anti-biglycan reduced the effect of oxLDL on the expression of BMP-2 and ALP while isotype-matching non-immune IgG had no effect. To further determine the role of biglycan in oxLDL-induced BMP-2 and ALP expression, we treated cells with biglycan siRNA. Figure 7B shows that silencing of biglycan reduced the levels of BMP-2 and ALP proteins following oxLDL stimulation. These results indicate that biglycan contributes to the mechanism by which oxLDL induces these osteogenic mediators in human AVICs.

Figure 7. Neutralization and silencing of biglycan suppress oxLDL-induced osteogenic response in human AVICs.

Figure 7

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A. Representative immunoblots and densitometric data show that treatment with a polyclonal antibody against human biglycan (BGN) markedly reduced BMP-2 and ALP levels in AVICs stimulated with oxLDL (20 μg/ml, 48 h). B. Representative immunoblots and densitometric data show that silencing of biglycan reduced cellular biglycan levels and attenuated the effect of oxLDL (20 μg/ml, 48 h) on BMP-2 and ALP expression. n=3 in each group; *P<0.05 vs. untreated control; #P<0.05 vs. oxLDL alone; †P<0.05 vs. oxLDL + Scrambled siRNA; ‡P<0.05 vs. oxLDL + non-immune IgG.

Discussion

In the present study, we found that AVICs of stenotic valves express greater levels of biglycan than cells of normal valves. Importantly, soluble biglycan is capable of inducing BMP-2 and ALP expression, and this effect of biglycan on human AVICs is mediated primarily by TLR2 and involves activation of the ERK1/2, p38 MAPK and NF-κB pathways. Further, we demonstrate that oxLDL promotes biglycan expression and release by human AVICs and that biglycan plays a crucial role in oxLDL-induced expression of BMP-2 and ALP in human AVICs. These findings provide novel insights into the molecular mechanisms by which an endogenous agent induces the osteogenic response in human AVICs. These findings highlight the possibility of pharmacological intervention for treatment of calcific aortic valve disease in its early stage.

Soluble biglycan is potent to induce the expression of BMP-2 and ALP in human AVICs

Macromolecules of the extracellular matrix are commonly thought to function as pure structural components. However, there is growing evidence that the extracellular matrix exerts more complex actions than being a mere scaffold for cells to attach to, including direct regulation of cellular functions 8, 29-32. In this regard, several studies in macrophages have found that biglycan induces cytokine production 9. Moreover, biglycan modulates BMP signaling and is involved in the regulation of osteoblast differentiation 33-36.

Several studies have reported biglycan accumulation in calcific, stenotic areas of aortic valves 11. However, the role of soluble biglycan in the pathogenesis of calcific aortic stenosis is unclear, and the effect of soluble biglycan on valve cells remains to be determined. The results of this study show that soluble biglycan induces the expression of osteogenic mediators BMP-2 and ALP in human AVICs. However, biglycan at the concentrations tested had no effect on the expression of Runx2, Msx2, Pit-1 and Sox9, and only moderately up-regulated the levels of Osx. It appears that BMP-2 and ALP responses in human AVICs are sensitive to soluble biglycan. BMP-2 is a potent osteogenic factor and plays an important role in vascular and aortic valve calcification. In this regard, high levels of BMP-2 expression have been found in calcified human atherosclerotic plaque, and smooth muscle cells isolated from the atherogenic aortic wall express BMP-2 in vitro and forms calcification nodules with prolonged culture 37. In addition, recombinant BMP-2 promotes phosphate uptake and calcification in human vascular smooth muscle cells in vitro 38. More importatntly, over-expression of BMP-2 accelerates atherosclerotic intimal calcification in apoE knockout mice 39. In calcific aortic valve disease, BMP-2 accumulation in valve leaflets is evident in the early stage of this desease 40. We have found that stimulation of human AVICs with recombinant BMP-2 in vitro upregulates ALP expression and induces calcification nodule formation 6. Since soluble biglycan is potent to up-regulate BMP-2 expression in human AVICs, it may contribute to the mechanism of aortic valve calcification. The results of the present study show that prolonged exposure to soluble biglycan causes AVIC calcification. These findings indicate a pathobiological role of soluble biglycan in the cell-based development and progression of calcific aortic valve disease.

It is likely that BMP-2 plays a critical role in mediating the pro-osteogenic changes induced by soluble biglycan as recombinant BMP-2 upregulates ALP expression and induces calcification nodule formation in human AVICs 6. Runx2 interacts with Smad in osteoblasts, and the Runx2-Smad complex mediates BMP-promoted osteoblast differentiation and bone formation 41. Osx-Nfatc1 complex has also been found to play an important role in the transcriptional program of osteoblasts associated with bone formation 42. The transcriptional mechanism of the pro-osteogenic programming of AVICs downstream of the novel biglycan:TLR2:BMP2 axis remains unknown from the present study. However, biglycan at the concentrations tested did not affect the expression of Runx2, Msx2, Pit-1 and Sox9, and only moderately up-regulated the levels of Osx. It is possible that modulation of macromolecular assembly of the transcriptional complexes and/or their nuclear redistribution is involved in mediating AVIC pro-osteogenic programming. Further studies are needed to elucidate the molecular mechanism downstream of the biglycan:TLR2:BMP2 axis.

The results of this study also show that AVICs isolated from stenotic valves express greater levels of biglycan in vitro. As biglycan is involved in tissue repair during and after injury, it is likely that valvular damage associated with the disease process results in a phenotypic change in AVICs of stenotic valves that leads to up-regulated expression of biglycan. The augmented expression of biglycan mRNA and protein in vitro in diseased AVICs after greater than 3 passages indicates that the phenotypic change is long-lasting. One of the limitations of this study, however, is that the diseased AVICs studied were from valves with severe stenosis. Although AVICs were isolated from tissues with relatively minor pathological changes, the findings may not reflect the pathobiology of AVICs in the early stage of calcific aortic valve disease. Nevertheless, the results show that AVICs of normal and diseased aortic valves are morphologically comparable and both exhibit a myofibroblastic phenotype. It is likely that the augmented expression of biglycan by AVICs of diseased valves is due to an alteration of cellular signaling mechanisms that regulate biglycan expression.

TLR2 plays a major role in mediating the effect of biglycan

In the present study, we observed that both TLR2 and TLR4 are co-immunoprecipitated with biglycan following treatment of cells with biglycan although the level of TLR2 in the co-immunoprecipitants is higher. The results indicate a molecular interaction of biglycan with these two innate immune receptors. However, the potency of biglycan for the induction of BMP-2 and ALP expression in human AVICs is markedly attenuated by knockdown of TLR2, and knockdown of TLR4 has no effect. These results demonstrate that soluble biglycan up-regulates BMP-2 and ALP expression in human AVICs primarily through the TLR2 pathway and are consistent with a previous study in which the effect of biglycan on the expression of a phospholipid transfer protein in AVICs is suppressed by a TLR2-neutralizing antibody 11. Further evidence that biglycan-induced calcium deposit formation is reduced by a TLR2-neutralizing antibody (Figure 5B) confirmed an important role of TLR2 in mediating the pro-osteogenic effect of soluble biglycan. TLR2 signaling is linked to multiple pro-inflammatory pathways 26, 43. The results of this study show that biglycan induces the phosphorylation of ERK1/2, p38 MAPK and NF-κB in human AVICs. All of the three pathways appear to be involved in mediating the induction of BMP-2 and ALP expression by soluble biglycan since a specific inhibitor for each of these pathways attenuates the up-regulation of BMP-2 and ALP expression following biglycan stimulation. It is noteworthy that inhibition of ERK1/2 with PD98059 results in a greater reduction in BMP-2 and ALP levels, with 65.2% reduction in BMP-2 and 61.2% reduction in ALP in the presence of 20 μM of PD98059. Thus, the ERK1/2 pathway appears to have a major role in mediating the TLR2-dependent pro-osteogenic effect of soluble biglycan. To confirm the link between TLR2 and ERK1/2 in human AVICs, we determine the effect of a TLR2-neutralizing antibody on biglycan-stimulated ERK1/2 phosphorylation. As shown in Figure 5A, TLR2-neutralizing antibody suppressed biglycan-induced ERK1/2 phosphorylation. Therefore, the TLR2-ERK1/2 pathway occupies a major role in mediating the up-regulation of BMP-2 and ALP expression in human AVICs by soluble biglycan. ERK1/2 can modulate NF-κB activity 20. The moderate effect of NF-κB inhibition on BMP-2 and ALP expression indicates the NF-κB-dependent action of ERK1/2, if any, is a partial mechanism underlying the role of ERK1/2 in mediating the osteogenic response in human AVICs. Further studies are necessary to determine the downstream molecular mechanisms.

Biglycan plays an important role in oxLDL-induced BMP-2 and ALP expression in human AVICs

Some pathological characteristics of calcific aortic valve disease resemble those of atherosclerosis, but there are also several distinctions. Although both diseases involve lipid entrapment within the tissue, atherosclerosis exhibits foam cell accumulation 44, whereas aortic valve disease progresses with leaflet calcification 1. Our recent study found that oxLDL, but not native LDL, up-regulates BMP-2 expression in human coronary artery endothelial cells 20. Further, oxLDL was reported to induce vascular smooth muscle cell calcification 45. To understand the role of oxLDL in human AVIC osteogenic response, we stimulated cells with oxLDL and examined cellular levels of BMP-2 and ALP. We observed in this study that oxLDL up-regulates the expression of BMP-2 and ALP. The findings are consistent to those obtained in coronary artery endothelial cells and vascular smooth muscle cells, and indicate that oxLDL is pro-osteogenic to human AVICs and may be involved in the cell-based disease process of calcific aortic stenosis.

OxLDL and biglycan interact in vascular disease. In this regard, biglycan is shown to retain oxLDL, and oxLDL is found to up-regulate biglycan expression in vascular smooth muscle cells 21. Our recent study found that oxLDL up-regulates BMP-2 expression in human coronary artery endothelial cells through a mechanism involving TLR2 and TLR4 20. In the present study, we found oxLDL not only induces the expression of biglycan mRNA and protein, but also promotes biglycan release in human AVICs. Since soluble biglycan up-regulates the expression of BMP-2 and ALP via TLR2, it is likely that biglycan plays a role in mediating the pro-osteogenic effect of oxLDL in human AVICs. Indeed, we found that either knockdown or neutralization of biglycan reduced the effect of oxLDL on the expression of BMP-2 and ALP in human AVICs. Thus, biglycan may play a role in mediating the effect of oxLDL on aortic valve calcification.

Conclusion

Human AVICs of stenotic valves express greater levels of biglycan. Soluble biglycan induces the expression of BMP-2 and ALP in human AVICs primarily through interaction with TLR2 and activation of the ERK1/2 pathway. Moreover, oxLDL promotes biglycan expression and release in human AVICs, and biglycan mediates the pro-osteogenic effect of oxLDL on human AVICs. These findings suggest that biglycan and oxLDL may contribute to the mechanism underlying the development and progression of calcific aortic valve disease through up-regulation of AVIC expression of BMP-2 and ALP.

Supplementary Material

Acknowledgments

Sources of Funding

This study was supported in part by National Institutes of Heart, Lung and Blood Grant HL106582 and American Heart Association grant 11GRNT7900016.

Footnotes

This study is presented at the 2012 ATVB Scientific Sessions.

Disclosures

None.

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