Author + information
- Received July 22, 2008
- Revision received September 22, 2008
- Accepted September 29, 2008
- Published online February 10, 2009.
- Xiaoping Yang, PhD,
- David A. Fullerton, MD,
- Xin Su, MD,
- Lihua Ao, BS,
- Joseph C. Cleveland Jr, MD and
- Xianzhong Meng, MD, PhD⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Xianzhong Meng, Department of Surgery, Box C-320, 12700 East 19th Avenue, Aurora, Colorado 80045
Objectives Our aim was to determine whether aortic valve interstitial cells (AVICs) and pulmonary valve interstitial cells (PVICs) differ in expression of toll-like receptor (TLR)2 and TLR4, response to TLR agonists, and osteogenic phenotypic changes.
Background Calcific stenosis occurs frequently in aortic valves but rarely in pulmonary valves. Studies have implicated AVICs in the inflammation associated with calcification and progression to stenosis. We previously reported that human AVICs express functional TLR2 and TLR4 and that stimulation of these receptors induces pro-osteogenic factor expression.
Methods Human aortic and pulmonary valve leaflets from the same heart were collected and interstitial cells isolated.
Results Aortic valves express more TLR2 and TLR4, in both tissue and isolated interstitial cells, than pulmonary valves. After stimulation with TLR2 and TLR4 agonists, AVICs express higher levels of pro-inflammatory and pro-osteogenic mediators (bone morphogenetic protein [BMP]-2, runt-related transcription factor 2) and greater osteogenic phenotypic changes (alkaline phosphatase [ALP] activity, calcified nodule formation) than PVICs. Silencing TLR2 and TLR4 in AVICs reduced BMP-2 expression and ALP activity to PVIC levels. ALP activity in AVICs induced by TLR2 and TLR4 agonists was abolished by BMP antagonism with Noggin and mimicked by stimulation with recombinant BMP-2. AVICs isolated from stenotic valves had greater expression of TLR2 and TLR4 and a greater BMP-2 response than AVICs from normal valves.
Conclusions 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.
Aortic valve calcification, which affects 27% of the U.S. population older than age 60 years, often progresses to calcific aortic stenosis (1). Despite its poor prognosis, the only therapy currently available for severe, symptomatic aortic stenosis is valve replacement. Several mechanisms for heart valve calcification have been proposed, including matrix remodeling, lipid accumulation in the valve, and dysfunction of the renin-angiotensin system (2). Clinical trials of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are underway, but none has yet shown a significant benefit (3).
Several studies have demonstrated an association between inflammation and aortic valve calcification (4). Macrophages, T-lymphocytes, and pro-inflammatory mediators such as interleukin-1, transforming growth factor-beta, and tumor necrosis factor-alpha have all been found in calcified human heart valves (2). But it is not yet clear that inflammation causes valve calcification or what molecular mechanisms may be involved. We recently found that human aortic valve interstitial cells (AVICs) express functional toll-like receptor (TLR)2 and TLR4 (5), important mediators of the innate immune response and inflammation.
Valve interstitial cells (VICs), the major cellular components of heart valve leaflets, have the dual ability to secrete matrix components and to maintain valvular contractile function (6). AVICs stimulated with TLR agonists up-regulate bone morphogenetic protein (BMP)-2 and runt-related transcription factor 2 (Runx2) (5), which have both been found in calcified valve leaflets (7,8). In addition, AVICs stimulated with BMP-2 up-regulate alkaline phosphatase (ALP) (9), and formation of calcified nodules in VICs is dependent on ALP activity (10). But the mechanisms by which AVICs up-regulate these pro-osteogenic factors are unclear. Because calcification occurs frequently in aortic valves but rarely in pulmonary valves, comparing pro-osteogenic signaling in AVICs and pulmonary valve interstitial cells (PVICs) may provide mechanistic insights.
We hypothesize that differential TLR expression and differential response to TLR agonists distinguish AVICs from PVICs and are associated with a pro-osteogenic phenotype. In this study we will examine whether: 1) human aortic and pulmonary valves, both tissue and isolated interstitial cells, express different levels of TLR2 and TLR4; 2) stimulation of TLR2 and TLR4 results in differential expression of pro-inflammatory and pro-osteogenic factors in AVICs and PVICs; 3) reducing cellular TLR2 and TLR4 levels via silencing influences the pro-osteogenic response in AVICs; 4) BMP-2 links TLR2 and TLR4 to osteogenic phenotypic changes; and 5) TLR2, TLR4, and BMP-2 expression differ in AVICs isolated from normal versus stenotic valves.
Cell isolation and culture
Normal aortic and pulmonary valves were collected from 4 explanted hearts of heart transplant recipients and 2 unusable donor hearts (from 4 males and 2 males, mean age 58.3 ± 6.6 years). Explanted hearts were from patients of the University of Colorado Hospital or the Denver Veterans Administration Medical Center with cardiomyopathy but no history of heart valve disease. Stenotic aortic valves were collected from 3 patients who underwent valve replacement surgery for calcific aortic stenosis (1 male/2 female patients, mean age 54.7 ± 16.2 years). All patients gave written informed consent, and this study was approved by the Colorado Multiple Institutional Review Board.
After harvest, aortic and pulmonary valve leaflets were processed as previously described (5). All stenotic valves had overt calcium deposits, and interstitial cells were isolated from soft tissue adjacent to calcium deposits.
AVICs and PVICs were isolated and cultured as previously described (5). Cells from passages 3 to 7 at approximately 90% confluence were used for all experiments. For certain experiments, cells were cultured in conditioning medium (growth medium with 10 mmol/l beta-glycerophosphate, 10 nmol/l vitamin D3, and 10 nmol/l dexamethasone), a modification of the osteogenic medium used by Osman et al. (9). The medium was changed every 3 days. All comparative experiments on AVICs and PVICs used cells isolated from the same heart. Cells were treated with peptidoglycan (Staphylococcus aureus, 10 μg/ml), lipopolysaccharide (Escherichia coli 0111:B4, 200 ng/ml), recombinant human BMP-2 (100 ng/ml, R&D Systems, Minneapolis, Minnesota), and/or Noggin (470 ng/ml, R&D Systems) for the time course indicated. Unless otherwise stated, reagents were purchased from Sigma (St. Louis, Missouri).
Protein samples were separated on 4% to 20% minigels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, California). Proteins were detected using primary antibodies to TLR2 and TLR4 (Imgenex, San Diego, California), BMP-2 (ProSci, Poway, California), BMP-4 (Assay Designs, Ann Arbor, Michigan), or Runx2 (Novus Biologicals, Littleton, Colorado) and corresponding peroxidase-linked secondary antibodies (Cell Signaling Technology, Danvers, Massachusetts). Blots were developed with enhanced chemiluminescence reagent and a computerized densitometer (Molecular Dynamics, Sunnyvale, California) was used to measure band density.
Cell-surface expression of TLR2 and TLR4 was detected using phycoerythrin-conjugated TLR2 and TLR4 antibodies (Imgenex) as previously described (11).
Chemokine levels in cell culture medium were determined using a BioPlex immunoassay (Bio-Rad Laboratories).
Silencing TLR2 and TLR4
Silencing TLR2 and TLR4, using TLR2 or TLR4 small interfering ribonucleic acid (60 nmol/l, Dharmacon, Lafayette, Colorado), was performed as previously described (11). Table 1 lists sequences of sense and antisense siRNA nucleotides.
ALP activity assay and staining
For the assay, cells were lysed with lysis buffer (20 mmol/l Tris-HCl, 150 mmol NaCl, 0.2% NP40, and 10% glycerol), pH 8.0, and cell lysates (<5 μg protein) were incubated in a buffer (0.1 mol/l glycylglycine, 10 mmol/l MgCl2, 10 mmol/l p-nitrophenyl phosphate), pH 9.5, for 1 h at 37°C. Color development was stopped with 0.5 N NaOH, and absorbance was measured at 405 nmol/l. ALP activity was calculated against a standard curve and normalized by protein concentration. Results are expressed in 10−6 Sigma units/mg protein (μU/mg protein). For the stain, cells were fixed and stained as previously described (12) and photographed with a Nikon Eclipse TS100 microscope (Melville, New York).
Alizarin red S stain
Cells were fixed and stained with alizarin red S as previously described (13) and photographed with a Nikon Eclipse TS100 microscope. Cryosections (5-μm thick) of calcified human aortic valves, stained by the same method, served as positive controls.
Data are presented as mean ± standard error of the mean. Analysis of variance with a post-hoc Bonferroni/Dunn test was performed to analyze differences between experimental groups, and differences were confirmed with Kruskal-Wallis and Mann-Whitney U tests. Statistical significance was accepted within a 95% confidence limit.
Expression of TLR2 and TLR4 is greater in aortic valves than in pulmonary valves
We compared TLR2 and TLR4 protein levels in aortic and pulmonary valves from the same human heart. TLR2 and TLR4 levels in aortic valve tissue homogenate were 1.7 and 2.4× (both p < 0.05), respectively, those in pulmonary valve tissue homogenate (Fig. 1A). TLR2 and TLR4 expression in AVIC lysates were 1.9 and 2.6× (both p < 0.05), respectively, those in PVIC lysates (Fig. 1B). Cell-surface levels of TLR2 and TLR4 in AVICs were 2.0 and 1.8× (both p < 0.05), respectively, those in PVICs (Fig. 1C).
AVICs exhibit a stronger pro-inflammatory response to TLR2 and TLR4 stimulation than PVICs
We then assessed whether greater TLR2 and TLR4 expression in AVICs results in a more robust inflammatory response. After stimulating AVICs and PVICs for 24 h with the TLR2 and TLR4 agonists peptidoglycan and lipopolysaccharide, respectively, chemokine release from AVICs was greater than from PVICs (Table 2). Release of interleukin-8, monocyte chemoattractant protein-1, interferon-inducible protein-10, and RANTES (regulated upon activation, normal T-cell expressed and secreted) was significantly higher from AVICs than PVICs after stimulation with either peptidoglycan or lipopolysaccharide, whereas release of macrophage inflammatory protein-1α and MIP-1β was significantly greater after lipopolysaccharide but not peptidoglycan stimulation. Therefore, AVICs have a stronger pro-inflammatory response to TLR2 and TLR4 activation than PVICs.
Stimulating TLR2 and TLR4 induces BMP-2 and Runx2 in AVICs, but not in PVICs
Next we assessed whether greater TLR2 and TLR4 expression in AVICs results in greater expression of the pro-osteogenic factors BMP-2, BMP-4, and Runx2. Stimulating AVICs with peptidoglycan and lipopolysaccharide for 24 h up-regulated BMP-2 expression to 4.7 and 4.1× control, and Runx2 expression to 5.3 and 4.7× control, respectively (Fig. 2A). Stimulation with peptidoglycan and lipopolysaccharide did not greatly influence BMP-2 or Runx2 expression in PVICs (Fig. 2A), nor did it influence BMP-4 expression in either AVICs or PVICs (Fig. 2B).
Silencing TLR2 and TLR4 in AVICs attenuates peptidoglycan- and lipopolysaccharide-induced BMP-2 expression
Since the higher TLR2 and TLR4 levels in AVICs are associated with greater BMP-2 expression, we then examined whether reducing TLR2 and TLR4 levels in AVICs by silencing would reduce the BMP-2 response to peptidoglycan and lipopolysaccharide stimulation. Treating AVICs with siRNA reduced TLR2 and TLR4 levels by 41% and 57%, respectively (Fig. 2C). Silencing TLR2 reduced peptidoglycan-stimulated BMP-2 expression 42%, and silencing TLR4 reduced lipopolysaccharide-stimulated BMP-2 expression 62%, compared with controls (Fig. 2D). Therefore, reducing TLR2 and TLR4 levels in AVICs to their levels in PVICs also reduced BMP-2 expression to PVIC levels.
Stimulating TLR2 and TLR4 promotes ALP activity in AVICs
Because peptidoglycan and lipopolysaccharide up-regulated BMP-2 and Runx2 in AVICs, we assessed whether they up-regulate ALP activity, a biomarker of early osteogenesis (14). After stimulating AVICs and PVICs with peptidoglycan and lipopolysaccharide for 14 days, ALP activity did not increase (Fig. 3A).
Others have reported that culturing AVICs in conditioning medium can induce ALP activity (9,10). Indeed, culturing our AVICs and PVICs in conditioning medium increased ALP activity, and stimulation with peptidoglycan and lipopolysaccharide increased ALP activity further (Fig. 3B). ALP activity in AVICs cultured in conditioning medium was significantly greater than in AVICs cultured in growth medium (1.10 ± 0.07 μU/mg vs. 0.47 ± 0.03 μU/mg protein, p < 0.05) and further increased by stimulation with peptidoglycan and lipopolysaccharide (2.07 ± 0.17 μU/mg and 2.11 ± 0.15 μU/mg protein, respectively, both p < 0.01 vs. AVICs cultured in conditioning medium alone) (Fig. 3B). Stimulation with peptidoglycan and lipopolysaccharide also enhanced ALP activity in PVICs grown in conditioning medium but not significantly (p > 0.05). More importantly, ALP activity in peptidoglycan- and lipopolysaccharide-stimulated AVICs was significantly greater (p < 0.01) than in PVICs (1.06 ± 0.19 μU/mg and 1.06 ± 0.18 μU/mg protein, respectively) (Fig. 3B).
Silencing TLR2 and TLR4 attenuates peptidoglycan- and lipopolysaccharide-induced ALP activity
Our earlier results and our previous report (5) demonstrate that stimulating AVICs with TLR2 and TLR4 agonists is associated with expression of pro-osteogenic mediators. Therefore, we reduced TLR2 and TLR4 levels via silencing to determine whether TLR2 and TLR4 are linked to ALP expression and whether the ALP response correlates to TLR2 or TLR4 levels in AVICs. Peptidoglycan and lipopolysaccharide stimulation increased ALP activity to twice that of cells grown in conditioning medium alone (Fig. 3B), but silenced TLR2 and TLR4 reduced ALP activity by one-half, to the level of cells grown in conditioning medium alone (Fig. 3C).
Stimulating TLR2 and TLR4 in AVICs promotes calcified nodule formation
To determine whether TLR stimulation increases cellular calcification, we stimulated cells grown in conditioning medium with peptidoglycan or lipopolysaccharide for 28 days. Peptidoglycan and lipopolysaccharide promoted calcified nodule formation in AVICs and PVICs cultured in conditioning medium, but more and larger nodules were formed in AVICs than in PVICs (Fig. 4). Neither peptidoglycan nor lipopolysaccharide induced calcification in cells cultured in growth medium (not shown).
BMP-2 is involved in the enhancement of ALP activity by TLR stimulation in AVICs
Because peptidoglycan- and lipopolysaccharide-stimulated AVICs up-regulate both BMP-2 protein expression and ALP activity, we assessed whether BMP-2 is involved in up-regulating peptidoglycan- and lipopolysaccharide-induced ALP activity in AVICs. When AVICs stimulated with peptidoglycan and lipopolysaccharide were treated with the BMP antagonist Noggin, ALP activity decreased to 1.13 ± 0.03 μU/mg and 0.96 ± 0.03 μU/mg protein, respectively, (both p < 0.05 vs. stimulated AVICs without Noggin treatment), which is similar to the level detected in unstimulated AVICs (1.10 ± 0.07 μU/mg protein in AVICs cultured in conditioning medium alone) (Fig. 5A).
Then we examined whether BMP-2 is sufficient to up-regulate ALP activity in AVICs. Treatment with recombinant BMP-2 did not increase ALP activity in AVICs cultured in growth medium, but significantly increased ALP activity in cells grown in conditioning medium (1.98 ± 0.19 μU/mg vs. 1.10 ± 0.07 μU/mg protein, p < 0.01) (Fig. 5B), to a level comparable to that induced by peptidoglycan or lipopolysaccharide stimulation (2.07 ± 0.17 μU/mg and 2.11 ± 0.15 μU/mg protein, respectively) (Fig. 3B).
AVICs isolated from stenotic valves exhibit greater TLR2, TLR4, and BMP-2 expression
Because our earlier results suggest that differences in TLR2 and TLR4 expression play a critical role in aortic valve calcification, we assessed TLR2 and TLR4 expression in AVICs isolated from calcified stenotic valves. TLR2 and TLR4 levels in AVICs from stenotic valves are 2.8 and 2.0×, respectively, those in normal AVICs (Fig. 6A). Basal BMP-2 expression is greater in AVICs from stenotic valves than in AVICs from normal valves. In addition, after 24-h stimulation with peptidoglycan or lipopolysaccharide, BMP-2 expression is 2.6 and 4.1× greater, respectively, in AVICs from stenotic valves than those from normal valves (Fig. 6B). AVICs from stenotic valves tend to spontaneously cluster and form nodules in long-term culture (data not shown).
It is well known that calcification occurs more frequently in aortic valves than in pulmonary valves, but the underlying mechanisms are unclear. It has been proposed that aortic valve calcification is related to the higher oxygen tension and greater transvalvular pressure present on the left side of the heart. A few studies have found differences in vimentin distribution (15) and smooth muscle alpha-actin expression (16) in aortic and pulmonary valves. In this study, using aortic and pulmonary valves from the same heart, we identified a novel difference between the valves: greater expression of TLR2 and TLR4 in aortic valves than pulmonary valves, whether in valve tissue, in isolated interstitial cells, or on cell surfaces. There were variations in TLR2 and TLR4 levels in different hearts, but in each heart, TLR2 and TLR4 levels were consistently greater in aortic valve tissue and cells than in pulmonary valve tissue and cells.
Not only do AVICs express more TLR2 and TLR4 than PVICs, but AVICs produce a stronger pro-inflammatory response to TLR2 and TLR4 stimulation than PVICs. Several studies have suggested that inflammation is involved in the pathogenesis of aortic valve calcification (17) and that VICs participate in the inflammatory process (6). Our results, which are the first to demonstrate that AVICs have greater pro-inflammatory potential than PVICs, suggest that AVICs play an important role in the inflammatory response in aortic valves and support the importance of inflammation in the pathogenesis of aortic stenosis.
We previously reported that stimulating human AVICs with TLR2 and TLR4 agonists up-regulates BMP-2 and Runx2 (5), which have both been found in calcified aortic valves (7,8). In this study, we found that stimulation with TLR2 and TLR4 agonists induced BMP-2 and Runx2 expression in AVICs but not in PVICs. And although TLR2 and TLR4 expression in AVICs is only twice that in PVICs, peptidoglycan- and lipopolysaccharide-induced BMP-2 and Runx2 expression is 4 to 5× greater in AVICs. Silencing TLR2 and TLR4, which halved receptor levels, reduced peptidoglycan- and lipopolysaccharide-induced BMP-2 and Runx2 expression by a factor of 2 to 3. Therefore, peptidoglycan- and lipopolysaccharide-induced BMP-2 and Runx2 expression is mainly mediated by TLR2 and TLR4.
In VICs, ALP activity and calcified nodule formation are markers of a change to an osteogenic phenotype (10,18). Stimulation with TLR2 and TLR4 agonists induced greater ALP activity and calcified nodule formation in AVICs than PVICs cultured in conditioning medium. Therefore, AVICs are more likely to be activated to a pro-osteogenic phenotype than PVICs, and pro-osteogenic phenotypic changes in human AVICs are promoted by TLR2 and TLR4 stimulation. In addition, because silencing TLR2 and TLR4 reduced ALP activity to the level in cells cultured in conditioning medium, peptidoglycan- and lipopolysaccharide-induced ALP activity is mainly mediated by TLR2 and TLR4. It should be noted that neither peptidoglycan nor lipopolysaccharide could increase ALP activity or calcified nodule formation in VICs cultured in growth medium, which does not have the additional osteogenic factors present in conditioning medium. This indicates that pro-osteogenic changes in VICs are complex and multifactorial.
AVICs stimulated with TLR2 and TLR4 agonists up-regulate both BMP-2 expression and ALP activity. When AVICs were stimulated with recombinant BMP-2, ALP activity in AVICs cultured in conditioning medium increased to a level similar to that induced by peptidoglycan or lipopolysaccharide. This confirmed the results of Osman et al. (9) under our experimental conditions. In addition, the BMP antagonist Noggin attenuated the lipopolysaccharide- and peptidoglycan-induced augmentation of ALP activity. Together, these results indicate that BMP-2 is involved in the peptidoglycan- or lipopolysaccharide-stimulated augmentation of ALP activity in AVICs. We should note that Noggin antagonizes other BMPs also, including BMP-4 (19). It is unlikely that Noggin's effects on ALP activity were mediated through BMP-4 because neither peptidoglycan nor lipopolysaccharide stimulation up-regulated BMP-4. But it is possible that other BMPs contribute to the effects of these TLR agonists on ALP activity, and that attenuation of these effects by Noggin is not only due to antagonism of BMP-2.
A comparison of AVICs from normal and stenotic valves reveals that AVICs from stenotic valves express even more TLR2 and TLR4 than normal AVICs. In addition, basal BMP-2 expression is greater in AVICs from stenotic valves than those from normal valves, and is further increased by stimulation with TLR agonists. This is consistent with studies that found greater expression of BMP-2 in calcified stenotic heart valves (7). It also demonstrates that AVICs from stenotic valves are phenotypically altered. Indeed, those AVICs tend to spontaneously form nodules in culture. Therefore, parallel experiments to examine ALP expression in response to long-term TLR stimulation are not feasible under our experimental conditions.
Among the limitations of this study are its relatively small sample size (6 normal aortic and pulmonary valves, 3 stenotic aortic valves). In addition, we examined only VICs. Although their role in valve calcification and stenosis continues to emerge, some studies suggest that endothelial damage or dysfunction initiates the process of valve calcification in vivo (20). Our study did not address the possible role of endothelial cells or other cell types in valve calcification. In addition, we used cultured VICs, which, like all cultured cells, might have changed some of the characteristics they had in vivo. We should note, however, that in our earlier study we found that levels of TLR2 and TLR4 in human AVICs were similar in newly isolated and repeatedly passaged cells (5).
Further, stimulating these cells with peptidoglycan and lipopolysaccharide is in itself an artificial experimental construct, since it is unknown whether VICs are exposed to pathogenic components in vivo. And although we used low concentrations of peptidoglycan and lipopolysaccharide, we cannot rule out TLR-independent effects of these agonists on the expression of pro-inflammatory and pro-osteogenic factors. In addition, recent studies have suggested that contaminants in a commercially prepared S. aureus-derived peptidoglycan preparation may contribute to TLR2 activation (21); however, it is agreed that this preparation indeed activates TLR2.
In this study, we demonstrated that TLR2 and TLR4 expression are greater in human aortic valves than in pulmonary valves, and that after stimulation with TLR2 and TLR4 agonists, AVICs demonstrate greater expression of pro-inflammatory and pro-osteogenic mediators and greater osteogenic phenotypic changes than PVICs. Although TLR2 and TLR4 levels in AVICs are only twice those in PVICs, stimulation with TLR agonists induces changes in pro-osteogenic mediator expression and osteogenic phenotype that are many times greater in AVICs than PVICs. Because these changes are attenuated by reducing TLR2 and TLR4 levels in AVICs to their levels in PVICs, and because TLR2 and TLR4 expression in AVICs from stenotic valves is even greater than their expression in normal AVICs, this study demonstrates that TLR2 and TLR4 play a critical role in mediating the pro-inflammatory and pro-osteogenic responses and osteogenic phenotypic changes in AVICs in vitro. Many other factors likely contribute to aortic valve calcification in vivo, including lipid infiltration, matrix protein remodeling, aging, and shear stress. But this study suggests that the TLR2- and TLR4-mediated inflammatory and osteogenic responses play an important role in the pathogenesis of aortic valve calcification and stenosis.
The authors are grateful to Dr. Helen Kim for critically reading this manuscript.
Supported by American Heart Association grant 0850148Z and the Academic Enrichment Fund of University of Colorado Denver.
- Abbreviations and Acronyms
- alkaline phosphatase
- aortic valve interstitial cell
- bone morphogenetic protein
- pulmonary valve interstitial cell
- runt-related transcription factor 2
- toll-like receptor
- valve interstitial cell
- Received July 22, 2008.
- Revision received September 22, 2008.
- Accepted September 29, 2008.
- American College of Cardiology Foundation
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