Abstract
Background.
Proinflammatory activation of toll-like receptor-4 (TLR4) drives phenotypic changes in aortic valve interstitial cells (AVICs) and produces a fibrogenic phenotype that mediates valvular fibrosis and contributes to aortic stenosis. Prior work identified upregulated Wnt signaling in AVICs taken from valves affected by aortic stenosis. Our purpose was to determine the contribution of Wnt signaling to TLR4-dependent fibrogenic activity in isolated human AVICs.
Methods.
Human AVICs were isolated from hearts explanted for cardiac transplantation (N = 4). To test whether Wnt signaling contributed to TLR4-dependent fibrogenic activity, AVICs were treated with Wnt inhibitor (Dkk1) prior to TLR4 activation (LPS) and fibrogenic markers assessed. To determine the mediator of TLR4-to-Wnt signaling, expression of the key Wnt ligand, Wnt3a, was assessed after TLR4 activation and neutralizing antibodies confirmed the identity of the mediator. Fibrogenic activity was assessed after AVICs were treated with recombinant Wnt3a. Statistics were by analysis of variance (P < .05).
Results.
TLR4 activation upregulated in vitro collagen deposition, type IV collagen and MMP2 expression, and Dkk1 inhibited these responses (P < .05). Expression of Wnt3a was upregulated after TLR4 activation (P < .05). Anti-Wnt3a neutralizing antibodies abrogated TLR4-dependent type IV collagen and MMP2 expression (P < .05). Wnt3a upregulated type IV collagen and MMP2 expression independent of TLR4 activation (P < .05).
Conclusions.
This study found that TLR4-dependent fibrogenic activity was mediated through Wnt signaling. The mediator of profibrogenic TLR4-to-Wnt signaling was a key Wnt ligand, Wnt3a. The abrogation of TLR4-induced fibrogenic activity in human AVICs by Wnt blockade illustrates a potential therapeutic role for Wnt inhibition in treatment and/or prevention of aortic stenosis.
Although aortic stenosis has heretofore been considered a degenerative disease of aging, recent data suggest that it is an active disease of chronic inflammation. The pathology of aortic stenosis is characterized by aortic valve leaflets that are fibrotic and heavily calcified. Histologic evaluation of aortic valve leaflets demonstrates the presence of chronic inflammatory cells1 and bone-forming proteins.2 The aortic valve interstitial cell (AVIC) has been implicated as central to this inflammatory disease. In response to proinflammatory stimuli, the AVIC undergoes a phenotypic change from that of a myofibroblast to that of an activated-fibroblast and an osteoblast-like cell.2–4 This osteoblast-like phenotype is characterized by the production of bone-forming proteins and the production of calcium phosphate granules.2,5 The activated-fibroblast phenotype is characterized by the production of collagen and matrix metalloproteinases (MMPs).6 Our laboratory has previously demonstrated that isolated human AVICs express receptors that mediate the actions of such inflammatory stimuli.1 In particular, toll-like receptor 4 (TLR4) plays an especially important role in mediating these phenotypic changes.1–4
Collagen is a protein with great tensile strength, a key component of connective tissue, and is dramatically upregulated in fibrotic processes. Similarly, MMPs are upregulated in diseases that involve fibrosis. MMPs facilitate extracellular matrix (ECM) turnover via degradation of proteinaceous structures. In fibrotic processes, upregulation of MMPs weakens the vascular basement membrane and enables immune cells to infiltrate involved tissues and incite additional inflammation and fibrosis.7 Further, in fibrotic processes heightened MMP activity is accompanied by exaggerated production of ECM components such as collagen.6,7 In the case of aortic stenosis, these processes eventuate in the valvular fibrosis that is characteristic of the disease.6,7
Wnt/β-catenin signaling is upregulated in AVICs isolated from diseased human aortic valves.5 In other tissues, Wnt/β-catenin signaling serves diverse functions, including regulation of cellular proliferation and bone development.8 It has been identified as a critical pathway that mediates fibrosis in a myriad of organ systems and disease processes.8,9 In addition to calcification, fibrosis of valvular leaflets is a hallmark of aortic stenosis. Interestingly, the mechanism underlying Wnt signaling and fibrosis appears to involve Wnt-induced phenotypic changes, resulting in cells with elevated fibrogenic activity.9
Given that Wnt/β-catenin signaling is important for fibrosis of other tissues, we hypothesized that it has a role in the fibrosis of aortic stenosis. We further hypothesized that TLR4 signaling interacts with Wnt signaling. The purpose of this study was to determine the mechanism of interaction between TLR4 and Wnt signaling pathways in the pathogenesis of fibrosis of aortic stenosis in isolated human AVICs.
Material and Methods
Overview of Methodology and Results
Dickkopf Wnt signaling pathway inhibitor 1 (Dkk1) is an endogenous secreted protein that plays a regulatory role in embryogenesis and modulates bone formation in adults.10 Dkk1 binds a key component of the Wnt receptor complex (LRP6) at the cell surface, resulting in its internalization, and consequently inhibits Wnt signaling.10 Accordingly, this protein was used as the Wnt signaling inhibitor in experiments (Figure 1). In this study, lipopolysaccharide (LPS) was applied to human AVICs in vitro and fibrogenic markers were assessed and found to be elevated. To test whether Wnt signaling mediated these results, AVICs were treated with Wnt inhibitor (Dkk1) prior to treatment with LPS and fibrogenic markers did not increase as before. Because Wnt inhibition abrogated the expression of fibrogenic markers induced by LPS, we probed for a key Wnt ligand, Wnt3a, to determine the role that it played in mediated LPS-induced expression of fibrogenic markers and found that LPS upregulated the expression of this ligand in AVICs. To confirm Wnt3a was the mediator of LPS-induced expression of fibrogenic markers, AVICs were treated with recombinant human Wnt3a and results demonstrated elevated expression of fibrogenic markers in AVICs, similar to the effects of LPS. Finally, as a second confirmatory test to prove Wnt3a was the mediator of LPS-induced expression of fibrogenic markers, AVICs were treated with anti-Wnt3a neutralizing antibodies prior to stimulation with LPS and fibrogenic markers again did not increase as before. Overall, these findings suggested that AVICs upregulated Wnt3a expression after treatment with LPS and Wnt3a mediated the observed LPS-induced increase in expression of fibrogenic markers in AVICs.
Figure 1.
Overview of methodology and results. From left to right in the figure: Lipopolysaccharide (LPS) was applied to human aortic valve interstitial cells (AVICs) in vitro and fibrogenic markers were assessed and found to be elevated. To test whether Wnt signaling mediated these results, AVICs were treated with Wnt inhibitor (Dickkopf Wnt signaling pathway inhibitor 1 [Dkk1]) prior to treatment with LPS and fibrogenic markers did not increase as before. Because Wnt inhibition abrogated the expression of fibrogenic markers induced by LPS, we probed for a key Wnt ligand, Wnt3a, to determine the role that it played in mediated LPS-induced expression of fibrogenic markers and found that LPS upregulated the expression of this ligand in AVICs. To confirm Wnt3a was the mediator of LPS-induced expression of fibrogenic markers, AVICs were treated with recombinant human Wnt3a and results demonstrated elevated expression of fibrogenic markers in AVICs, similar to the effects of LPS. Finally, as a second confirmatory test to prove Wnt3a was the mediator of LPS-induced expression of fibrogenic markers, AVICs were treated with anti-Wnt3a neutralizing antibodies prior to stimulation with LPS and fibrogenic markers again did not increase as before. Overall, these findings suggested that AVICs upregulated Wnt3a expression after treatment with LPS and Wnt3a mediated the observed LPS-induced increase in expression of fibrogenic markers in AVICs.
Cell Isolation and Culture
All patients gave informed consent for use of their aortic valves for this study, approved by the University of Colorado Denver institutional review board. Aortic valve interstitial cells were isolated and cultured as described previously1 with the following modifications: (1) valve leaflets were subjected to an initial digestion with a high concentration of collagenase (2.5 mg/mL) to remove endothelial cells (Sigma-Aldrich, St. Louis, MO); (2) then the remaining tissue was treated with a low concentration of collagenase (0.8 mg/mL) to free the interstitial cells; (3) finally, cells were collected by centrifugation and cultured in M199 growth medium (Lonza, Walkersville, MD) containing penicillin G, streptomycin, amphotericin B, and 10% fetal bovine serum (Aleken Chemicals, Nash, TX). Aortic valve interstitial cell isolates obtained by using this modified protocol are free of endothelial cells as verified by Von Willebrand factor staining.1 Each isolate from a separate aortic valve is used as a cell line. Cells of passages 3–6 were used for this study and were treated when they reached 80% to 90% confluence.
Immunoblotting
Immunoblotting was applied to analyze levels of type IV collagen (abcam, Cambridge, MA), MMP2 (Cell Signaling, Danvers, MA), Wnt3a (R&D Systems, Minneapolis, MN), and β-actin (Cell Signaling). Cells were lysed in sample buffer (100 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 0.02% bromophenol blue, and 10% glycerol). Protein samples were separated on gradient (4%−20%) mini-gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% skim milk solution for 1 hour at room temperature, incubated with a primary antibody against a protein of interest, and subsequently incubated with a peroxidase-linked secondary antibody specific to the primary antibody applied (Sigma-Aldrich). Chemiluminescent reagents (Thermo Scientific, Rockford, IL) were used to expose protein bands in membranes and Image J (Wayne Rasband, National Institutes of Health, Bethesda, MD) was used to analyze band density. See Supplemental Materials for additional in depth detail regarding experiments.
Statistical Analysis
Data have been normalized to control groups and presented as bar graphs in which bars represent mean values + standard error. Statistical analysis was performed using GraphPad Prism software (La Jolla, CA). Analysis of variance with the post hoc Bonferroni/Dunn test was used to analyze differences between experimental groups, and a Student t test was applied to compare data between groups. Normal distribution of data was confirmed by the Pearson test and, if not met, a nonparametric Mann-Whitney test was used instead of the t test. Normal and non-normal data are presented as bar graphs in which bars represent mean values + standard error. Statistical significance was defined as P less than .05.
Results
Donor Information
Normal aortic valve leaflets were obtained from 4 patients with the diagnosis of idiopathic dilated cardiomyopathy who underwent heart transplant at the University of Colorado Hospital. At the time of excision, valve leaflets were grossly normal: thin, pliable, and with no evidence of calcification or fibrosis. AVICs isolated from donor valves were found to be phenotypically “normal” and did not display upregulated expression of collagen or MMP production.
Effect of Dkk1 on TLR4-dependent Type IV Collagen and MMP2 Expression
To test whether Wnt inhibitor Dkk1 downregulated TLR4-induced type IV collagen and MMP2 expression, AVICs were treated with TLR4 agonist LPS (0.2 μg/mL) for 72 hours with or without pretreatment with Dkk1 (0.2 μg/mL). Activation of TLR4 resulted in elevated expression of type IV collagen and MMP2 (P < .05) and Dkk1 inhibited this response (P < .05) (Figure 2A).
Figure 2.
Dickkopf Wnt signaling pathway inhibitor 1 (Dkk1) inhibited toll-like receptor 4 (TLR4)-dependent fibrogenic activity. (A) Human aortic valve interstitial cells (AVICs) were treated with TLR4 agonist (lipopolysaccharide [LPS], 0.2 μg/mL) for 72 hours with and without Wnt inhibitor (Dkk1, 0.2 μg/mL). Type IV collagen and matrix metalloproteinase 2 (MMP2) expression were assessed by immunoblotting. TLR4 activation upregulated type IV collagen and MMP2 expression (*P < .05 vs Control, N = 4 donors). Dkk1 inhibited TLR4-dependent type IV collagen and MMP2 expression (#P < .05 vs LPS, N = 4 donors). (B) Human AVICs were treated with TLR4 agonist (LPS, 0.2 mg/mL) every 3 days for 10 days with and without Wnt inhibitor (Dkk1, 0.2 μg/mL). Picrosirius red staining was used to assess in vitro collagen deposition via a 10x objective lens on a light microscope. TLR4 activation upregulated collagen deposition (*P < .05 vs Control, N = 4 donors) and Dkk1 inhibited TLR4-dependent in vitro collagen deposition (#P < .05 vs LPS, N = 4 donors).
In Vitro Collagen Deposition Assay
To confirm Wnt inhibition downregulates TLR4-dependent in vitro collagen deposition, AVICs were incubated in cell culture media (2.5% fetal bovine serum) with LPS (0.2 μg/mL) with or without Dkk1 (0.2 μg/mL) for a period of 10 days. In vitro collagen deposition was assessed by Proics-irius red staining. Activation of TLR4 increased collagen deposition (P < .05) and Dkk1 blocked TLR4-mediated in vitro collagen deposition (P < .05) (Figure 2B).
Wnt3a Expression After TLR4 Activation
To determine whether TLR4 activation affected expression of a Wnt ligand, AVICs were treated with LPS (0.2 μg/mL) for 72 hours and expression of a key Wnt ligand, Wnt3a, was assessed at various time points (30 minutes, 2 hours, 4 hours, 6 hours, and 8 hours). Activation of TLR4 upregulated Wnt3a expression at all time points (P < .05) with peak expression of Wnt3a occurring at 4 hours after TLR4 activation (Figure 3).
Figure 3.
Toll-like receptor 4 (TLR4) activation induced Wnt3a expression. Human aortic valve interstitial cells were treated with TLR4 agonist (lipopolysaccharide, 0.2 mg/mL) and expression of Wnt3a was assessed by immunoblotting at various time points. TLR4 activation induced Wnt3a expression (*P < .05 vs Control [C], N = 4 donors).
Effect of Anti-Wnt3a Neutralizing Antibodies on TLR4-dependent Type IV Collagen and MMP2 Expression
To confirm Wnt3a was the mediator of TLR4-dependent type IV collagen and MMP2 expression, AVICs were treated with anti-Wnt3a neutralizing antibodies (5 μg/mL) vs IgG control (5 μg/mL) prior to TLR4 activation. TLR4 activation in the presence of IgG control upregulated type IV collagen and MMP2 levels (P < .05), while anti-Wnt3a neutralizing antibodies abrogated TLR4-dependent type IV collagen and MMP2 expression (P < .05) (Figure 4).
Figure 4.
Neutralization of Wnt3a abrogates toll-like receptor 4 (TLR4)-dependent type IV collagen and matrix metalloproteinase 2 (MMP2) expression. Human aortic valve interstitial cells were treated with TLR4 agonist (lipopolysaccharide [LPS], 0.2 μg/mL) and expression of type IV collagen and MMP2 were assessed by immunoblotting at various time points. TLR4 activation upregulated type IV collagen and MMP2 expression (*P < .05 vs Control, N = 4 donors). Anti-Wnt3a neutralizing antibodies (5 ug/mL) inhibited TLR4-dependent type IV collagen and MMP2 expression (#P < .05 vs LPS + IgG, N = 4 donors). Abbreviations: Anti-(Wnt3a Ab, Anti-Wnt3a neutralizing antibodies; IgG, Immunoglobulin G.)
Influence of Recombinant Wnt3a on Type IV Collagen and MMP2 Expression
To determine whether Wnt signaling upregulates type IV collagen and MMP2 expression independent of TLR4 activation, AVICs were treated with recombinant Wnt3a (0.5 μg/mL) for 72 hours. Wnt3a upregulated type IV collagen and MMP2 expression (P < .05) (Figure 5).
Figure 5.
Recombinant Wnt3a induced type IV collagen and matrix metalloproteinase 2 (MMP2) expression in aortic valve interstitial cells AVICs. Human AVICs were treated with recombinant Wnt3a (0.5 μg/ml) for 72 hours and type IV collagen and MMP2 expression were assessed by immunoblotting. Recombinant Wnt3a induced type IV collagen and MMP2 expression in human AVICs independent of toll-like receptor 4 activation (*P < .05 vs Control, N = 4 donors).
Comment
The results of this study demonstrated that profibrogenic signaling interaction occurred between TLR4 and Wnt signaling pathways in isolated human AVICs. Signaling from TLR4 to Wnt was mediated by TLR4-dependent expression of a key Wnt ligand: Wnt3a. Further, activation of Wnt signaling by human recombinant Wnt3a upregulated type IV collagen and MMP2 expression in AVICs independent from TLR4 signaling (Figure 5). The results of the present study therefore demonstrate a role for Wnt signaling in the pathogenesis of fibrosis in aortic stenosis.
Wnt signaling has been shown to play a role in the pathogenesis of several cardiovascular diseases.11–15 Fu and colleagues15 demonstrated that Wnt inhibition played a key role in myocardial recovery after myocardial infarction, and others have reported evidence of interaction between TLR4 and Wnt signaling. For example, inhibition of Wnt signaling by a Wnt inhibitor, Dkk1, blocked TLR4-mediated inflammatory phenotypic changes in human cells16 and osteoblastic differentiation in bone progenitor cells.17,18 Interestingly, inflammatory and osteogenic phenotypic changes represent 2 key pathobiological processes in AVICs that are believed to contribute to development of aortic stenosis.
Therapeutic potential for Wnt inhibitors in the treatment of aortic stenosis lies in the fact that Wnt signaling has been found to be heightened in diseased tissue. Deng and associates5 reported that LRP6 and β-catenin, 2 important components of the Wnt signaling pathway, were upregulated in AVICs taken from valves of patients diagnosed with aortic stenosis. These AVICs were also found to have undergone osteoblastic phenotypic changes and exhibited elevated expression of bone-forming proteins, such as Runt-related transcription factor-2 and bone morphogenetic protein-2. A large body of prior work demonstrated a key role for TLR4 in mediating inflammatory and osteoblastic phenotypic changes in human AVICs.3,4,19,20 In light of the findings in the present study, Wnt signaling may play a role in TLR4-dependent osteogenic and inflammatory activity in AVICs as well.
With the availability of a myriad of US Food and Drug Administration–approved Wnt inhibitors for various noncardiovascular indications, it is possible that 1 of these drugs may have therapeutic potential for aortic stenosis. A number of Wnt inhibitors are involved in clinical trials for treatment of various cancers.21 However, Wnt signaling functions ubiquitously throughout the human body and has a wide range of effects on different tissues. Accordingly, systemic delivery of a Wnt inhibitor may have unwanted effects that are specific to a drug target.22 For example, a number of oral Protein-serine O-palmitoleoyltransferase porcupine (PORCN) inhibitors are involved in clinical trials to treat cancer.22 PORCN is a signaling protein in the Wnt pathway. These drugs have side effects specific to inhibition of PORCN signaling: increased bone resorption resulting in decreased bone mass. PORCN regulates bone remodeling in the skeleton. Nonetheless, the results of the present study suggest that Wnt signaling may be a therapeutic target for aortic stenosis.
This study has several limitations. First, this in vitro study of isolated human AVICs cannot approximate the dynamic microenvironment of the aortic valve in vivo. Second, AVICs taken from “normal” valve leaflets were harvested from patients who had dilated cardiomyopathy. Although valves had no gross evidence of disease at time of excision, it is possible AVICs taken from these valves may not be normal. Third, this study is an in vitro semiquantitative study that requires several repeats, commonly 2 to 3, to validate the reproducibility of observations. To normalize for potential donor variability, experiments were repeated with cell isolates from 4 different donors. Nevertheless, small sample size is a limitation of this study. Finally, the cellular and molecular pathobiology underlying development of aortic stenosis is complex. Although Wnt-inhibition in this study was found to prevent TLR4-mediated expression of specific markers that represent phenotypic changes in AVICs, the true pathobiology that results in aortic stenosis likely involves additional signaling pathways and mechanisms. Toll-like receptor 2 (TLR2) has also been found to be a potential mediator of some pathophysiology underlying aortic stenosis: Oxidized low-density lipoprotein has been found to induce elevated expression of bone-forming proteins in AVICs via TLR2 signaling.23 Further, other pathways such as the mitogen-activated protein kinase (MAPK) pathway, have been implicated in aortic stenosis.24 However, many of these pathways (including TLR2 and MAPK) have been found to interact with the TLR4 pathway downstream to affect gene transcription. Hence, this study focused on Wnt signaling and its interactions with the TLR4 pathway. More complex mechanisms may govern development of aortic stenosis, however, including genetics, flow dynamics across the valve, and immune cells.23 Ultimately, in vivo studies will need to be done to determine the true impact of potential pathophysiologic mechanisms discovered in vitro. Accordingly, future studies will include an in vivo animal model of aortic stenosis in which US Food and Drug Administration–approved Wnt inhibitors are used both in a preventative fashion and in effort to treat established disease.21 The in vivo model may involve a mouse with induced renal failure to accelerate formation of aortic stenosis. Wnt inhibitors that target LRP6, β-catenin, and Wnt3a should be included to confirm the in vitro findings of the current study.
In summary, this study found profibrogenic TLR4-to-Wnt signaling, mediated by Wnt3a, is the mechanism that governs TLR4-dependent type IV collagen and MMP2 expression in human AVICs. These data provide mechanistic insights into the pathobiology underlying development of aortic stenosis, specifically the fibrotic component of the disease, and suggest a potential therapeutic role for Wnt inhibition in the treatment and/or prevention of aortic stenosis.
Supplementary Material
Acknowledgments
This work was supported by the National Institutes of Health (grant no. NIH RO1 HL106582).
Footnotes
The Supplemental Material can be viewed in the online version of this article[https://doi.org/10.1016/j.athoracsur.2020.08.068] on http://www.annalsthoracicsurgery.org.
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