Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification
Abstract
Background:
Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling.
Methods:
Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/β receptor alpha chain (Ifnar1)–deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans.
Results:
Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn−/−, Tlr3−/−, and Ifnar1−/− mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1–BGN–TLR3–interferon-α/β receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans.
Conclusions:
This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.
Clinical Perspective
What Is New?
•
Toll-like receptor 3 (Tlr3), biglycan (Bgn), and interferon-α/β receptor alpha chain (Ifnar1) mutant mice are protected from aortic valve calcification.
•
Maturation of the extracellular matrix protein biglycan (BGN) via xylosyltransferase 1 (XYLT1) is crucial for TLR3 activation.
•
Human genetic association analysis reveals that aortic valve calcification associates with variants in the XYLT1-BGN-TLR3 pathway.
What Are the Clinical Implications?
•
Our data identify the XYLT1-BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway of morphogenesis and calcification, paving the way for novel therapeutic strategies for detecting and treating calcific aortic valve disease in humans.
Calcific aortic valve disease (CAVD) is the third leading cause of cardiovascular related disease, constituting a major socioeconomic burden in the Western world.1 Age is the principal risk factor for CAVD, and the aging of the population predicts an increased prevalence of CAVD.2 Valvular interstitial cells (VICs), the predominant cell type within the aortic valve, are specialized fibroblasts responsible for maintaining valvular function.3 Progressive aortic calcification occurs when VICs acquire the phenotype of bone-forming osteoblasts.4 At present, there are no pharmacological options for slowing or preventing the progression of CAVD.
The aortic valve is relentlessly subjected to high levels of mechanical strain and cellular injury, in turn promoting inflammation and the formation of calcific lesions.5 Endogenous alarm signals, including the release of RNA and protein from injured cells, activate Toll-like receptors (TLRs),6,7 which are typically involved in detecting pathogens.8 The first described TLRs expressed on VICs were TLR2 and TLR4. Their activation by bacterial membrane components such as lipopolysaccharide or peptidoglycan induces expression of pro-osteogenic factors, including BMP-2 and Runx2 through NOTCH1 and nuclear factor-κB.9 VICs from stenotic valves express higher levels of TLR2 and TLR4 and are more responsive to inflammatory stimuli, resulting in osteogenic activity.10 Interleukin-37 suppresses the osteogenic response on TLR2 and TLR4 activation. TLR2- and TLR4-deficient mice are protected from high-fat diet–induced changes of the aortic valve.11 Besides TLR2 and TLR4, aortic valves express TLR9.12 TLR9 recognizes CpG motifs in nucleic acids but also bind endogenous ligands, including extracellular matrix (ECM) components and lipids, and is possibly involved in sustained inflammation and subsequent valvular calcification.13 Here, we show that TLR3 is abundantly expressed in aortic valves, exhibiting a potential distinct role in the calcification process of aortic valves attributable to its unique signaling features.
TLR3 can be activated by extracellular dsRNA intermediates or byproducts of viral infection and by unknown endogenous agonists. Its activation on VICs was previously shown to induce osteogenic response through the nuclear factor-κB and extracellular signal-regulated kinase 1/2 pathways.9 Besides nuclear factor-κB, TLR3 activation appears to be a major pathway resulting in an IRF3-dependent induction of type I interferons (IFNs),8 therefore aiding in the control of physiological type I IFN levels.14
Type I IFNs have been implicated not only in the host response to viruses but also in CAVD, physiological calcification, and bone formation. Consistent with this role, Ifnb−/− and Ifnar1−/− mice, which have compromised type I IFN signaling, display osteoporosis.15,16
Human mendelian disorders associated with increases in the production of type I IFNs are known as type I interferonopathies.17 The causal genes and the cells displaying enhanced type I IFN production differ between disorders, accounting for their clinical heterogeneity. Gain-of-function mutations in the human IFIH1/MDA5 gene, which regulates the antiviral response, cause pathological vascular and valvular calcification with osteopenia in children with Singleton-Merton syndrome.18 Aicardi-Goutières syndrome, which is genetically heterogeneous, is characterized by pathological calcifications of the basal ganglia in the brain.19 Last, ADAR-related type I interferonopathy has been linked to valvular calcifications in children.20 The impact of human inherited deficiencies of type I IFNs on calcification in physiology and disease is unknown.
Here, we show that the activation of TLR3 by endogenous ligands leads to type I IFN production, promoting bone formation in young mice but also triggering the phenotypic switch to osteoblast-like cells and contributing to the pathogenesis of CAVD in aged mice. We identify biglycan (BGN) as an endogenous TLR3 ligand. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. We propose that the evolutionary advantage of TLRs for improved defense against infections might come at the cost of increased risk for cardiovascular disease, representing a model of pleiotropic antagonism at a later age.
Methods
Institutional Approval
Ethics committee approval for the use of human material was obtained from the Medical University of Innsbruck (institutional review board no. AN2014-026 340/4.34), and all subjects gave informed consent. Ethics committee approval was obtained for all animal experiments (BMWFW 66.011/0152-WF/V/3b/2014 and BMWF-66.011/0101-V/3b/2018). Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, 1996, revised 201121). All genetic analyses in GERA (Genetic Epidemiology Research on Adult Health and Aging) were approved by the appropriate review boards at Kaiser Permanente Northern California and the McGill University Health Centre (2015-1292). The UK Biobank was approved by the North West Multi-Center Research Ethics Committee (11/NW/0382) as a research tissue bank. Analyses performed on the UK Biobank data as part of application 41025 were approved by the internal review board at the McGill University Health Centre (2015-1292).
Availability of Data and Materials
All high-throughput data are available through GEO. Accession numbers are GSE138360 and GSE223543. The data sets generated or analyzed during this study are available from the corresponding author on reasonable request except for GERA and UK Biobank data, which are available directly from those cohorts on application.
Cell Culture
VICs from patients undergoing aortic valve replacement or heart transplantation were isolated by collagen digestion, as previously described.22 Wild-type and TLR3−/− human dermal fibroblasts were kindly provided by Jean-Laurent Casanova.
Mouse Models
Experiments were performed by blinded investigators on C57BL/6N mice (Charles River Laboratories, Wilmington, MA) and Tlr3−/− mice (C57BL/6N background). Tlr3−/− mice were crossed with ApoE−/− mice obtained from Jackson Laboratories (Bar Harbor, ME). C57BL/6N, Tlr3−/−, Bgn−/− (C3.129S4[B6]-Bgntm1Mfy/Mmmh; MMRRC), and Ifnar1−/− (B6.129S2-Ifnar1tm1Agt/Mmjax; Jackson Laboratory) mice were used. For the induction of aortic valve stenosis, mice were switched onto a proatherogenic high-fat/high-carbohydrate diet without added cholesterol (No. F3282, BioServ, Frenchland, NJ), as previously described.
Echocardiography
Cohorts for Genetic Associations With Aortic Stenosis in Humans
After the exclusion of individuals with congenital valvular heart disease (International Classification of Diseases [ICD], Ninth Revision codes 746–747), aortic stenosis cases in the GERA cohort25 were defined as participants with ICD-9 code 424.1 or a procedure code for aortic valve replacement in their electronic health records between January 1996 and December 2015 inclusive; all other participants were deemed controls. Analyses in the GERA cohort were limited to unrelated individuals of self-reported European ancestry ≥55 years of age (n=55 192 [3469 aortic stenosis cases]).
In the UK Biobank, after the exclusion of participants with congenital valvular heart disease (ICD-9 codes 746–747 or ICD-10 codes Q20–Q23), aortic stenosis cases were defined as participants with ICD-9 code 424.1 or ICD-10 code I35.0 in their hospital inpatient records or death records or OPCS Classification of Surgical Operations and Procedures, Fourth Revision code K26.1, K26.2, K26.3, K26.4, K31.2, K32.2, or K35.2 in their hospital inpatient records.26 The remaining UK Biobank participants were classified as controls. Analyses were restricted to unrelated participants with genetically confirmed White British ancestry (n=257 231 [2213 aortic stenosis cases]).
Statistical Analysis
Results are presented as mean±SEM. Statistical comparisons between 2 groups were performed by Student t tests or Mann-Whitney tests, whereas comparisons between multiple groups were performed by 1-way ANOVA with Tukey post hoc analysis for statistical significance. Values of P<0.05 were considered statistically significant.
A detailed description of the methods used is included in the Supplemental Appendix.
Results
Tlr3 Deficiency Protects Against Age-Related CAVD
To investigate a potential role for TLR3 in CAVD in vivo, we assessed its expression in the aorta and aortic valve. TLRs display a vessel-specific pattern of expression within the human cardiovascular system, depending on their anatomic site.27 In mice, Tlr3 was expressed at high levels in the ascending aorta and aortic valve and at much lower levels in the peripheral arteries (Figure 1A). TLR3 expression levels were also high in human aortic valves (Figure 1A), which have the same embryonic origin as the ascending aorta.28 VICs isolated from human aortic valves maintained high levels of TLR3 expression in culture (Figure 1A), consistent with previous findings of TLR3 expression by other fibroblasts, including dermal fibroblasts.14 TLR3 and IFN-β levels were higher in the aortic valves from patients with CAVD compared with healthy controls (Figure 1B and 1C), suggesting a role of the TLR3-IFN axis in disease. Age is the principal risk factor for CAVD, the prevalence of which increases significantly at >80 years of age.29 As a proxy for age, we passaged human VICs in vitro and found that TLR3 protein levels increased markedly with the number of passages (Figure 1D). To confirm our results, we have studied a second aging model. For this purpose, we treated human VICs with hydroxyurea and measured senescence markers. Not only was the senescence marker p21 found to be upregulated 3-fold, but we observed a higher amount of β-galactosidase–positive cells on treatment, indicating induction of senescence. Whereas TLR3 mRNA expression was not affected in this model, aging with hydroxyurea was associated with markedly increased IFN-β levels (Figure S1A–S1D).

Moreover, aortic valve leaflet thickness and area, as well as the levels of valvular TLR3, increased with age in wild-type mice (Figure 1E–1H). Our data indicate that aging, TLR3 levels in the aortic valve, and the occurrence of CAVD are associated in humans and mice. To prove a direct causality in disease development, we studied CAVD development in Tlr3−/− mice. It is well documented that TLR2/4 und TLR3 signaling may converge through TRIF and TRAF adapter molecules, therefore enabling the cell to respond (besides nuclear factor-κB) with an adequate IFN signaling and ultimately calcification. It appears in our experiments that a lack of TLR3 in mice is compensated for by an increase of TLR2/TLR4 throughout the murine aorta (Figure S1E). Aged wild-type mice displayed aortic valve thickening and calcification, whereas age-matched Tlr3−/− mice did not display these CAVD phenotypes (Figure 1I–1L). Transthoracic echocardiography confirmed increased leaflet thickness and revealed high-pressure gradients and transvalvular velocities in aged wild-type animals but not in aged Tlr3−/− mice (Figure 1M–1P).
TLR3 Induces Osteoblast-Like Phenotype Through IFN-β
We tested the hypothesis that high-pressure gradients in heart valves and the associated mechanical strain may lead to increased TLR3-IFN signaling in VICs. Cultured human VICs subjected to mechanical strain had increased levels of TLR3 and of its downstream signaling components TRIF and IRF3 (Figure S1F and S1G). VICs subjected to mechanical strain also displayed increased levels of both BMP2 and RUNX2 (Figure S1G), which are known to promote osteoblast differentiation.30,31 Moreover, the supernatants of mechanically stretched VICs activated TLR3 signaling in HEK293 reporter cells that express a Renilla luciferase reporter under the transcriptional control of the IFN-stimulated response element and TLR3 (Figure S1H).
To investigate whether TLR3-RUNX2 signaling in VICs induces an osteoblast expression program, we performed RNA-sequencing analyses in untreated VICs, VICs treated with the specific TLR3 agonist polyinosinic:polycytidylic acid [Poly(I:C)], and osteoblasts. Compared with untreated VICs, the Poly(I:C)-treated VICs and human osteoblasts shared 118 upregulated genes and 199 downregulated genes (Figure 2A and 2B). Besides TLR3, human VICs expressed TLR1, TLR4, and TLR6, and treatment with Poly(I:C) caused upregulation of TLR1 through TLR4 and, to a smaller extent, TLR5 through TLR7 and TLR9 (Figure S2A).

Gene set enrichment analysis on Poly(I:C)-treated VICs revealed that many of the upregulated genes were involved in calcification (Figure 2B), more specifically in CAVD (Figure S2B–S2D). Our data clearly show activation of TLR3 in VICs to induce an osteoblastic signature.
To gain more mechanistic insights into the phenotypic switch observed in TLR3-activated VICs, we next set up experiments aiming to better delineate the involved signaling cascade. As observed for mechanical strain, treatment of VICs with Poly(I:C) resulted in increased TLR3 and IFN-β expression (Figure 2C and 2D). Immunoblot analysis revealed phosphorylation of IRF3, upregulation of interferon-α/β receptor alpha chain (IFNAR) 1, and associated phosphorylation of JAK1/STAT3, resulting in increased levels of the essential osteoblastic transcription factor RUNX2 (Figure 2E). To further corroborate the newly identified TLR3-RUNX2 pathway, a set of experiments was designed using either LY294002, a compound abolishing IFN-β synthesis,32 or a specific IFNAR1-blocking antibody (Figure 2F). As shown in Figure 2G, both LY294002 and a specific IFNAR1-blocking antibody abolished RUNX2 upregulation in VICs induced by Poly(I:C).
Last, stimulation of TLR3 enhanced the production of calcific nodules in VICs, which was paralleled by increased activity of the osteoblastic enzyme alkaline phosphatase,33 whereas TLR3 inhibition decreased calcification and alkaline phosphatase activity (Figure 2H–2J). These findings strongly suggest that TLR3 stimulation of VICs activates an osteoblast-related pathway and promotes calcification. This finding is consistent with the established notion that osteoblasts are essentially sophisticated fibroblasts.34
TLR3 Governs Bone Development In Vivo Through a Conserved Mechanism of Calcification
We next considered the possibility that TLR3 is involved in the maturation of fibroblasts into osteoblasts beyond the aortic valve. TLRs are highly conserved among species and have been shown to drive dorso-ventral polarity in Drosophila melanogaster.35 Specifically, the TLR3 analog tollo is of central importance for the axial development in Drosophila melanogaster by activating the transcription factor runt, the analog of vertebrate RUNX2.36 To test whether TLR3 would play an evolutionarily conserved role in morphogenesis, we next targeted Tlr3 in zebrafish (Danio rerio), a well-established model organism to study calcification and bone formation (Figure 2K). In zebrafish, the first bones develop in the head, including the opercle and the branchiostegal rays. It was previously shown that at this early larval stage (8–10 days after fertilization), premature calcification of these hyomandibular bones can be experimentally hyperinduced on vitamin D3 supplementation.37 Accordingly, in our experiments, an early and strong calcification of landmark bones in zebrafish morphogenesis was observed in vitamin D3–treated larvae. Coincubation with a TLR3 inhibitor abolished vitamin D3–mediated calcification of the hyomandibular bones, indicating that TLR3 activation represents an evolutionarily conserved mechanism in development, promoting cellular calcification in vertebrates (Figure 2L and 2M).
Ifnb−/− mice have a distinctive bone phenotype, with impaired bone formation and osteoporosis.15,16 Our data suggest that TLR3 stimulation of VICs induces calcification through IFN, IFNAR1, and JAK1/STAT3 signaling, yet no specific bone phenotype has been described for Tlr3−/− mice. Here, we show that wild-type and Tlr3−/− mice display a similar size and skeletal development (Figure S3A and S3B). However, microcomputed tomography revealed a distinct osteoporotic phenotype with decreased bone volume and bone density in Tlr3−/− mice compared with wild-type mice (Figure 3A–3C). The number of trabecles and trabecular networks was decreased, resulting in higher intertrabecular distances and in increased trabecular thickness in Tlr3−/− mice compared with wild-type mice (Figure 3D–3G). Histological analysis confirmed the osteoporotic phenotype in Tlr3−/− mice, with decreased trabecular volume (Figure 3H and 3I). As an incidental finding, we noted a shorter femur length in Tlr3−/− mice (Figure 3J and 3K). Together, these findings suggest that Tlr3 plays a central role in bone development and calcification in both zebrafish and mice.

BGN Is an Endogenous TLR3 Ligand
To systematically investigate the endogenous TLR3 ligands potentially involved in CAVD and VIC calcification, we first considered modified bioactive lipids, including oxidized low-density lipoprotein and lipoprotein(a), which have been associated with CAVD in humans.38,39 In our in vitro experiments, neither oxidized low-density lipoprotein nor lipoprotein(a) caused direct activation of TLR3 (Figure S4A and S4B). Nevertheless, both upregulated TLR3 protein expression in human VICs (Figure 4A), a finding corroborated in vivo in aortic valves of ApoE−/− mice that are characterized by high plasma levels of oxidized low-density lipoprotein40 (Figure S4C and S4D). It is important to note that ApoE−/− and Tlr3−/− double-knockout mice were protected from hyperlipidemia-induced CAVD (Figure S4E–S4N).

We screened other putative ligands previously found to accumulate in human calcific aortic valves (Figure S5) and found that the proteoglycan BGN, a structural ECM protein, activated TLR3 signaling in reporter cells in a dose-dependent manner (Figure 4B). Signaling was dependent on TLR3; it was abolished in reporter cells treated with a pharmacological TLR3 inhibitor (Figure 4C).
The human TLR3 ectodomain forms a horseshoe structure that is connected to the transmembrane helix through its C-terminal domain. Binding of a dsRNA ligand leads to human TLR3 homodimer formation and initiates the TLR3 signaling cascade.41,42 The BGN core protein forms obligate dimers,43 suggesting that TLR3 dimerization may be induced on binding to a BGN dimer (Figure 4D). We identified the most plausible interaction between human TLR3 and human BGN by modeling the complex while maintaining the collinearity of the dimer axes as a restraint in subsequent protein-protein docking and local refinement44,45 (Figure 4E). Binding-site analysis and the absence of steric interference with glycan decorations supported the proposed interaction model (Figure S6A).
We tested the physical interaction between BGN and TLR3 by incubating purified human TLR3 ectodomain with BGN and performing size-exclusion chromatography followed by immunoblotting, which demonstrated the coelution of these 2 proteins (Figure 4F). The coimmunoprecipitation of BGN and human TLR3 ectodomain confirmed this interaction (Figure 4G).
To substantiate the interaction of the 2 proteins, we have performed double staining of both TLR3 and BGN in murine aortic valves. Costaining showed colocalization of both proteins, confirming our results previously performed in prespecified buffers, further indicating a physical interaction (Figure S6B). These data provide additional evidence in support of a direct interaction between BGN and TLR3.
Maturation of BGN Through XYLT1 Is Crucial for TLR3 Activation
The direct interaction of BGN with other proteins is mediated by gylcosaminoglycan chains.46 Indeed, BGN lacking chondroitin sulfate chains (Figure S6C) did not activate TLR3 in reporter cells (Figure S6D).
XYLTs add a xylose linker to BGN, which is the crucial step for further BGN maturation. The protein levels of both BGN and XYLT1 increased with higher passage number of cultured VICs (Figure 4H and 4I). Moreover, XYLT1-deficient VICs showed reduced transcription of Xylt1, Tlr3, and IFN-β (Figure S7A–S7C), and supernatant from XYLT1-deficient VICs did not activate TLR3-signaling reporter cells (Figure 4J) independently of applied mechanical strain (Figure S7D). These experiments not only uncovered that maturation of BGN is crucial for TLR3 activation but also indicated that mechanical strain in heart valves may induce the release of BGN from VICs, resulting in a transcellular activation of this repair and defense mechanism. Overall, our data suggest that the XYLT1-mediated addition of xylose sulfate chains during BGN maturation is essential for TLR3 activation. XYLT1 mutations appear to impair calcification and chondrocyte maturation,47 supporting a role for the XYLT1-BGN axis in this process.
The BGN-TLR3-IFNAR1 Axis Induces Calcification Both In Vitro and In Vivo
To assess the generality of these effects, we examined human dermal fibroblasts, which have a slightly different expression profile in response to TLR3 stimulation compared to VICs (Figure S8A–S8E). Both BGN and Poly(I:C) treatments induced the transcription of TLR3 and IFN-β in wild-type and empty vector–transformed fibroblasts but not in Tlr3−/− fibroblasts (Figure 5A and B). Both Poly(I:C) and BGN treatments led to IRF3 phosphorylation, IFNAR1 upregulation, phosphorylation of JAK1/STAT3, and increased levels of RUNX2 in wild-type and empty vector–transformed fibroblasts but not in Tlr3−/− fibroblasts (Figure 5C).

To probe the physiological role of the BGN-TLR3-IFNAR1 axis in vivo, we analyzed gene expression of the pathway in human tissue. Both IFN expression and RUNX2 expression were increased in tissue from patients with CAVD (Figure S8F). To further explore the role of the axis in vivo, we used hypercholesterinemia to induce CAVD in wild-type mice, as well as in Tlr3−/−, Bgn−/−, and Ifnar1−/− mice. We fed animals a high-fat diet for 4 months and then analyzed their valvular phenotype.48 Mean weight gain and serum triglyceride levels were similar between groups (Figure 5D and 5E), but Tlr3−/−, Bgn−/−, and Ifnar1−/− mice displayed significantly reduced aortic valve leaflet thickness compared with wild-type mice (Figure 5F and 5G). In addition, hypercholesterinemia induced aortic valve calcification in wild-type but not Tlr3−/−, Bgn−/−, and Ifnar1−/− mice (Figure 5H). A functional analysis of the aortic valves by transthoracic echocardiography confirmed that deficiencies of Tlr3, Bgn, or Ifnar1 protected the mice from the valvular thickening and hemodynamic consequences of CAVD; these animals did not display the hypercholesterinemia-induced changes in leaflet thickness, aortic valve opening, mean transvalvular gradient, or peak velocity observed in wild-type animals (Figure 5I–5M). These results provide strong support for the involvement of the BGN-TLR3-IFNAR1 axis in CAVD development.
Last, we investigated the effect of the BGN-TLR3-IFNAR1 axis on mouse bone structure by performing microcomputed tomography on femurs from Bgn−/− and Ifnar1−/− mice. Knockout animals had a clear osteoporotic phenotype, with a lower bone volume and bone density compared with wild-type animals (Figure S9).
Genetic Association of TLR3 Signaling With CAVD
To determine whether genetic variations at genes implicated by the uncovered TLR3 signaling pathway are associated with aortic stenosis in humans, we examined 2 large-scale cohorts (GERA, n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases; genotyping methods are available in the Supplemental Material). We discovered 307 variants that were nominally significant (P≤0.05) for aortic stenosis in a meta-analysis of the UK Biobank and GERA, including 16 variants in the JAK1, TLR3, IFNB1, IFNA1, XYLT1, and IFNAR1 loci, representing 13 independent signals, that demonstrated strong associations (P≤1×10−3) or ≥2-fold (up to 5.86-fold) odds of aortic stenosis (Figure 6A and Table 1). Of the 16 variants, all were rare (minor allele frequency <0.01) except for 3 variants at the XYLT1 locus. Variants throughout the XYLT1 locus demonstrated significant associations with aortic stenosis in the meta-analysis (Figure 6B). Mendelian randomization analysis identified that XYLT1 expression in the aorta was associated with greater odds of aortic stenosis, but this was significant only with the penalized weighted median method (Tables S1 and S2). Mendelian randomization consistently indicated that XYLT1 expression in the aorta was associated with greater odds of aortic valve replacement (indicating progression to severe aortic stenosis) among aortic stenosis cases in a meta-analysis of the GERA and UK Biobank cohorts (odds ratio per unit of normalized expression, 2.42 [95% CI, 1.53–3.83]; P=1.7×10−4; Tables 2 and 3 and Figure 6C).
Gene | Position (GRCh37) | Variant | Meta-analysis risk allele (frequency) | GERA odds ratio per risk allele (95% CI) | GERA P value | UKB odds ratio per risk allele (95% CI) | UKB P value | Meta-analysis odds ratio per risk allele (95% CI) | Meta-analysis P value |
---|---|---|---|---|---|---|---|---|---|
JAK1 | 1:65335640 | rs143732508 | G (0.0087) | 1.23 (0.89–1.68) | 0.21 | 1.75 (1.29–2.37) | 3.1E-04 | 1.47 (1.18–1.83) | 5.2E-04 |
JAK1 | 1:65342993 | rs564691204 | T (0.9984) | 2.11 (0.57–7.82) | 0.26 | 8.80 (1.14–67.77) | 0.037 | 3.20 (1.06–9.64) | 0.039 |
JAK1 | 1:65347527 | rs528952911 | C (0.0012) | 3.06 (1.53–6.09) | 1.5E-03 | 0.23 (0.03–1.67) | 0.15 | 2.31 (1.20–4.42) | 0.012 |
JAK1 | 1:65380580 | rs146653955 | C (0.0013) | 1.20 (0.47–3.04) | 0.70 | 2.67 (1.49–4.78) | 9.8E-04 | 2.13 (1.30–3.49) | 2.7E-03 |
TLR3 | 4:186953463 | rs548870644 | G (0.9981) | 2.38 (0.96–5.90) | 0.062 | 6.09 (1.14–32.50) | 0.035 | 2.94 (1.32–6.54) | 8.1E-03 |
TLR3 | 4:187028029 | rs184106700 | G (0.9983) | 2.84 (1.08–7.47) | 0.035 | 1.59 (0.60–4.17) | 0.35 | 2.12 (1.07–4.20) | 0.031 |
IFNB1 | 9:21119979 | rs569915578 | T (0.9986) | 2.45 (0.79–7.59) | 0.12 | 2.76 (0.74–10.28) | 0.13 | 2.58 (1.09–6.07) | 0.030 |
IFNB1 | 9:21120058 | rs755535058† | T (0.9986) | 2.48 (0.79–7.76) | 0.12 | 2.74 (0.73–10.24) | 0.13 | 2.59 (1.09–6.13) | 0.031 |
IFNA1 | 9:21457591 | rs551992948 | C (0.9980) | 13.34 (1.49–119.69) | 0.021 | 4.35 (1.16–16.32) | 0.029 | 5.86 (1.89–18.20) | 2.2E-03 |
XYLT1 | 16:17153381 | rs118001479 | A (0.0064) | 1.56 (1.16–2.10) | 3.0E-03 | 1.44 (1.04–1.99) | 0.027 | 1.51 (1.21–1.87) | 2.4E-04 |
XYLT1 | 16:17283730 | rs550834189 | A (0.0054) | 1.48 (1.05–2.09) | 0.026 | 1.66 (1.19–2.31) | 2.6E-03 | 1.57 (1.24–2.00) | 2.0E-04 |
XYLT1 | 16:17289368 | rs531295111 | C (0.0018) | 1.71 (0.98–2.97) | 0.059 | 2.48 (1.54–4.01) | 2.0E-04 | 2.11 (1.47–3.04) | 5.2E-05 |
XYLT1 | 16:17342509 | rs62033189 | C (0.1530) | 1.10 (1.02–1.17) | 0.010 | 1.09 (1.01–1.18) | 0.032 | 1.09 (1.04–1.15) | 8.4E-04 |
XYLT1 | 16:17345488 | rs34588333‡ | A (0.2161) | 1.07 (1.01–1.14) | 0.028 | 1.10 (1.02–1.18) | 0.011 | 1.08 (1.03–1.14) | 8.5E-04 |
XYLT1 | 16:17376126 | rs936346‡ | C (0.5907) | 1.05 (1.00–1.11) | 0.059 | 1.10 (1.03–1.16) | 3.5E-03 | 1.07 (1.03–1.11) | 8.9E-04 |
IFNAR1 | 21:34683984 | rs554831417 | T (0.9986) | 4.19 (0.96–18.22) | 0.056 | 2.99 (0.69–12.84) | 0.14 | 3.53 (1.25–9.95) | 0.017 |
GERA indicates Genetic Epidemiology Research on Adult Health and Aging; and UKB, UK Biobank.
*
Associations with either P≤1×10−3 or P≤0.05 and odds ratio ≥2 are provided.
†
In linkage disequilibrium with rs569915578 (r2=1).
‡
In linkage disequilibrium with rs62033189 (r2≥0.098).
Method | Odds ratio per unit of normalized expression (95% CI) | P value |
---|---|---|
Inverse-variance weighted | 2.42 (1.53–3.83) | 1.7×10-4 |
Penalized weighted median | 2.55 (1.51–4.32) | 5.0×10-4 |
Chromosomal position (GRCh37) | Variant | Expression-increasing allele | Other allele | Expression in normalized effect size (95% CI) | Expression P value | Aortic valve replacement odds ratio (95% CI) | Aortic valve replacement P value |
---|---|---|---|---|---|---|---|
16:17415463 | rs6416675 | C | T | 0.19 (0.09–0.28) | 1.3E-04 | 1.21 (1.10–1.34) | 1.3E-04 |
16:17436344 | rs12924407 | T | C | 0.19 (0.09–0.28) | 8.4E-05 | 1.16 (1.06–1.28) | 1.7E-03 |
16:17499765 | rs8054100 | G | A | 0.18 (0.09–0.28) | 2.1E-04 | 1.19 (1.07–1.32) | 1.1E-03 |
Variants are correlated (0.23≤r2≤0.84; see Figure S7). Estimates for aortic valve replacement were from the meta-analysis of the GERA (Genetic Epidemiology Research on Adult Health and Aging) and UK Biobank cohorts.

Discussion
This study identifies TLR3 as a key element in a conserved pathway of aortic valve calcification. Here, we show that a central component of the ECM, BGN, induces the TLR3 signaling cascade, resulting in a phenotypic switch of VICs to bone-forming osteoblast-like cells. BGN is a proteoglycan consisting of a core protein with 2 glycosaminoglycan side chains. It stabilizes the ECM and is released from cells subjected to stress,49 leading to the recruitment of macrophages and dendritic cells through TLR2/TLR4, potentially contributing to inflammaging and CAVD.50
We provide the first evidence for a direct interaction between BGN and TLR3 in the cell type primarily responsible for aortic valve calcification. Our results suggest that BGN may govern the TLR3-dependent levels of basal type I IFN in human fibroblasts. They also demonstrate that only a mature form of BGN can activate TLR3, substituted with glycosaminoglycan side chains playing a key role in the biological activity of this proteoglycan. Enzymes responsible for the modification of BGN side chains such as XYLT1 may therefore play a crucial role in determining the fate of BGN and the initiation and progression of CAVD. From an evolutionary perspective, with the aim of understanding the involvement of the newly identified XYLT1-BGN-TLR3-IFNAR1 pathway in the context of calcification per se, it is important to note that the loss of XYLT1 results in impaired chondrocyte maturation and skeletal defects.47 Consistent with this observation, we found that Tlr3−/− mice and zebrafish treated with a Tlr3 inhibitor displayed severe impairments of skeletal calcification. Moreover, we found that variants of XYLT1 significantly associate with CAVD in humans.
A correlation between cardiovascular calcification and osteoporosis has been suggested in some side-by-side clinical studies. Bioactive modified lipoproteins promote inflammation and the production of inflammatory cytokines, facilitating aortic valve calcification and bone demineralization at the same time.51 A recent study showed that the antiresorptive treatment with denosumab and alendronate successfully inhibited bone resorption but had no effect on the progression of aortic valve calcification.52 The potential effect of a medical therapy targeting the uncovered BGN-TLR3-IFN pathway on bone metabolism appears promising and has to be determined.
If the fate of an ECM-derived protein is connected to type I IFN signaling, the uncovered calcification pathway is at the interface of innate immunity and bone formation. Moreover, our results suggest that type I IFN may be involved in the physiological and pathological calcification of connective tissues in general, including musculoskeletal tissue and the cardiovascular system. This finding is consistent with recent reports linking interferonopathies with elevated basal IFN levels to pathological calcifications in the brain and heart.17
We suggest that pharmacological inhibition of the discovered XYLT1-BGN-TLR3-IFNAR1 axis might be a potentially promising approach to the treatment of CAVD. TLR3 inhibition has been achieved successfully in experimental settings with small-molecule inhibitors or blocking antibodies.53 Besides the direct inhibition of TLR3, inhibition of its ligand BGN, for example, by the use of monoclonal antibodies directed against its Toll-binding site, might represent a therapeutic strategy.
An alternative approach would involve the inhibition of type I IFN. Both TLR3 inhibition and inhibition of IFN signaling have been used in clinical studies for patients with asthma, inflammatory bowel disease, or systemic lupus without reporting serious adverse events in their setting.54,55 However, targeting of TLR3 or IFNAR1 signaling might entail increased risk for viral infections, leading to Herpes simplex encephalitis, influenza, or coronavirus disease 2019 (COVID-19), which has yet to be assessed.56 It certainly remains a major challenge to identify which part of the pathway can be tackled to effectively inhibit aortic stenosis with the least adverse events.
Conclusions
We reveal a novel mechanism driving CAVD in which mature BGN, modified by XYLT1, constitutes a selective and potent endogenous TLR3 ligand, inducing valvular calcification through a type I IFN–dependent switch of phenotype in VICs from a fibroblast to an osteoblast-like phenotype. Our data identify the XYLT1-BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway of morphogenesis and calcification, paving the way for novel therapeutic strategies for detecting and treating CAVD in humans.
Article Information
Supplemental Material
Expanded Methods
Supplemental Figures S1–S10
Supplemental Tables S1 and S2
References 52–95
Uncropped gel blots
Acknowledgments
Conception and design of the research: C.G.-T, M. Graber, F.K., D.M., B.R., J.-L.C., I.T., J.H. Data acquisition: C.G.-T., M. Graber, J.H., A.N., L.P., F.N., E.K., G.D., E.D., R.H., D.L., D.L., C.F., S.T., G.F.V., M.S., S.C., M.B., A.W., H.H., A.N., S.M., S.-Y.Z., V.S., J.E. Analysis and interpretation of the data: C.G.-T., M. Graber, D.M., P.P., I.T., J.H., R.K., J.-L.C., S.-Y.Z., M.K., J.C.E., G.T. Statistical analysis: C.G.-T., M. Graber, H.-Y.C., I.T., J.H. Obtaining funding and supervising the work: I.T., J.H., C.G.-T., J.C.E., G.T. Drafting the manuscript: C.G.-T., M. Graber, I.T., J.H., A.N., B.R., M.K., S.T. Critical revision of the manuscript for important intellectual content: Z.T., J.T., L.A.H., P.P., M. Grimm, S.T., H.-Y.C., J.C.E., M.K., G.T., S.-Y.Z., J.-L.C. Structure analysis and modeling: A.N. and B.R. The authors thank Dr Shizuo Akira for providing the Tlr3−/− animals.
Footnote
Nonstandard Abbreviations and Acronyms
- BGN
- biglycan
- CAVD
- calcific aortic valve disease
- COVID-19
- coronavirus disease 2019
- ECM
- extracellular matrix
- GERA
- Genetic Epidemiology Research on Adult Health and Aging
- ICD
- International Classification of Diseases
- IFN
- interferon
- IFNAR
- interferon-α/β receptor alpha chain
- Poly(I:C)
- polyinosinic:polycytidylic acid
- TLR
- Toll-like receptor
- VIC
- valvular interstitial cell
- XYLT1
- xylosyltransferase 1
Supplemental Material
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Received: 6 December 2022
Accepted: 8 March 2023
Published online: 4 April 2023
Published in print: 16 May 2023
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This work was supported by grants from the “Gesellschaft zur Förderung der Herz-Kreislaufforschung in Tirol“ (to Dr Gollmann-Tepeköylü), Bayer Grants4Targets (No.2014-08-1162), the Austrian Science Fund (FWF) to Drs Gollmann-Tepeköylü, Holfeld, and Tancevski (P 32821), “Medizinischer Forschungsfond Tirol” (No. 257) to Drs Gollmann-Tepeköylü and Holfeld. It was also supported in part by the Austrian Science Fund (FWF) under project P28395-B26 to Dr Rupp, project I-3321 grants to Dr Weiss, the City of Vienna Competence Team Signal Tissue (MA23#18-08), and the City of Vienna Competence Team Aging Tissue (MA23#29-07). This study is supported by VASCage–Research Centre on Vascular Ageing and Stroke. VASCage is a COMET Centre within the Competence Centers for Excellent Technologies (COMET) program and funded by the Federal Ministry for Climate Action, Environment, Energy, Mobility, Innovation and Technology, the Federal Ministry of Labour and Economy, and the federal states of Tyrol, Salzburg, and Vienna. This work was partially supported by funding to Dr Thanassoulis from the Canadian Institutes of Health Research, National Institutes of Health/National Heart, Lung, and Blood Institute (HL128550), the Heart and Stroke Foundation of Canada, and the “Fonds de Recherche Québec–Santé.”
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