Toll-like receptor 9 promotes aortic chondrogenesis and calcification in apolipoprotein E-deficient mice

Abstract

Vascular calcification represents a significant clinical challenge, leading to cardiovascular disease, though its underlying mechanisms remain incompletely understood. Recent studies indicate that Toll-like receptor 9 (TLR9), a key element of innate immunity, plays a pathogenic role in vascular inflammation and atherogenesis. Therefore, we hypothesized that TLR9 signaling promotes vascular chondrogenesis and calcification. We compared apolipoprotein E-deficient (ApoE^−/−) mice and Tlr9^−/− ApoE^−/− mice after 24 -weeks high-cholesterol diet feeding. There were no differences between the groups in body weight gain, blood pressure, or plasma glucose levels, although total cholesterol levels were significantly lower in the Tlr9^−/− ApoE^−/− mice. The genetic deletion of TLR9 attenuated vascular calcification as determined by von Kossa staining (5.83 ± 1.14% vs. 3.04 ± 0.68%; P < 0.05), alkaline phosphatase (ALP-1) activity (P < 0.05), and chondroid matrix deposition as determined by Alcian blue staining (P < 0.05) in aortic arch compared with control mice. Immunohistostaining revealed that TLR9 deletion also decreased bone morphogenetic protein (BMP)-2 expression in aortic plaques (P < 0.05). In vitro experiments revealed that TLR9 activation by ODN1826, a TLR9 agonist, stimulated BMP-2 expression in murine peritoneal macrophages, but not in Tlr9-deficient macrophages. Although TLR9 agonists had no direct effect on vascular smooth muscle cells (VSMCs), the culture supernatants of macrophages stimulated with TLR9 agonist increased BMP-2 expression in VSMCs. TLR9 signaling promotes vascular chondrogenesis and calcification in ApoE^−/− mice. Our analyses suggest that TLR9 pathway contributes to bone morphogenic activation of macrophages and VSMCs at least partially, participating in the development of vascular calcification.

Introduction

Atherosclerosis remains a critical pathology, significantly contributing to morbidity and mortality worldwide [1]. As a recognized chronic inflammatory condition [2–4], atherosclerosis is the primary cause of cardiovascular disease and the main underlying factor in coronary artery disease, peripheral artery disease, aortic aneurysms, as well as numerous cases of heart failure and stroke. The progression of arteriosclerosis leads to arterial calcification [5–9], which presents a major clinical challenge due to its poorly understood mechanisms and the lack of effective treatment options.

Toll-like receptors (TLRs) are essential pattern recognition receptors that play a central role in innate immunity by recognizing molecular patterns on diverse pathogens, thus triggering inflammatory and adaptive immune responses to maintain homeostasis and eradicate infections [10–13]. However, the inappropriate activation of TLR signaling by pathogenic components and endogenous harmful molecules has been implicated in atherosclerosis [14–17]. Our laboratory previously demonstrated that TLR9, which recognizes bacterial DNA and initiates inflammation in response to fragmented DNA, promotes vascular inflammation and atherosclerosis by inducing the proinflammatory activation of macrophages in response to endogenous DNA, contributing to cardiometabolic disorders [18–20]. We have also confirmed similar properties for stimulator of interferon genes (STING), which is also a DNA sensor [21–24]. Interestingly, recent literature has reported the association between TLR2 and vascular calcification [25]. Although TLR9 plays a pathogenic role in the development of vascular diseases, its role in vascular calcification remains unclear.

Therefore, we hypothesized that TLR9 signaling promotes vascular chondrogenesis and calcification. The aim of the study was to assess the role of TLR9 on the development of vascular calcification and chondrogenesis in apolipoprotein E-deficient (ApoE^−/−) mice. Bone morphogenic responses by TLR9 agonist were also investigated in macrophages, because the activation of macrophage is central to the pathobiology of TLR9 response. Our findings demonstrate that genetic deletion of TLR9 attenuates vascular calcification and chondrogenesis. Furthermore, in TLR9-deficient mice, BMP-2 expression in atherosclerotic plaques were significantly lower compared with control mice. Additionally, our in vitro experiments demonstrated that TLR9 agonist promoted bone morphogenic activation of macrophages, leading to subsequent activation of vascular smooth muscle cells (VSMCs). The results of our study suggest a novel mechanism of vascular calcification that could serve as a potential therapeutic target.

Methods

Animal experiments

Male ApoE^−/− mice (C57BL/6 J background) and Tlr9^−/− mice (C57BL/6 J background) were originally purchased from the Jackson Laboratory and Oriental BioService, Inc, respectively. Tlr9^−/− ApoE^−/− mice were generated by crossing ApoE^−/− mice and Tlr9^−/− mice. Male Tlr9^−/− ApoE^−/− mice were fed a high-fat, high-cholesterol diet (D12108; Research Diets) for 24 weeks from 8 weeks of age. Sex- and age-matched nontreated ApoE^−/− mice served as the control. All mice were housed under a 12-h light/dark cycle, with food and water available ad libitum. All experimental procedures conformed to the guidelines for animal experimentation of Tokushima University. The protocol was reviewed and approved by our institutional ethics committee.

Blood pressure and plasma lipid level measurement

The blood pressure of each mouse was measured using a tail-cuff system (BP-98A, Softron) in conscious animals. The average value of 3 measurements was used for comparison. At the time of sacrifice, blood was collected from the heart into K2-EDTA-containing tubes. After centrifuge, plasma was stored at −80 °C until required. Plasma lipid levels (total cholesterol, high density lipoprotein cholesterol (HDL-C), and triglyceride) were measured at the Sanritsu Zelkova examination center (Japan).

Preparation of aortas and atherosclerotic lesion analysis

Mice were sacrificed by administration of an overdose of pentobarbital and perfused with 0.9% sodium chloride solution at a constant pressure via the left ventricle. Both the heart and whole aorta were immediately removed. Each aortic arch was snap-frozen in optimal cutting temperature compound (TissueTeck) for histological analyses.

Histology and immunohistochemical analyses

Histological and immunohistochemical staining were performed as previously described [26]. Each aortic arch was snap-frozen in optimal cutting temperature compound (TissueTeck). The aortic arch was then sectioned longitudinally and serially at 5-μm intervals, starting from the point where the brachiocephalic artery emerges to the ascending aorta and continuing until the three branches of the arch were no longer visible.

Calcium deposition within the aortic walls was assessed using von Kossa staining (Polysciences, Inc.). Endogenous alkaline phosphatase activity in the aorta was visualized using a Vector Red substrate kit (Vector Laboratories). Chondroid matrix deposition in the aortic arch was stained with Alcian blue (Muto Pure Chemicals). BMP-2 expressions were detected by using an anti-BMP-2 (Bioss, bs-1012R) antibody followed by the avidin–biotin complex technique and stained with Vector Red substrate kit (Vector). Each section was counterstained with hematoxylin. Each section was counterstained with nuclear fast red or hematoxylin.

Cell culture experiments

Thioglycolate‐induced or non-induced (resident) peritoneal macrophages were collected from female ApoE^−/− mice and Tlr9^−/− ApoE^−/− mice at 8 to 10 weeks of age and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Isolated peritoneal macrophages were used for experiments 24 h after seeding. Bone marrow (BM)-derived macrophages were also used in this study. BM cells were obtained from femurs and tibias of 8-week-old ApoE^−/− mice. Cells were cultured in DMEM supplemented with 10 ng/mL macrophage colony-stimulating factor (R&D Systems) for 7 days. Rat VSMCs were isolated and cultured as described previously [27]. BM-derived macrophages and VSMCs were used after 24-h serum starvation. These cells were stimulated with CpG‐ODN1826 (ODN1826), a specific oligonucleotide that activates TLR9, or its control (Ctrl‐ODN1826) (Gene Design Inc., Osaka, Japan) for 4 to 24 h to examine gene expression. BMP-2 (Mouse BMP-2 ELISA Kit; ab119582, abcam) in macrophage culture supernatant were measured using commercially available kits.

Reverse transcription, real-time polymerase chain reaction

Total RNA was extracted from tissues and cells using illustra RNAspin RNA Isolation Kit (GE Healthcare). Reverse transcription was performed using a QuantiTect Reverse Transcription kit (Qiagen) from 1 μg of the extracted total RNA. Quantitative real-time PCR (qPCR) was performed on Mx3000P (Agilent Technologies) using gene-specific primers (Supplement Table) and Power SYBR Green PCR Master Mix (Applied Biosystems). Data are expressed in arbitrary units that were normalized by β-actin. The base sequences of each primer used in PCR are summarized in Table 1.

Table 1.

Primer sequences

	Forward primer sequence	Reverse primer sequence
For mice
β-actin	5’-cctgagcgcaagtactctgtgt-3’	5’-gctgatccacatctgctggaa-3’
BMP-2	5’-tggaagtggcccatttagag-3’	5’-tgacgcttttctcgtttgtg-3’
RANKL	5’-tggaaggctcatggttggat-3’	5’-atggtgaggtgtgcaaatgg-3’
For rat
β-actin	5’-ggccaaccgtgaaaagatga-3’	5’-gaccagaggcatacagggacaa-3’
BMP-2	5’-tagtgacttttggccacgacg-3’	5’-gcttccgctgtttgtgtttg -3’
RANKL	5’-gggctggtgaggaaattagc-3’	5’-gaaagccccaaagtacgtcg-3’

BMP; bone morphogenetic protein, RANKL; Receptor activator of Nuclear Factor-kappa B ligand

Statistical analysis

Respective statistical analyses were performed using StatView software. All numerical values are expressed as means ± standard error of the mean (SEM). Comparison of parameters between 2 groups was performed with unpaired Student’s t test when data followed a normal distribution or with Mann–Whitney U test when data did not Differences between multiple groups were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test when data followed a normal distribution or with Brown-Forsythe test when data did not. Comparison of dose–response curves was performed by two-factor repeated measures ANOVA, followed by Dunnett’s post hoc test for comparison between groups, and P-value < 0.05 was considered significant.

Results

Genetic deletion of TLR9 attenuated vascular calcification and osteogenesis in ApoE^−/− mice

After 24 weeks of high-fat, high-cholesterol diet (D12108; Research Diets) feeding, von Kossa staining of the aortic arch showed a significant reduction of advanced calcification in Tlr9^−/−ApoE^−/− mice compared with ApoE^−/− mice (5.83 ± 1.14% versus 3.04 ± 0.68%, P < 0.05) (Fig. 1A). Genetic deletion of TLR9 demonstrated a significant reduction of alkaline phosphatase (ALP-1) activity (6.64 ± 1.26% versus 3.24 ± 0.65%, P < 0.05) (Fig. 1B) in aortic arch in ApoE-/- mice. There was no difference in the cross-sectional area of the intima-media of the aortic arch between the two groups (0.27 ± 0.03 mm² vs 0.25 ± 0.04 mm², p = 0.67). These results indicated that genetic deletion of TLR9 in ApoE^−/− mice attenuates vascular calcification and osteogenesis.

Fig. 1.

Effect of genetic deletion of toll-like receptor 9 (Tlr9) on aortic calcification and osteogenesis in apolipoprotein E-deficient (ApoE-/-) mice. A von Kossa staining of aortic arch. Advanced calcification in aortic arch was decreased in Tlr9^−/−ApoE^−/− mice compared with that in ApoE^−/− mice, B immunostaining against alkaline phosphatase (ALP-1) in aortic arch. Osteogenic activity in aortic arch was decreased in Tlr9^−/−ApoE^−/− mice compared with that in ApoE^−/− mice. Scale bar: 1 mm (Upper Panels), 100 μm (Lower Panels). n = 20 (per group), *p  < 0.05. All values are mean ± SEM

Genetic deletion of TLR9 attenuated aortic Chondrogenesis in ApoE^−/− mice

Genetic deletion of TLR9 in ApoE^−/− mice fed a high-fat, high-cholesterol diet for 24 weeks significantly reduced chondroid matrix deposition as determined by Alcian blue staining (8.70 ± 0.86% versus 6.25 ± 0.70%, P < 0.05) (Fig. 2) in aortic arch compared with control mice. These results indicated that genetic deletion of TLR9 in ApoE^−/− mice attenuates aortic chondrogenesis.

Fig. 2.

Effect of genetic deletion of toll-like receptor 9 (Tlr9) on aortic chondrogenesis in apolipoprotein E-deficient (ApoE^−/−) mice. A Alcian blue staining of aortic arch. Chondroid matrix deposition in aortic arch was decreased in Tlr9^−/−ApoE^−/− mice compared with that in ApoE^−/− mice. Scale bar: 1 mm (Upper Panels), 100 μm (Lower Panels). n = 20 (per group), *p < 0.05. All values are mean ± SEM

Genetic deletion of Tlr9 did not changed body weight gain and blood pressure in ApoE^−/− mice

There were no significant differences in body weight gain, blood pressure, plasma glucose level, and plasma triglyceride levels between ApoE^−/− mice and Tlr9^−/−ApoE^−/− mice (Table 2). The total cholesterol levels were significantly lower in the Tlr9^−/−ApoE^−/− mice, while the HDL-C value was significantly higher in Tlr9^−/−ApoE^−/− mice, respectively.

Table 2.

Effect of genetic deletion of Tlr9 on metabolic parameters

	ApoE−/−	Tlr9−/− ApoE−/−	p-value
Body weight, g	34.9 ± 3.6	35.7 ± 3.1	0.21
Heart rate, bpm	693.1 ± 58.8	678.5 ± 61.7	0.22
Systolic blood pressure, mm Hg	108.9 ± 9.5	105.2 ± 9.0	0.11
Diastolic blood pressure, mm Hg	56.6 ± 10.7	59.6 ± 13.0	0.21
Total cholesterol, mg/dl	1153.3 ± 43.0	849.4 ± 57.2	< 0.0001
Triglyceride, mg/dl	98.2 ± 5.4	112.5 ± 9.2	0.17
HDL-C, mg/dl	6.5 ± 0.6	9.7 ± 1.2	0.02

All values are mean ± SEM. ApoE^−/− indicates apolipoprotein E–deficient; HDL-C, high-density lipoprotein cholesterol; and Tlr9, toll-like receptor. n = 20–25, per group

Genetic deletion of TLR9 decreased BMP-2 expression in aorta in ApoE^−/− mice

BMP-2 like other bone morphogenetic proteins, plays an important role in the development of bone and cartilage [28, 29]. We also compared the expression of BMP-2 in the atherosclerotic aorta using immunohistochemistry staining between ApoE^−/− mice and Tlr9^−/−ApoE^−/− mice. Consistent with the results of histological study, Tlr9^−/−ApoE^−/− mice demonstrated reduced expression of the BMP-2 (P < 0.05) compared with ApoE^−/− mice (Fig. 3). These results indicated that genetic deletion of TLR9 in ApoE^−/− mice decreased BMP-2 expressions in aortic arch.

Fig. 3.

Effect of genetic deletion of toll-like receptor 9 (Tlr9) on BMP-2 expression in aorta in apolipoprotein E-deficient (ApoE-/-) mice. Immunohistochemistry staining against BMP-2 of aortic arch. TLR9 deletion decreased BMP-2 expression of aortic arch in Tlr9^−/−ApoE^−/− mice compared with that in ApoE^−/− mice. Scale bar: 100 μm. n = 10 (per group), *p < 0.05. All values are mean ± SEM

TLR9 signaling enhanced the BMP-2 expression of macrophage

In general, TLR9 was known as expressed in macrophages, monocytes, plasmacytoid dendritic cells, B lymphocytes, and microglia [30–32]. We investigated the role of TLR9 in macrophage activation, particularly focused on the functions related to vascular calcification. TLR9 activation by ODN1826, a TLR9 agonist, consistently promoted the expression of BMP-2 in several in vitro macrophage models, such as thioglycolate-induced or non-induced (resident) peritoneal macrophages and BM-derived macrophages obtained from ApoE^−/− mice (Fig. 4A), but not in Tlr9^−/− ApoE^−/− macrophages (Fig. 4B). Additionally, the TLR9 agonist significantly increased the BMP-2 concentration in the culture supernatant of thioglycolate-induced peritoneal macrophages (Fig. 4C). Although TLR9 agonist did not directly altered the BMP-2 expression of VSMCs, the culture supernatants of macrophages stimulated with TLR9 agonist significantly increased BMP-2 expression in VSMCs (Fig. 4D). In addition, TLR9 agonists significantly increased RANKL (Receptor activator of nuclear factor-kappa B ligand) expression in thioglycolate-induced macrophages obtained from ApoE^−/− mice (Fig. 4E). Culture supernatants from macrophages stimulated with TLR9 agonists also tended to increase RANKL expression in VSMCs (Fig. 4F). The base sequences of each primer used in PCR are summarized in Table 1. These results suggest that TLR9 signaling in macrophages directly increases BMP-2 and RANKL expression, and that activated macrophage-derived cytokines indirectly increase BMP-2 and RANKL expression in VSMCs, suggesting that it is at least partially involved in the effects of TLR9 on vascular calcification.

Fig. 4.

Role of toll-like receptor (Tlr9) in macrophage activation. A Quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated that ODN1826, TLR9 agonist, significantly increased the expression of BMP-2 in (a) thioglycolate (TG)-induced peritoneal macrophages, (b) resident peritoneal macrophages, (c) and BM-derived macrophages. Each macrophage was obtained from ApoE^−/− mice, B the expression of BMP-2 in TG-induced peritoneal macrophages was examined by real-time qPCR. Stimulation of TLR9 by ODN1826 enhanced the expression of BMP-2 in ApoE^−/− macrophages but not in Tlr9-deficient macrophages, C BMP-2 concentrations in the culture supernatant of TG-induced peritoneal macrophages were quantified by ELISA. TLR9 stimulation with ODN1826 significantly enhanced BMP-2 expression in macrophages, D Culture medium from ODN1826-treated macrophages increased BMP-2 expression in VSMCs, E qPCR analysis demonstrated that ODN1826, TLR9 agonist, significantly increased the expression of RANKL in thioglycolate (TG)-induced peritoneal macrophages obtained from ApoE^−/− mice. F, Culture medium from ODN1826-treated macrophages tended to increase RANKL 2 expression in VSMCs. (n = 6, per group) * P < 0.05, ** P < 0.01, *** P < 0.0001. All values are mean ± SEM

Discussion

Accumulating evidence suggests that the TLR9 signaling pathway promotes proinflammatory responses of innate immune cells and contributes to the development of inflammatory diseases including atherogenesis [18, 33–35], although little is known about its role in arterial calcification. In this study, genetic deletion of TLR9 attenuated the development of vascular calcification and chondrogenesis in ApoE^−/− mice. TLR9 deletion also reduced BMP-2 expression in atherosclerotic plaques in these mice. In vitro experiments demonstrated that TLR9 signaling contributes to pro-inflammatory activation and increased BMP-2 and RANKL expression of macrophages, leading to calcification formation by VSMCs. These results suggest that TLR9 signaling promotes the development of vascular calcification and chondrogenesis, at least in part through enhanced BMP-2 and RANKL expression in macrophages.

Several past studies have suggested a relationship between vascular calcification and TLRs, such as TLR2 [25], TLR3 [36], and TLR4 [37–39], however little has been known about the effect of TLR9 on vascular calcification. Previously, Zhao et al. reported that genetic deletion of TLR9 reduced vascular calcification by using ex vivo vascular calcification assay, incubating aortic rings derived from wild-type or Tlr9^−/− mice fed 8-weeks high-casein plus adenine diet in high-Pi medium (3 mmol/L) for 7 days [40]. There were many fundamental differences between their study and ours in study design, such as dietary content and administration period, presence or absence of genetic ApoE deficiency, and whether it was an in vivo or an ex vivo model, although both studies consistently observed the reduction of vascular calcification by TLR9 deletion.

BMP-2, a well-known osteogenic protein required for osteoblast differentiation and bone formation, plays a significant role in vascular calcification [29]. BMP-2 signaling cascade involves interaction with receptors such as BMPR1A (ALK3), BMPR1B (ALK6), and BMPRII [41, 42]. BMPR1A facilitates the trans differentiation of VSMCs into osteoblast-like or chondrocyte-like cells, acting as a critical bridge in vascular calcification [43]. Nakagawa et al. [44] highlighted the role of BMP-2 in accelerating atherosclerotic intimal calcification in ApoE^−/− mice, while Nguyen-Yamamoto et al. [45] demonstrated that BMP-2 inhibition in CKD models reduced VSMC osteogenic differentiation and calcification. Additionally, Sun et al. [46] identified endogenous BMP-2 as a driver of IL-6-induced VSMC calcification. In this study, we further demonstrated that TLR9 signaling increased BMP-2 expression of macrophages. RANKL expressed in osteoblasts functions as a receptor that recognizes RANK released from osteoclasts, contributing to the promotion of osteoblast differentiation and increased bone formation [47, 48]. In this study, we also demonstrated that TLR9 signaling increases RANKL expression in macrophages. Pro-inflammatory activation of macrophages by TLR9 stimulation also recruits more monocytes/macrophages into inflamed vasculature, leading to the further secretion of BMP-2 and RANKL in plaques, resulting in VSMC osteogenic differentiation and calcification. Thus, blockade of TLR9 signals may provide a novel therapeutic target for the attenuation of vascular calcification.

In this study, regarding the serum lipid profile, total cholesterol levels were significantly lower and HDL-C levels were higher in the Tlr9^−/− ApoE^−/− group compared with ApoE^−/− group. Interestingly, similar trend of lower total cholesterol and higher HDL-C levels in Tlr9^−/−ApoE^−/− mice was observed in our previous study [18]. Although there was no statistically significant difference in our previous study, the dietary content (D12108; Research Diets in current study) and dietary period was quite different (6 weeks in previous one vs 24 weeks in current study), which might affect these significances. Even in previous publications, the effects of pharmacological or genetic blockade of TLR9 on serum total cholesterol levels were highly controversial, with no consistency depending on the dietary period or dietary composition of each study [35, 49–51]. TLR9 is also expressed in liver sinusoidal endothelial cells and has been suggested to be involved in chronic hepatitis and hepatic steatosis, which might be one of the reasons for the complex effects on long-term cholesterol metabolism in TLR9-deficient mice [52–55]. While differences in cholesterol levels could potentially affect chronic vascular inflammation and atherogenesis, although in this study, which evaluated relatively advanced stages of atherosclerosis with calcification, there was no significant difference in the intima-media area of the aortic arch between the two groups (p = 0.67). Our in vitro experiments provided a novel mechanism by which TLR9 signaling directly increases BMP-2 and RANKL expression in macrophages and contributes, at least in part, to vascular calcification, without mediated cholesterol metabolism.

There are several limitations to this study that must be considered. First, although we proposed BMP-2 as one of the mechanisms by which TLR9 is involved in vascular calcification, we have not yet verified its exact contribution or all other pathways. Second, our mouse model showed some statistically significant differences in the lipid profile between the two groups, and we cannot completely rule out the possibility that changes in lipid metabolism due to TLR9 deficiency or not may modify vascular calcification. Third, this study used a vascular calcification model induced by long-term administration of a high-cholesterol diet to ApoE-deficient mice, but no verification was conducted with other vascular calcification models, so caution is required when interpreting the results.

This study underscores the critical role of TLR9 in the pathogenesis of vascular calcification, a hallmark of advanced atherosclerosis. Among the TLR family, TLR9 emerges as a pivotal contributor, promoting calcification through macrophage-driven upregulation of BMP-2 and RANKL, a potent osteogenic protein. The findings demonstrate that TLR9 deficiency significantly reduces vascular calcification, highlighting its potential as a therapeutic target. Additionally, the study provides evidence linking BMP-2 and RANKL signaling with VSMC osteogenic differentiation, emphasizing the interplay between inflammatory pathways and calcification. These insights reinforce the importance of exploring TLR9-targeted therapies to mitigate vascular calcification and associated cardiovascular complications. Future research should focus on elucidating the molecular pathways involved, the role of dietary and temporal factors, and their implications for long-term vascular health. This line of investigation holds promise for developing innovative interventions to address calcification-related cardiovascular diseases and improve patient outcomes.

Funding

This work was partially supported by JSPS Kakenhi Grants (Number 22H03069 and 25K02646 to M.S., Number 24K02451 to D.F., Number 23K15132 to T.H.), Bristol-Myers Squibb Research Grants (D.F.), Bayer Vascular Frontiers Research Grant (M.S. and D.F.), and Japan Agency for Medical Research and Development (M.S.). The funders had no role in the study design, data collection and analysis, or manuscript preparation.

Data availability

The data that support the findings of this study are available from the corresponding author, T.H., upon reasonable request.

Declarations

Conflict of interest

The other authors declare that they have no conflict of interest.