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Introduction

Periodontitis is a chronic oral inflammatory response of the periodontium that affects almost 50% of US adults ≥30 years of age (1,2). This chronic, multifactorial disease is characterized by gingival inflammation and alveolar bone loss (3). Periodontitis can trigger an immune-inflammatory response that ultimately leads to a non-reversible change of bone supporting tissues, resulting in progressive destruction of alveolar bone and finally tooth loss (4). Periodontitis has also been reported to be associated with the development of several systemic diseases, such as rheumatoid arthritis, psoriasis, systemic sclerosis, Alzheimer's disease and cardiovascular diseases (CVD) (5). Among the systemic diseases associated with periodontitis, CVD has received the most attention, as a number of epidemiological and clinical studies have indicated a strong association between periodontitis and CVD (6,7). Periodontitis clearly imparts adverse risks for CVD in clinical evidence in addition to animal models (8,9).

Atherosclerosis is a degenerative vascular disease accompanying chronic and progressive vascular inflammatory conditions, which results in fatal consequences such as myocardial infarction and stroke due to the development of atherosclerotic plaque (10). Although hyperlipidemia is known as a major risk factor for the development of atherosclerosis, previous studies have indicated that systemic inflammation plays a notable role in the initiation and progression of atherosclerosis and is also one of the precipitating factors for atherogenesis (11,12). Systemic inflammation (such as increased serum proinflammatory cytokine levels) is known to induce vascular inflammation, resulting in vascular endothelial cell dysfunction, an early important step of atherogenesis. Also, vascular inflammation causes atheroma progression and complications by inducing the proliferation and migration of vascular smooth muscle cells, resulting in thrombosis (13).

There are several systemic inflammatory diseases that are known to increase the risk of atherosclerosis development, such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus (SLE) and systemic sclerosis (14). Periodontitis, a local and systemic condition, has also been suggested to be one of the putative risk factors for atherogenesis (15–18). We previously reported that ligature-induced periodontitis induces severe systemic inflammation and exacerbates atherosclerosis in hyperlipidemic Apolipoprotein E-deficient (ApoE−/−) mice (19); however, to the best of our knowledge, the effect of severe periodontitis on atherogenesis in normolipidemic condition remains unknown. To understand whether hyperlipidemic condition is necessary for the development and exacerbation of atherosclerosis associated with periodontitis, the present study investigated the effect of ligature-induced periodontitis on atherogenesis in normolipidemic wild-type (WT) and hyperlipidemic ApoE−/− mice.

Materials and methods

Animals

A total of 20 male WT and ApoE−/− C57BL/6 mice (Jackson Laboratory) that were 7 weeks old (mean weight, 19 g), were housed in the vivarium at The University of California (Division of Laboratory Animal Medicine, Los Angeles, USA). All mice were housed in a pathogen-free animal experimental facility of University of California, Los Angeles, under a 12 h light/dark cycle at 20°C, with 40% humidity, and proper ventilation and air circulation. The mice were fed with a high-fat diet (HFD) for 14 weeks (cat. no. D12079B; Research Diets, Inc.) to facilitate the development of atherosclerosis (19). All mice had free access to the food and drinking water. Mice health and behavior were monitored every other day throughout the whole duration of the experiment (14 weeks). No mice were found dead during the experimental period. Isoflurane (99.9%) and a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg) were used as anesthetics during ligature placement. Carprofen (3 mg/kg), a pain relief drug, was also used once a day for 3 days after ligature placement to minimize pain of the mice. All mice were euthanized under general anesthesia using isoflurane (99.9%) to minimize suffering. Euthanasia was performed via cardiac perfusion, and the heartbeats of the mice were assessed for 5 min to verify death. All experiments were approved by the Chancellor's Animal Research Committee of the University of California, Los Angeles (approval no. ARC # 2019-057-01A).

Induction of periodontitis in mice

At 1 week after starting the HFD, the mice were divided into four groups (five mice per group) as follows: i) WT control mice (WT/no lig.); ii) ligature-placed WT mice (WT/with lig.); iii) ApoE−/− mice (ApoE−/−/no lig.); and iv) ligature-placed ApoE−/− mice (ApoE−/−/with lig.). The 6–0 silk ligature (MilliporeSigma) was placed around the second molars under general anesthesia using Ketamine/Xylazine (100 and 5 mg per kg, respectively) via intraperitoneal injection as described previously (20).

Tissue collection and histological analysis

Whole blood was collected from mice by cardiac puncture under general anesthesia with isoflurane (Abbott Laboratories). The mice were then perfused and fixed with 4% paraformaldehyde at room temperature in phosphate-buffered saline (PBS) via the left ventricle for 5 min. After the perfusion, the heart, the carotid artery and the full-length of the aorta-to-iliac bifurcation were exposed and dissected carefully from any surrounding tissues. The heart and artery samples were embedded in Scigen Tissue-Plus O.C.T. Compound (Thermo Fisher Scientific, Inc.), and sectioned at 7-µm thickness. A total of 12 sections at 100 µm intervals were collected from each mouse and stained with Oil red O (Sigma-Aldrich; Merck KGaA) at room temperature for 15 min to quantify atherosclerotic burden at the sinus lesion. The maxillae were excised and half of the palatal tissues were harvested using a blade for gene expression analysis. For micro-computed tomography (µCT) analysis, the maxillae were fixed with 4% paraformaldehyde in PBS, pH 7.4, at 4°C overnight and stored in 70% ethanol solution at 4°C.

After µCT scanning, the maxillae were decalcified using 5% EDTA and 4% sucrose in PBS (pH 7.4) for 3 weeks at 4°C. The decalcification solution was changed daily. Decalcified maxillae were processed for paraffin embedding blocks at the UCLA Translational Procurement Core Laboratory. Blocks were sectioned at 5-µm intervals using a microtome (Thermo Fisher Scientific, Inc.). The sections were dewaxed at 60°C for 30 min, and were then rehydrated at room temperature by incubating the sections twice in xylene for 5 min, twice in 100% ethanol for 2 min, twice in 95% ethanol for 2 min and in 70% ethanol for 2 min. After the sections were rinsed with running tap water for 1 min at room temperature, the sections were stained with hematoxylin and eosin for 30 min at room temperature (Sigma-Aldrich; Merck KGaA). For tartrate-resistant acid phosphatase staining, the sections were stained using an acid phosphatase kit (cat. no. 378A; Sigma-Aldrich; Merck KGaA), according to the manufacturer's protocol, and then counterstained using hematoxylin for 20 min at room temperature. The digital images of the stained sections were obtained using the DP72 light microscope (Olympus Corporation). The clinical attachment loss was observed under the microscope by measuring cement-enamel junction (CEJ) to the base of the pocket depth by an investigator (JS). The reading was confirmed in a double blind manner by another individual (N-HP).

µCT and histological analysis of maxillae

The fixed maxillae were subjected to µCT scanning (Skyscan1275; Bruker Corporation) using a voxel size of 20 µm3 and a 0.5 mm aluminum filter at 55 kVp and 145 µA. Two-dimensional slices from each maxilla were combined using NRecon and CTAn/CTVol programs (Bruker Corporation) to form a three-dimensional reconstruction. Alveolar bone loss was quantified by measuring the distance from the palatal and mesiobuccal CEJ to the alveolar bone crest (ABC) of the second molars by an investigator (JS). The reading was confirmed in a double blind manner by another individual (N-HP).

En face analysis

The full-length of the aorta-to-iliac bifurcation was opened along the ventral midline and dissected free of the animal under a stereomicroscope (Stemi 305; Zeiss AG). For en face analysis, the aorta was stained with Sudan IV (Sigma-Aldrich; Merck KGaA) as previously described (9) and pinned out flat, intimal side up, between cover slides. Aortic images were captured using a Nikon digital camera (Nikon Corporation) and the intensity of lipid staining was analyzed using ImageJ software version 1.48 (National Institutes of Health).

Serum lipid and cytokine measurements

Levels of total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low-density lipoprotein (LDL) were measured using a Cholesterol Assay kit (Abcam) in the UCLA Cardiovascular Core Facility (21). The serum levels of TNF-α, IL-1β and IL-6 were measured by ELISA using Ready-SET-go kits [TNF-α Mouse Uncoated ELISA kit with Plates (cat. no. 88732422); IL-6 Mouse Uncoated ELISA Kit with Plates (cat. no. 88706422); IL-1β Mouse Uncoated ELISA kit with Plates (cat. no. 88701322); Thermo Fisher Scientific, Inc.] according to the manufacturer's protocol. The color reaction was stopped with the addition of Stop solution (BioLegend, Inc.), and absorbance was read immediately using a plate reader at 450 nm (Bio-Rad Laboratories, Inc.). The standard curve was calculated by plotting the standards against the absorbance values, and the cytokine levels were measured in pg/ml.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA from mouse tissues was extracted using RNeasy micro kit (cat. no. 74106; Qiagen GmbH) and reverse-transcribed with the following cycle: 5 min at 65°C, 2 min at 25°C and 50 min at 45°C using SuperScript® III Reverse Transcriptase Synthesis kit (cat. no. 18064014; Thermo Fisher Scientific, Inc.). Subsequently, qPCR was performed under the following conditions: Pre-incubation at 95°C for 2 min; amplification at 95°C for 15 sec, 57°C for 15 sec and 72°C for 1 min, for 55 cycles; melting curve at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec using PowerUp™ SYBR Green Master Mix (cat. no. A25741; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The following primers were used for qPCR: TNF-α forward, 5′-TCAGGTTGCCTCTGTCTCAG-3′, and reverse, 5′-GCTCTGTGAGGAAGGCTGTG-3′; IL-1β forward, 5′-CACAGCAGCACATCAACAAG-3′, and reverse, 5′-GTGCTCATGTCCTCATCCTG-3′; IL-6 forward, 5′-TGGGACTGATGCTGGTGACA-3′, and reverse 5′-GCCTCCGACTTGTGAAGTGGT-3′; and β-actin forward, 5′-CATTGCTGACAGGATGCAGAAGG-3′, and reverse 5′-TGCTGGAAGGTGGACAGTGAGG-3′. β-actin served as control and the fold induction was calculated using the comparative ΔCq method (22). Data were presented as relative transcript levels (2−ΔΔCq).

Immunohistochemical (IHC) staining

The slides containing the maxilla sections were submerged in a citric acid-based antigen unmasking solution (Vector Laboratories) at 65°C overnight for antigen retrieval and then incubated with anti-CD68 antibody (cat. no. ab125212; Abcam) or p65 (cat. no. ab32536; Abcam) as primary antibodies. The tissue sections were visualized using 3-amino-9-ethylcarbazole (AEC; cat. no. SK-4205; Vector Laboratories, Inc.) at room temperature for 1 min, followed by counterstaining with hematoxylin for 1 min at room temperature, then sealed with ImmunoHistoMount™ solution (Agilent Technologies, Inc.). The digital images of the stained sections were obtained using the DP72 light microscope (Olympus Corporation).

Statistical analysis

All graphs were created and statistical analyses were calculated using GraphPad Prism 9.3.1 (GraphPad Software, Inc.), and for multiple comparisons, one-way ANOVA with Tukey's post hoc test was used. P<0.05 was considered to indicate a statistically significant difference. All in vitro results were confirmed by at least three independent experiments. Error bars represent mean ± SEM.

Results

Development of periodontitis is induced by the ligature placement in WT and ApoE−/− mice

To investigate the effect of periodontitis on atherogenesis in both normolipidemic and hyperlipidemic conditions, periodontitis was induced in WT and ApoE−/− mice by placing ligature for 14 weeks as described previously (19). Histological examination revealed that the ligature placement induced alterations in the organization of the junctional epithelium and deep subgingival pockets, such as clinical epithelial attachment loss (Fig. 1A) and alveolar bone loss (Fig. 1B) in both WT and ApoE−/− mice compared with the control mice without ligature placement. The µCT analysis revealed a similar alveolar bone loss in both WT and ApoE−/− mice with ligature compared with the control mice, as measured by the distance between the CEJ and the ABC (Fig. 1B and C). These data indicate that long-term ligature placement induced a similar degree of periodontitis in both groups of mice.

Figure 1. Evaluation of periodontitis induced by ligature placement in WT and ApoE-/- mice fed with HFD. (A) Hematoxylin and eosin staining on the periodontium of maxillary second molar (scale bar, 50 µm). Black arrows indicate the CEJ. There was notable epithelial detachment around the molars of mice receiving ligature placement. (B) Representative 2D or 3D µCT images of mouse maxilla (scale bar, 1 mm). a, alveolar bone. There was severe alveolar bone loss around the second molars of mice receiving ligature placement. (C) Alveolar bone loss measured at the distobuccal and palatal roots of the maxillary second molars from CEJ to ABC. ****P<0.0001. CEJ, cement-enamel junction; ABC, alveolar bone crest; WT, wild-type; ApoE-/-, Apolipoprotein E-deficient; HFD, high-fat diet.

As local inflammation of periodontium induces alveolar bone loss (23), the presence of osteoclasts, macrophages and inflammation-specific markers, such as p65, were evaluated to examine the status of local inflammation in both WT and ApoE−/− mice. Ligature placement significantly increased the numbers of osteoclasts around the ligated tooth in both WT and ApoE−/− mice experimental groups, but not in the non-ligated groups (Fig. 2A and B). Furthermore, IHC staining of CD68, a macrophage surface marker, revealed that ligature placement similarly increased the recruitment of macrophages in both WT and ApoE−/− mice (Fig. 2C). Ligature placement also upregulated the expression of p65, a subunit of the major inflammatory transcriptional factor, NF-κB (24) in the alveolar bone of both WT and ApoE−/− mice (Fig. 2D). These data indicated that ligature placement induced similar degree of periodontitis and local inflammation in both WT and ApoE−/− mice.

Figure 2. Evaluation of osteoclasts and immunohistochemistry on maxillary tissues placed ligature in WT and ApoE-/- mice fed with HFD. (A) TRAP-stained tissues at the maxillary second molar areas. Black stars indicate osteoclasts. Scale bar, 50 µm. (B) Average number of TRAP+ osteoclasts per mm2 of alveolar bone. ****P<0.0001. Immunohistochemistry of (C) CD68 antibody and (D) p65 antibody on the periodontium of maxillary second molar. Scale bar, 50 µm. TRAP, Tartrate-resistant acid phosphatase; WT, wild-type; ApoE-/-, Apolipoprotein E-deficient; HFD, high-fat diet.

Differential systemic status of lipid and systemic inflammation in WT and ApoE−/− mice by ligature placement

To determine whether the ligature placement altered lipid content in the blood stream, enzymatic assays were performed to detect the levels of several types of lipids. As expected, the control ApoE−/− mice without ligature placement had significantly higher levels of TG, TC and LDL levels in the serum compared with WT control mice, although WT control mice revealed significantly higher HDL levels compared with the ApoE−/− control mice (Fig. 3A-D). Notably, the long-term ligature placement did not alter the lipid profiles in both experimental WT and ApoE−/− mice when compared with those of control mice without ligature placement (Fig. 3). By contrast, ligature placement significantly increased the serum levels of proinflammatory cytokines, such as, IL-1β, IL-6 and TNFα in the serum of both WT and ApoE−/− mice compared in the mice that did not receive ligature placement, although the cytokine levels were markedly higher in ApoE−/− mice compared with WT mice (Fig. 4). These data indicated that ligature-induced periodontitis induced systemic inflammation in both WT and ApoE−/− mice without altering lipid profiles.

Figure 3. Cholesterol levels in WT and ApoE-/- mice fed with HFD. Mice sera were used and analyzed using ELISA for (A) TG, (B) TC, (C) LDL and (D) HDL. Results represent the means ± SEM from 5 different samples. ***P<0.001, ****P<0.0001. WT, wild-type; ApoE-/-, Apolipoprotein E-deficient; TC, total cholesterol; LDL, low density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; TG, triglyceride.

Figure 4. Levels of pro-inflammatory cytokines in WT and ApoE-/- mice fed with HFD. Mice sera was analyzed using ELISA for (A) IL-1β, (B) IL-6 and (C) TNF-α. Results represent the means ± SEM from 5 different samples. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. WT, wild-type; ApoE-/-, Apolipoprotein E-deficient; HFD, high-fat diet.

Ligature induced periodontitis enhances arterial lipid deposition and vascular inflammation in ApoE−/− mice, not in WT mice

To examine the atherosclerosis development, en face analysis was performed to quantify the lipid deposition on the arterial wall. WT mice did not develop notable lipid depositions in both with or without ligature placement groups. By contrast, ApoE−/− mice revealed significantly increased lipid deposition in ligatured mice compared with the non-ligatured group (Fig. 5A and B). Similarly, the Oil Red-O staining of aortic roots also demonstrated increased deposition of lipid in the vessel walls of ApoE−/− mice receiving ligature placement compared with those in ApoE−/− mice without ligature placement, but no notable lipid deposition was observed in the WT mice regardless of ligature placement (Fig. 5C).

Figure 5. Development of vascular inflammation and atherosclerosis in WT and ApoE-/- mice fed with HFD. (A) Images of mice aortas from the en face preparation after staining with Sudan IV (scale bar, 5 mm). (B) Quantification of the en face analysis. The intensity of lipid staining was compared: Results represent the means ± SEM from 5 different samples. ****P<0.0001 vs. ApoE-/- with no ligation. (C) Representative examples of cross sections from Oil Red O-stained aortic root (scale bar, 200 µm). Gene expression levels of (D) TNF-α, (E) IL-1β and (F) IL-6 from aortic tissue determined using RT-qPCR. β-actin served as loading control. Results represent the means ± SEM from 5 different samples. *P<0.05. WT, wild-type; ApoE-/-, Apolipoprotein E-deficient; HFD, high-fat diet; AL, aortic leaflet; AW, aortic wall; L, lumen.

When gene expression levels of the pro-inflammatory cytokines were measured from the aorta using RT-qPCR, the present study revealed negligible levels in WT mice whether the ligature was placed or not (Fig. 5D-F). By contrast, there was a significant increase in the expression levels of TNF-α, IL-1β and IL-6 in the arterial wall in ApoE−/− mice with ligature placement compared with ApoE−/− mice without ligature placement (Fig. 5D-F). These data indicated that ligature-induced periodontitis increased vascular inflammation and aortic lipid deposition in ApoE−/− mice, but not in WT mice.

Discussion

In the present study, severe periodontitis was introduced in both WT and ApoE−/− mice by a long-term ligature placement around the second molars. WT mice did not develop atherosclerosis even under extreme conditions such as severe periodontitis and HFD Meanwhile, HFD alone moderately developed atherosclerosis in ApoE−/− mice, but the ligature-induced severe periodontal disease significantly exacerbated the development of atherosclerosis.

It is worth noting that WT mice seldomly develop atherosclerosis, which is attributed to their innate capacity to lowering lipid contents (25,26). In the present study, serum lipids in WT mice were significantly decreased compared with those in ApoE−/− mice when both groups were exposed to HFD. One of the major functions of ApoE protein is to serve as a ligand for the LDL receptor and to facilitate uptake and clearance of various lipoprotein complexes (27). Subsequently, the current study clearly demonstrated and verified that elevated lipid levels are a bona fide pre-requisite for atherosclerosis development.

Notably, severe periodontitis induced by the ligature placement did not alter the levels of lipids in WT mice when compared with WT mice without periodontitis, despite the significant increase of systemic inflammation by periodontitis. Previous clinical studies demonstrate rather conflicting results on the association between periodontitis and lipid levels; some studies have revealed a positive correlation while others demonstrated it to be insignificant (28–31). Based on the present data, it was hypothesized that ligature-induced periodontitis did not directly affect the lipid levels in serum and that ligature-induced periodontitis and lipid levels may be independent risk factors for the atherosclerosis development in mice.

The present study indicated that the levels of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α were all significantly elevated in ApoE−/− mice in the presence of severe periodontitis, which suggested that these cytokines were putative therapeutic targets for the atherosclerosis. Indeed, the utilization of anti-IL-6 or anti-TNF-α therapy such as infliximab or tocilizumab have been suggested in clinics; however, however, their negative side effects decrease their likelihood of being used (32,33). By contrast, a previous clinical trial, Canakinumab Anti-inflammatory Thrombosis Outcomes Study, demonstrated that Canakinumab, an anti-IL-1β antibody, effectively reduces recurrent major adverse cardiovascular events (34). Overall, these clinical studies underscore the importance of systemic inflammation in mediating the development of atherosclerosis.

Although the present study demonstrated an association between severe periodontitis and the development of atherosclerosis in ApoE−/− mice, the molecular mechanism of this link remains unknown. However, there are several explanations for this association. First, severe periodontitis induces chronic inflammation, which results in persistent pro-inflammatory cytokines at the local level and releases them systemically throughout the body, such that it ultimately affects atherosclerosis in hyperlipidemic condition. Second, the oral bacteria responsible for periodontal disease might induce the development of atherosclerosis. Indeed, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis have been revealed in atherosclerotic lesions in humans (35). In addition, oral inoculation of Porphyromonas gingivalis induces atherosclerosis in experimental hyperlipidemic mouse models (36,37). However, given that other ‘sterile’ inflammatory conditions, such as SLE, are also a risk factor for atherosclerosis (38), the sole role of bacteria requires further clarification. Lastly, chronic and persistent periodontal disease may trigger alterations in local immunity (via epigenetics) such that it permanently changes the behaviors of the key players in atherosclerosis (39,40). The fundamental mechanisms of periodontitis-induced atherosclerosis development need to be further investigated.

There are several limitations to the present study. First, although ApoE−/− mice are well-established to represent studies on atherosclerosis, global knockout of ApoE in other cells make it challenging to rule out involvement of confounding cells (41,42). As such, it may require additional validation of the present findings with other mouse models such as LDLR−/− mice. Second, 14 weeks of ligature placement were used to mimic chronic periodontal disease conditions. However, it remains to be determined whether 14 weeks of ligature in mice truly represents chronic periodontal disease in humans. Third, involvement of the oral microbiome cannot be ruled out because, as aforementioned, several known oral bacterial species are found in the atherosclerotic lesions (35–37) and play a direct pathologic role in atherosclerosis development.

In summary, the present study suggested that severe periodontal diseases were a significant risk factor for exacerbating atherosclerosis under specific conditions, such as hyperlipidemia. In addition, periodontal disease and high cholesterol were seemingly independent risk factors for atherosclerosis development, and the coupling of systemic inflammation and hyperlipidemia may be necessary for the development and exacerbation of atherosclerosis induced by periodontitis. Alleviating chronic periodontal disease could potentially be a novel therapeutic method to intervene in atherosclerosis development in high-risk groups.

Acknowledgements

Not applicable.

Funding

This work was supported in part by the research funds awarded from the UCLA Chancellor's Office and National Institutes of Health/National Institute of Dental and Craniofacial Research (grant no. R01DE 023348).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

NHP and RK were involved in the conceptualization of the study. JS, SK, and SHL performed the experiments and participated in data analysis. JS, SK and SHL confirmed the authenticity of all the raw data. JS, SK, SHL, RK and NHP were involved in the discussion and interpretation of the results. JS, SK, RK, and NHP drafted the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

All procedures were performed in compliance with the institution's policy and applicable provisions of the United States Department of Agriculture (USDA) Animal Welfare Act Regulations and the Public Health Service (PHS) Policy. The experimental protocols were approved by the Animal Research Committee of the University of California, Los Angeles (approval no. ARC# 2019-057).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References