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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 Mar 19;35(5):1123–1133. doi: 10.1161/ATVBAHA.115.305324

Resolvin E1 Attenuates Atherosclerotic Plaque Formation in Diet and Inflammation Induced Atherogenesis

Hatice Hasturk 1, Rima Abdallah 2, Alpdogan Kantarci 1, Daniel Nguyen 1, Nicholas Giordano 4, James Hamilton 3,4, Thomas E Van Dyke 1
PMCID: PMC4415167  NIHMSID: NIHMS671682  PMID: 25792445

Abstract

Objective

Epidemiological and recent clinical studies implicate periodontitis as an independent risk factor for cardiovascular disease. Previously, we demonstrated that rabbits with experimental periodontitis and cholesterol diet exhibit more aortic plaque compared to diet alone. We also showed that a proresolution mediator, Resolvin E1 (RvE1), reverses the experimental periodontitis. Here, we determined whether oral/topical application of RvE1 attenuates aortic atherosclerosis induced by both diet and periodontal inflammation.

Approach and Results

Thirty-nine rabbits on a 13-week regimen of 0.5% cholesterol diet were included. Periodontitis was induced by P. gingivalis in 24 rabbits and 15 rabbits were placed in no-periodontitis groups. Interventions were no-treatment, vehicle, and RvE1 treatment (4μg/site or 0.4 μg/site) topically applied 3-times/ week. At 13 weeks, both periodontitis and atherosclerosis were quantified. Atherosclerotic plaques were assessed by Sudan IV staining, histology and ex vivo MRI. Serum levels of C-reactive protein (CRP) were evaluated as a measure of systemic inflammation.RvE1, used as an oral/topical agent, significantly diminished atherogenesis and prevented periodontitis (p<0.05). In the absence of periodontal inflammation, oral/topical application of RvE1 resulted in significantly less arterial plaque, a lower intima/media ratio, and decreased inflammatory cell infiltration compared to no-treatment (p<0.001). Local oral RvE1 application significantly reduced systemic levels of CRP (p<0.05).

Conclusions

The results suggest that oral/topical RvE1 attenuates enhanced atherogenesis induced by periodontitis and prevents vascular inflammation and atherogenesis in the absence of periodontitis. The inhibition of vascular inflammation with endogenous mediators of resolution of inflammation provides a novel approach in the prevention of atherogenic events.

Keywords: atherosclerosis, inflammation, vascular inflammation, animal model

Introduction

Atherosclerosis is a complex chronic vascular disease characterized by accumulation of plasma-derived lipids in large vessel walls. Cardiovascular diseases, including atherosclerosis, are a major health problem worldwide and account for 1 in every 4 deaths in the United States1. Despite efforts aimed at reducing modifiable risk factors such as cholesterol, smoking, hypertension and diabetes, atherosclerosis continues to increase in the population. Additional etiologic factors must therefore contribute to the pathogenesis of the disease 2.

Advances in basic and experimental science have elucidated the role of inflammation and the underlying cellular and molecular mechanisms that contribute to atherogenesis. It is now recognized that inflammation is a pathological component at all stages of aortic plaque formation from initiation to rupture2. Early plaques (type I) are characterized by accumulation of intimal macrophages and foam cells that progress to grossly visible fatty streaks (type II plaques). The factors that determine lesion progression to cause an atherothrombogenic event remain unclear; however, progression is strongly associated with persistent inflammation of the vascular wall, which suggests that chronic, unresolved inflammation may be an underlying mechanism for atherothrombogenesis3.

Resolution of inflammation is now understood to be a biochemically active process, and a mechanism that self-limits a normal inflammatory challenge. Resolvin E1 (RvE1) is a potent mediator of active resolution derived from the ω-3 fatty acid, eicosapentaenoic acid. RvE1 is protective in rabbit periodontal disease4, murine colitis5, murine peritonitis6, asthmatic airway inflammation7, bacterial pneumonia and acute lung injury in mice8. Accumulating evidence suggests an important role for resolution phase agonists derived from dietary ω-3 polyunsaturated fatty acids, resolvins (i.e., RvE1, RvD1), as well as lipoxins (i.e., LXA4), and protectins (PD1, PD2), in the return to tissue homeostasis (for review, see Serhan, Chiang and Van Dyke, 2008)9. These findings open a potential new avenue for the modulation of the host response in inflammatory diseases.

Periodontitis is an inflammatory disease of the tissues supporting the teeth (gingiva, alveolar bone, periodontal ligament and cementum) in which microbial etiologic factors induce innate immune-mediated destruction of these tissues. Periodontitis has been implicated as a risk factor systemic inflammatory diseases including atherosclerosis, myocardial infarction, and stroke10. While still controversial, recent systematic reviews suggest a significant risk association10. In addition, clinical studies demonstrate that people with periodontitis have elevated C-reactive protein, interleukin 6, haptoglobin, and fibrinogen. Post-acute myocardial infarction patients with periodontitis have significantly higher C-reactive protein than acute myocardial infarction alone, suggesting that periodontal disease is an independent contributor to systemic inflammation11, 12.

Rabbit atherosclerosis models are advantageous for studying therapies to attenuate plaque progression or to reverse atherosclerosis because of their potential for translation to human disease. Rabbits provide a range of plaque stages that more closely resemble those in humans without genetic modification of the animal, and the plaque development can be modulated by diet13, 14. The rabbit is also a unique and valuable model for experimental periodontitis, which is induced by topical application of a human periodontal pathogen, Porphyromonas gingivalis (P. gingivalis.), over 6 weeks4, 15. Similar to atherosclerotic plaque formation in the aorta, the periodontal disease closely resembles that in humans macroscopically and histologically14. In a previous study with simultaneous induction of periodontitis and atherosclerosis in New Zealand White (NZW) rabbits over 13 weeks, rabbits with experimentally induced periodontitis had more extensive accumulation of aortic lipids than did periodontitis-free animals16.

In this study, we examine the therapeutic impact of RvE1 on initiation of atherosclerosis in the rabbit model. Additionally, we assess the impact of local, topical treatment of periodontitis on enhanced atherogenesis associated with periodontitis,

Materials and Methods

Materials and methods are available in the online-only Supplement.

Results

Periodontitis modifies the course of early atherogenesis

A total of 26 male rabbits were utilized to assess the impact of local inflammation, in this case, periodontal disease, on atherosclerotic lesion development when induced by cholesterol-rich diet (Supplemental Figure 1). Periodontal disease and atherosclerosis were simultaneously developed in 20 of the rabbits with a 0.5% cholesterol diet (CD) and application of topical P. gingivalis to ligated teeth (+Pg). Four rabbits on CD received ligatures without P. gingivalis and served as the “atherosclerosis alone” group. Negative controls included two rabbits fed a normal chow diet and kept free of P. gingivalis (ND). P. gingivalis application was stopped after 6 weeks of induction according to the established disease model4, 15. Treatments continued for another 7 weeks as did the cholesterol diet. At the end of the 13-week period, the rabbits were euthanized and the mandibles as well as the entire aorta were dissected. The aorta was dissected from aortic arch to the iliac bifurcation, cut longitudinally, stained with Sudan IV and imaged en face for quantification of aortic plaque deposition using ProImage software as previously described15. Further, the aorta was embedded in paraffin, sectioned and stained with H&E for histopathological evaluation of intima, media and adventitia. Intima/media ratio was calculated by the linear measurements of intima and media thickness. Sequential sections were stained with Masson's trichrome for evaluation of collagen and the characteristics of atherogenesis including inflammatory cell infiltration, medial fibrosis and necrotic core as previously described17-19.

Macroscopic and microscopic evaluation of the mandibles revealed significant periodontal tissue destruction (both crestal and interproximal) similar to human periodontal disease in rabbits receiving tooth ligature and the human periodontal pathogen Porphyromonas gingivalis, (Figure 1A and B, +Pg compared to ND and CD). Evaluation of fatty streaking in the aortas of the animals that received both the cholesterol diet and P.gingivalis revealed that periodontal disease resulted in significantly more atheromatous plaque formation compared to CD alone (Figure 1C-aorta en face images). As previously shown20, rabbits do not develop natural atherosclerotic changes; the rabbits on normal diet did not present with any fatty streaks (Figure 1C-ND). Histopathological evaluation of aortic layers; (tunica), intima, media and adventitia, revealed atherosclerotic changes with intimal thickening and lipid accumulation in cholesterol-fed animals, which was significantly greater in animals receiving P. gingivalis (+Pg; n=5). Foam cell accumulation and invasion of smooth muscle cells within the tunica intima were obvious especially in animals with periodontal inflammation in addition to cholesterol-diet (Figure 1D; +Pg). Pathological thickening of the tunica intima and underlying atrophic media were also observed. A thin fibrous cap was visible overlying the smooth muscle cell layer facing the lumen containing macrophages and other inflammatory cells.

Figure 1. Impact of periodontal disease on atherosclerotic changes in aorta.

Figure 1

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A. Macroscopic assessments of alveolar bone loss in animals (n=5) received P. gingivalis compared to those that did not receive P. gingivalis (ND and CD). Arrows depict the area of interest B. Histological assessments on H&E stained sections depict the histological bone loss associated with periodontal disease compared to ND (n=2) and CD (n=4). C. Aortas were cut en-face, pinned down and stained with Sudan IV to detect fatty depositions. The top figure is depicting an aorta from an animal who received normal diet. D. Aortas were embedded in paraffin; thin sections were cut and stained with H&E for histomorphometric assessments of media and intima thickness. Red arrow depicts a thin fibrous cap. E. The quantitative measurements on defleshed mandibles and histological sections depict the bone loss associated with periodontal disease compared to CD and ND). The inserts are depicting the direct and histomorphometric measurements. Lipid covered area and intima/media ratio were greater in animals received P. gingivalis in addition to CD. Pg: P. gingivalis. TA: tunica adventitia; TM: tunica media; TI: tunica intima; L: lumen.*Statistically significant compared to CD and ND; #Statistically significant compared to ND (ANOVA with Bonferroni post hoc test).

Quantitative measurements confirmed these observations. For periodontal disease, infrabony defect formation was significantly greater in P.gingivalis induced cholesterol-fed animals (+Pg) compared to rabbits on cholesterol-diet alone (CD; n=5) (Figure 1E-upper left graph, p=0.0001). The histological bone loss measured is shown in the insert (Figure 1E-upper right graph). The animals that received P. gingivalis in addition to cholesterol diet had significantly greater bone loss compared to cholesterol diet alone (p=0.007) and normal diet (p=0.0001). For aortic changes, the area covered by lipid was quantified as percentage of the entire aortic surface. Periodontal disease significantly increased the fatty deposits stained by Sudan IV (>75%) (Figure 1E-lower left graph; p=0.01). Calculation of intima/media ratio revealed that periodontal disease markedly increased intimal thickening (Figure 1E-lower right graph), however the difference was not statistically significant compared to CD alone. As expected, cholesterol diet alone showed significant changes in lipid deposition and intima/media ratio compared to healthy rabbits (ND). It appears that CD negatively affects periodontal tissues as seen with infrabony defect and histological bone loss assessments; however the differences between ND were not statistically significant (Figure 1E).

RvE1 prevents periodontal inflammation and attenuates atheromatous plaque

Simultaneous treatments with vehicle or 0.4μg RvE1/site or 4μg RvE1/site were applied at the time of the disease induction (n=5/group). Periodontal disease induction was dramatic and clearly observed in all animals receiving P. gingivalis and ligature without treatment. The initiation of periodontal disease by P. gingivalis was unaffected by vehicle therapy and tissue destruction was at a level similar to the untreated rabbits (Figure 2A-upper panels). Oral topical application of RvE1 demonstrated a dose/response inhibition of development of periodontal destruction Descriptive histopathological evaluations (H&E staining) of periodontal tissues supported the clinical observations where RvE1- (4 μg/site) treated samples showed healthy histological architecture with intact bone and no inflammatory cell infiltration. Untreated (Figure 1B, +Pg) and vehicle-treated samples (Figure 2A-lower panels) showed classical histopathological characteristics of periodontal disease with crestal bone loss, collagen degradation and increased inflammatory cell infiltration. Quantitative morphological and histomorphological assessments revealed the statistically significant impact of topical RvE1 treatment on prevention of periodontitis (Figure 2B). The cholesterol diet alone group also exhibited mild interproximal bone loss compared to normal diet animals, but oral topical RvE1 completely prevented periodontal tissue destruction in all groups with periodontal disease.

Figure 2. Outcome of treatment with oral topical RvE1.

Figure 2

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A. Treatment with RvE1 in both doses resulted in apparent prevention from periodontal disease induction; this was also demonstrated by histological assessments (RvE1-treated) compared to vehicle treated animals. B. Bone loss was quantified as infrabony defect on defleshed jaws and as histological bone loss on H&E stained sections. C. En face evaluation of aortas dissected from animals treated with vehicle (n=5) or RvE1 low dose (n=5) or RvE1 high dose (n=5). For comparison, an aorta from untreated animal is depicted (+Pg; n=5). D. Bar graph shows quantification of percent lipid covered area in all groups. E. Histological assessments of H&E stained sections of aortas from groups treated with vehicle, RvE1 low dose and RvE1 high dose. Red arrow depict fibrous cap lining outward of intima. F. Comparison of intima/media ratio between groups. (*p<0.05 compared to +Pg and vehicle-treated groups; #p<0.05 compared to ND (n=2) and CD (n=4); ANOVA with Bonferroni post hoc test; ND: normal diet; CD: cholesterol diet; Pg: P. gingivalis; TM: tunica media; TI: tunica intima; TA: tunica adventitia; L: lumen).

Evaluation of the impact of oral topical RvE1 on atheromatous changes enhanced by periodontitis revealed that atherogenesis was prevented by RvE1 at both doses (Figure 2C). The animals on 0.5% cholesterol diet with induced periodontal disease developed significantly more lipid accumulation that was inhibited by RvE1 (Figure 2D; p<0.05). Vehicle treatment did not prevent these changes; animals in this group had large areas of lipid-rich plaque that covered the entire surface of aorta which was comparable to untreated (+Pg) group (Figure 2D). In fact, vehicle treatment tended to have worse outcomes. We do not think that 5% ethanol in saline would account for this effect. Although the results show more disease in vehicle-treated group, the difference between it and the untreated group was not statistically significant. Histopathological observations and histomorphometric measurements were similar to macroscopic assessments. Cholesterol diet alone, untreated and vehicle-treated groups all showed signs of early atheroma formation (fatty streaks), including lipid containing macrophages (foam cells). In both RvE1-treated groups, there was a marked decrease in plaque extent and thickness (Figure 2E-histology images). RvE1 (4μg/site) resulted in significantly less total intimal surface calculated as the percent of total area indicating that RvE1 decreases atherogenesis characterized by thickening of intima and anthropic media layers (Figure 2F; p<0.05).

Further, to characterize the atherosclerosis process, sequential sections of aorta were stained with Masson's trichrome. Figure 3 demonstrates further atherosclerotic changes induced by cholesterol diet and P. gingivalis. Inflammatory infiltration and medial fibrosis were significantly increased by the induction of P. gingivalis-mediated periodontal inflammation. Necrotic core occupied 30-50% of the lesions where smooth muscle disorientation was observed. Fibrous cap surrounded the necrotic areas and foam cells were evident. No sign of plaque rupture was observed in any specimen. Vehicle treatment did not impact the atherosclerotic or inflammatory changes associated with the cholesterol diet and P. gingivalis. Treatment with RvE1 dramatically prevented inflammatory and atherosclerotic changes with minimal signs of necrotic areas, fibrotic changes in media, substantially lower infiltration of inflammatory cells, no sign of foam cells, and no evidence of fibrous cap or smooth muscle cell disorientation. RvE1 prevention of medial fibrosis was dose dependent; the higher dose showed statistically significant reductions in medial fibrosis compared to lower dose (4 mg/site versus 0.4 mg/site; p=0.025).

Figure 3. Characterization of the atherosclerotic process.

Figure 3

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Aorta sections were stained with Masson's trichrome for further assessments of the changes. A. Panels represent histological images from study groups. B. Inflammatory infiltration and medial fibrosis were graded semiquantitatively as detailed in Material and Methods. Necrotic area was quantified as a percentage of the total surface area occupied by the lesion. In cholesterol diet (CD) sections, induced inflammation (*), fibrotic changes in media and evidence of necrotic areas are observed (N). Periodontal disease further and significantly increased these changes. Foam cells became evident (Insert, F). Fibrous cap (arrow) was observed around the necrotic core. (*p<0.05 compared to all other groups, #p<0.05 compared to RvE1 0.4 mg/site; ANOVA) CD: cholesterol diet; Pg: P.gingivalis; SM: smooth muscle; N: necrotic core; F: foam cells.

Oral-Topical RvE1 in the Absence of Periodontitis

To determine if the impact on periodontitis enhanced atherogenesis of the pro-resolving agonist RvE1 is mediated through resolution of local inflammation alone, oral-topical local application of RvE1 was assessed in the cholesterol diet model without concomitant periodontal disease. Simultaneous treatments with vehicle (n=3) or 0.4μg RvE1/site or 4μg RvE1/site were applied at the time of the cholesterol diet initiation (n=5/RvE1 group). The en face aortas of rabbits treated with oral-topical RvE1 showed smaller area coverage and less intense deposition of staining compared to aortas from untreated or vehicle treated rabbits (Figure 4A and B-aorta images and graph). Histopathological evaluations of thin slices confirmed these findings and showed lower intima/media ratios. There was also significant dose-dependent action of RvE1 on protection against atherogenesis (Figure 4C and D, histological images and graph; p<0.05). Results from this experimental arm demonstrated for the first time that locally applied oral-topical RvE1 has direct actions on the systemic inflammatory response and can prevent and/or control inflammation in the distant organ systems.

Figure 4. Impact of oral topical RvE1 treatment on atherosclerotic process in the absence of oral disease.

Figure 4

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Atherosclerosis were induced by 0.5% cholesterol diet for 13 weeks, while simultaneous treatments with oral topical RvE1 in low (n=5) and high (n=5) doses and vehicle (n=5) were applied. A group of rabbits did not received any treatment and served as untreated control (CD; n=4). A. Aortas were cut en-face, pinned down and stained with Sudan IV to detect fatty depositions. The top figure is depicting an aorta from an animal who received normal diet. B. Quantification of lipid covered area and comparison between groups. Please note that rabbits on normal diet do not develop aortic fatty deposits. C. Formalin-fixed aortas were embedded in paraffin, sectioned and stained with H&E staining for histological assessments. For ND, histological section was shown in Fig 1. D. Intima/media ratio was calculated and compared between groups. E. Magnitude of impact of treatment in the presence of local periodontal inflammation was compared to the impact of treatment without periodontal disease. Mean values of percent lipid covered area were calculated per group and subtracted from the mean value of percent covered area in CD group (n=4). (*p<0.05 compared to vehicle and CD; #p<0.05 compared to CD; ANOVA with Bonferroni post hoc test). ND: Normal Diet; CD: Cholesterol diet; TA: tunica adventitia; TM: tunica media; TI: tunica intima; L: lumen.

To determine the magnitude-of-impact of the RvE1 treatment on atherosclerotic changes as a result of cholesterol feeding with and without periodontitis, we compared percent lipid covered area in all treatment groups to the cholesterol diet alone (CD) (Figure 4E). 0.5% cholesterol-rich diet resulted in lipid accumulation that covered on average 50 ± 12% of the aorta surface (en face). When cholesterol-fed rabbits were simultaneously introduced to P. gingivalis-mediated periodontal inflammation, lipid accumulation was about 25 ± 8 % more (reached to >75% lipid covered area). Both doses of RvE1 without added P. gingivalis showed a dramatic preventive impact of up to 25% protection; the doses did not differ significantly. The group that received both P. gingivalis and RvE1 showed approximately 15% protection at the high dose; 10% at the low dose. These data suggest that periodontitis, as a local inflammatory insult, has significant systemic actions. In addition, as supported by the serum CRP levels, local oral-topical application of RvE1 has a significant impact on reducing vascular inflammation in the peripheral circulation.

High Field ex vivo MRI

Figure 5 shows representative high field MR images that demonstrate differences in atherosclerotic plaques in a subset of 3 groups of rabbits (CD, +Pg and +Pg with 4μg RvE1/site). CD (no periodontitis) images show a region without extensive plaque and no significant luminal narrowing. The magnetic transfer contrast (MTC) image reveals a modest amount of thickening with fibrous protein and DWI and a low amount of lipid in the intima (Figure 5A). In the cholesterol- fed rabbit with periodontal disease (Figure 5B), there is an increase in lipid in a foamy macrophage region (an early type of lesion without an organized fibrous cap) with a protein mesh (grey). The red arrow shows a dark band characteristic of a fibrous cap and the green arrow the lipid-enriched region. The very hyper intense (bright regions) fat (triglyceride) depots adherent to the adventitia that are not part of the plaque can be seen external to the plaque in the DWI (Figure 5B). The images of an aortic segment of a rabbit treated with RvE1 shows a remarkably normal vessel wall with no sign of significant plaque in either MRI sequence (Figure 5C).

Figure 5. Ex vivo MRI.

Figure 5

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High field (11.7T) ex vivo images of rabbit aortic segments. A. Cholesterol fed rabbit, early atherosclerotic changes are seen as thickening of intima and tunica media; B. Cholesterol fed rabbit with periodontal disease; the red arrow shows a lipid rich foamy macrophage region (an early type of lesion without an organized fibrous cap) within a protein mesh C. Cholesterol fed rabbit with periodontal disease with RvE1 treatment. No detectable lipid is shown within the plaque; the bright regions external to the plaque are fat depots adherent to the adventitia in the segment examined. In the magnetic transfer images (top row) the dark regions depict a dense protein structure while the DWI images (obtained on the same slice corresponding to the MT image) illustrates lipid (cholesteryl esters) as bright regions. Corresponding histology images from rabbits with or without treatment show similar findings. H&E stained sections show a thickened tunica media for the cholesterol fed rabbit with periodontal disease (B) and a thin vessel wall for the RvE1-treated rabbit (C). MT: magnetic transfer; DWI: diffusion-weighted images; CD: cholesterol diet; +P. gingivalis: cholesterol diet + periodontal disease.

Serum Lipid Level

The lipid profiles including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) at each time point throughout the experiment were assessed to evaluate potential shifts in serum lipid levels induced by various treatments. Every group demonstrated high levels of circulating lipids as a result of the cholesterol diet, which was statistically significant between baseline and 13 weeks (p<0.0001), except in the normal diet group. Although the serum lipid levels were similarly elevated in all groups (Supplemental Table 1), this did not translate into plaque formation in the RvE1 treated groups, which supports the current literature suggesting that inflammation is an important contributor to plaque formation and eventually cardiovascular disease. Interestingly, the periodontal disease group showed smaller changes in all lipids compared to other groups (not statistically significant), but the atherosclerosis level was significantly greater compared to cholesterol diet alone and RvE1-treated groups.

RvE1 Treatment reduces Serum CRP to Baseline Healthy Levels

CRP is accepted as the gold standard clinical marker of vascular inflammation, atheroma plaque burden and as a predictor of cardiovascular disease21-23. Elevated serum CRP levels with periodontitis were reported by several clinical and preclinical studies15, 24, 25. To evaluate the influence of local inflammation on the systemic inflammatory response and to determine whether proresolving actions of RvE1 alter systemic inflammation, serum CRP levels were measured at baseline, 6 weeks and 13 weeks. CRP levels were significantly elevated in all cholesterol-fed rabbits (Figure 6A and B), but more dramatically in those who additionally had periodontal inflammation (Figure 6A; p<0.05). Of interest, topical treatment with RvE1, in both doses, significantly reduced the serum CRP levels; the reduction was more pronounced in the rabbits without periodontal inflammation with significant differences between two doses of RvE1 at 13 weeks (p=0.013). This finding supports the results of the measurements of lipid-covered area as well as histological evaluations suggesting that local inflammation contributes to increased atheromatous plaque burden and systemic inflammation.

Figure 6. Local inflammation induces up-regulation of CRP in serum.

Figure 6

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Blood samples were collected at baseline, 6 and 13 weeks of disease/treatment. Serum CRP levels were measured using a rabbit-specific ELISA. CRP levels were elevated in all cholesterol-fed rabbits escalating significantly through 6 to 13 weeks (A and B; *p<0.05), but the up-regulation was more dramatic in animals with periodontal inflammation (A; *p<0.001). Of interest, topical treatment with RvE1 (n=5/ RvE1 group) significantly reduced the elevated CRP levels compared to untreated (n=5) and vehicle-treated (n=5) groups (A and B; #p<0.05). RvE1 dose dependently resulted in significant regression of CRP levels in cholesterol-fed rabbits without periodontal inflammation (B, Ψp<0.001 compared to RvE1 0.4 mg/site); CRP levels at 13 weeks were comparable to those on normal diet. CD=Cholesterol diet; n=4; ANOVA with Bonferroni correction.

Discussion

In this paper, we report the novel finding that preventive use of oral-topical RvE1, an endogenous mediator of resolution of inflammation, protected hypercholesterolemic rabbits against aortic atherosclerotic plaque formation. Additionally, local oral-topical RvE1 treatment significantly prevented the extensive atheromatous plaque formation induced by the presence of a chronic local inflammation, periodontal disease. The goal was to investigate whether RvE1, a resolution phase lipid mediator known to prevent and treat periodontitis, provides protection from increased atherogenesis induced by periodontal disease in a prospective study. As previously shown by our group and others20, 26, rabbits develop significant lipid deposition and atherogenic changes in the aorta over a 13-week period when fed a 0.5% cholesterol diet; local periodontal inflammation induced by P. gingivalis dramatically increases lipid deposition in this model20. The data support a role for periodontitis, as a local, chronic inflammatory insult in the progression of cardiovascular disease and the role of inflammation in the initiation and progression of both periodontitis and cardiovascular disease. The prevention of both diseases by topical RvE1 suggests a potential therapeutic benefit.

Proinflammatory lipid mediators such as leukotrienes have been implicated in the promotion of atherosclerosis; it is noteworthy that people with periodontal disease have elevated levels of leukotriene in their gingival fluid27. The LTB4 receptor BLT-1 was identified on human vascular smooth muscle cells (VSMCs) and co-localized with VSMCs in human atherosclerotic plaques. E series resolvins (RvE1 and RvE2) are antagonists for the leukotriene B4 receptor BLT-1, in addition to agonist signaling properties. Inhibition of LTB4 actions with BLT-1 receptor antagonists protects rabbits from intimal hyperplasia28. Moreover, RvE1 is an agonist for the ChemR23 (ERV1) receptor, which was recently demonstrated to be expressed by human VSMCs. Binding of RvE1 to chemR23 modulated VSMC migration via platelet-derived growth factor phosphorylation28. These findings support the actions of RvE1 in the resolution vascular inflammation and protection from atherosclerotic lesions.

Perhaps the most striking observation is the marked inhibition of atherogenesis in the absence of periodontitis by local oral-topical application of relatively small doses of RvE1. These data suggest that RvE1 is rapidly absorbed through the mucosa into the circulation and that these low doses have a major impact of vascular inflammation. The findings are supported by significantly reduced levels of CRP after RvE1 treatment in both scenarios (with or without periodontal disease). The impact of RvE1 was more pronounced in rabbits without periodontal inflammation. The study results support the previous reports indicating periodontitis as an independent modifier of systemic inflammation29, 30. The therapeutic actions of RvE1 is comparable to those reported with statins in a recent report 23 where CRP levels, but not LDL, were associated with atheroma plaque regression after statin therapy.

Homeostasis is a fundamental characteristic of living organisms and is often described as a process of balance. The physiologic resolution of a well-orchestrated inflammatory response is essential to maintain homeostasis at the cellular and tissue level. This is accomplished in large part by specialized lipid pathways generating specific mediators that dampen the magnitude of the leukocyte and macrophage infiltrate during inflammation and promote resolution. There is a substantial body of evidence that pro-resolution molecules derived from ω-3 polyunsaturated fatty acids, the resolvins and protectins, act to counter-regulate inflammation and promote resolution31-33. RvE1 specifically interacts with its receptor, ERV1, on monocytes and macrophages to regulate leukocytes during inflammation, as well as local cytokines. A failure in the actions of these natural regulatory molecules in the pathogenesis of atherosclerosis has been hypothesized34, 35 and a potential role in positively modifying the progression of disease has been suggested. For instance, Fredman et al. demonstrate that in addition for modifying macrophage inflammatory phenotype, as reported by Bannenberg et al.36, resolvins play a major role in prevention of platelet aggregation37. These findings when taken in the context of the experiments reported here are consistent with reduced macrophage accumulation in vessels and reduced lipid accumulation and inflammatory infiltration in vascular lesions.

Several classes of pro-resolving mediators exist, spanning from bioactive lipid mediators (like resolvins) to peptides (like annexin A1) and autacoids (like adenosine). In addition, therapies used for other inflammatory disease, particularly rheumatoid arthritis (RA), have the potential to have beneficial effects in cardiovascular diseases. For instance, activation of the melanocortin receptor type 3 has been linked to protection from periodontitis in mice38 and is associated with decreased CRP levels in humans24. Additionally, a recent retrospective study has shown that statins may ameliorate periodontal inflammation in the same individuals who suffer from cardiovascular disease and periodontal disease39. This is likely related to the anti-inflammatory mechanism of statins that is linked to lipoxin and resolvin generation40, 41. Conversely, NSAIDs for arthritis do not have the same outcomes as RvE1, because NSAIDs actually interfere with resolution of inflammation pathways blocking synthesis of resolvins and lipoxins9. Finally, IL-6 inhibition for treatment of RA has been suggested to provide cardiovascular protection, but this suggestion comes from in silico data analysis and has yet to be demonstrated in prospective studies in animals or man42. IL-6R antagonism in periodontal disease prevention is reported to have a positive effect in a single study, but the methods employed in that study make the observations far from conclusive43.

For brief period of time, an infectious etiology was considered as a potential co-factor for cardiovascular disease. The initial reports of an epidemiologic association of periodontal disease and cardiovascular disease coincided with the Chlamydia observations in atherosclerotic lesions and spurred the idea that periodontal bacteria could be etiologic in cardiovascular disease44-46. There are examples in the literature of studies demonstrating oral bacteria in atheromas47, but the logic suggesting that antibiotic therapy would be expected to have an impact on disease progression may be flawed. Richardson reported that an infectious agent anywhere in the body might lead to co-activation of the innate immune system and this co-activation accelerated atherosclerosis in hypercholesterolemic rabbits that were suffering from respiratory tract infections with Pasteurella multocida48. Hence, infectious agents might be an indirect etiologic factor for cardiovascular disease providing the necessary inflammatory stimulus. However, human atheromas often lack any indication of the presence of infectious agents, and even if an infectious particle is present in the lesion, a pathogenic role is far from established for that particular organism.

It is more likely that the link between periodontitis and atherosclerotic disease is inflammation. A body of evidence in the cardiology literature suggests that a local inflammatory nidus can increase vascular inflammation systemically49-51. Recent papers suggest that an elevated innate host response of any origin is a risk factor for cardiovascular disease as well as periodontitis, suggesting the inflammatory response as a common susceptibility determinant. A relatively recent study revealed that initiation of isolated inflammation in the murine dorsal air pouch model resulted in an up regulation of COX-2 mRNA in the heart and lung52 supporting the hypothesis that the magnitude of the inflammatory response to insult or injury is a major determinant in the pathogenesis of inflammatory diseases, including periodontitis and cardiovascular disease.

In this study, MRI was valuable to further confirm our novel findings that periodontal disease, as a model of local inflammation, was capable of increasing atherosclerotic changes in the aorta and that topical use of RvE1 significantly reduces the extent of atherosclerotic changes induced by atherogenic diet or both atherogenic diet and periodontal disease in rabbits.

A limitation of the rabbit model is the relatively short duration of the atherogenic diet. In rabbits, lesion morphology is altered by the percentage of cholesterol added to the diet and the duration of the diet. Short duration and a high percentage of cholesterol of more than 2% cause hypercholesterolemia, and atherogenic lesions rich in foam cells originating from macrophages14. In contrast, a diet with a low cholesterol content and long duration causes atherosclerotic lesions, which are rich in smooth muscle cells and contain cholesterol deposits leading to lesions more similar to those of humans14. In general, the typical diet induced atherosclerosis involves supplementation of 0.5% to 4% cholesterol per weight for approximately 8-16 weeks and resulting lesions primarily consist of macrophage-derived foam cells53. In our model of 0.5 % cholesterol over a 13-week period, atherosclerotic lesions rich in foam cells and macrophages were developed, in addition to an increase in the intima media ratio. The histomorphometric analysis showed significant differences in the intima media ratio and atherosclerotic changes including inflammatory infiltration, medial fibrosis and necrotic core formation between periodontitis and RvE1 treated groups indicating that the prevention of periodontal disease by RvE1 resulted in the prevention of atherogenesis. The interesting finding was that topical application of RvE1 in both doses protected the aortas from the atherogenic changes that are caused by the 0.5 % cholesterol diet in the absence of periodontal disease. Quantitatively, it appears that while periodontitis is a modifier of atherogenesis progression, prevention of inflammation is the overriding principle for the prevention of both diseases.

Conclusions

In an experimental model of periodontitis and atherogenesis, RvE1, used as a topical mono-therapeutic agent, prevented atherosclerotic changes as well as periodontal bone loss induced by periodontal inflammation. Topically administered RvE1 to the gingiva was capable of protecting from atherogenic changes in 0.5% cholesterol-fed rabbits in the absence of oral lesions. Significant reduction in the circulating levels of CRP confirmed the anti-inflammatory changes induced by RvE1. These results show the potential benefits of RvE1 treatment in the attenuation or prevention of atherogenic changes in the aorta indicating a role for RvE1 in macrophage infiltration into intima. The mechanism of action of RvE1 in this scenario warrants further investigation. The study also supports ex vivo MRI as a reliable method of imaging that shows early atherogenic changes and a clear distinction between the foamy lipid area and the fibrous cap.

Taken together, the data suggest that topically applied RvE1, absorbed through dental tissues and oral mucosa, presents a unique therapeutic approach to control local and systemic inflammatory responses.

Supplementary Material

1
2

Significance.

Epidemiological and recent clinical studies have implicated periodontitis as an independent risk factor for cardiovascular disease. Our data support a role for periodontitis in the progression of cardiovascular disease and the role of inflammation in the initiation and progression of both periodontitis and cardiovascular disease. We show that periodontal disease creates a chronic inflammatory state for local periodontal tissues that also complicates the pathogenesis of atherogenesis. The prevention of both diseases by topical RvE1 suggests a potential therapeutic benefit for resolution agonists for the prevention and treatment of these two inflammatory diseases. Topically applied RvE1, which is readily absorbed through dental tissues and oral mucosa, presents a unique therapeutic approach for the treatment of local and systemic inflammatory disease.

Acknowledgments

The authors of this study thank the veterinary and technical personnel of the Boston University Medical Campus Laboratory Animal Science Center for assistance during animal handling and experimental procedures; and the Organic Synthesis Core of P50-DE016191 (Charles N. Serhan, Brigham and Women's Hospital, Boston) for preparing synthetic RvE1.

Sources of Funding

This study was partly supported by USPHS National Institute of Dental and Craniofacial Research grants (DE19938, DE15566 to T.E. Van Dyke), (DE18917 to H. Hasturk) and a Boston University Nano Medicine grant (J.A. Hamilton).

Abbreviations

P. gingivalis

Porphyromonas gingivalis

RvE1

Resolvin E1

MRI

magnetic resonance imaging

LXA4

Lipoxin A4

DWI

Diffusion-weighted images

CRP

C-reactive protein

Footnotes

Disclosures

Boston University is partly assigned patents on resolvins that are subject to potential consultant agreements for Dr. T. E. Van Dyke. The other authors report no conflicts.

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Supplementary Materials

1
NIHMS671682-supplement-1.pdf (190.5KB, pdf)
2
NIHMS671682-supplement-2.pdf (140.3KB, pdf)

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