Resolvin E1 (RvE1) Attenuates Atherosclerotic Plaque Formation in Diet and Inflammation-Induced Atherogenesis

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 with 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 Porphyromonas gingivalis in 24 rabbits and 15 rabbits were placed in no-periodontitis groups. Interventions were no-treatment, vehicle, and RvE1 treatment (4 μg/site or 0.4 μg/site) topically applied 3× per 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 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 with no-treatment (P<0.001). Local oral RvE1 application significantly reduced systemic levels of C-reactive protein (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; models, animal.

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

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).

Figure 2. Outcome of treatment with oral topical RvE1

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).

Figure 3. Characterization of the atherosclerotic process

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.

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

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.

Figure 5. Ex vivo MRI

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.

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

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.