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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:1446.)
© 2005 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Porphyromonas gingivalis Bacteremia Induces Coronary and Aortic Atherosclerosis in Normocholesterolemic and Hypercholesterolemic Pigs

Nadine Brodala; Elizabeth P. Merricks; Dwight A. Bellinger; Damrong Damrongsri; Steven Offenbacher; James Beck; Phoebus Madianos; Daniela Sotres; Yu-Ling Chang; Gary Koch; Timothy C. Nichols

From the School of Dentistry and Department of Periodontology (N.B., D.D., S.O., J.B., P.M.), the Department of Pathology and Laboratory Medicine (E.P.M., D.A.B., T.C.N.), and the Department of Biostatistics (D.S., Y.-L.C., G.K.), University of North Carolina at Chapel Hill and the Center of Oral and Systemic Diseases, Chapel Hill, NC.

Correspondence to Timothy C. Nichols, MD, Francis Owen Blood Research Laboratory, Department of Pathology and Laboratory Medicine, UNC School of Medicine, CB#3114, 125 University Lake Rd, Chapel Hill, NC 27516-3114. E-mail tnichols{at}med.unc.edu

	Abstract

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Objectives— The aim of this study was to determine whether recurrent intravenous injections with Porphyromonas gingivalis (P gingivalis), mimicking periodontitis-associated bacteremia, promotes coronary artery and aortic atherosclerosis in pigs.

Methods and Results— Pigs (n=36) fed low- or high-fat chow were divided into P gingivalis–sensitized and P gingivalis–challenged groups or P gingivalis–sensitized controls and saline-treated controls. Pigs were sensitized with 10⁹ killed P gingivalis subcutaneously. Four weeks later all sensitized pigs in the group to be challenged started intravenous injections thrice weekly for 5 months with 10⁶ to 10⁷ colony forming units of P gingivalis while controls received saline. Anti–P gingivalis antibody, serum cholesterol, and complete blood counts were assayed monthly. Pigs were euthanized 2 weeks after the last injection, and coronary arteries and aortas were analyzed by histomorphometry and immunohistochemistry. Anti–P gingivalis antibody was increased by P gingivalis exposure. P gingivalis–challenged pigs developed a significantly greater amount of coronary and aortic atherosclerosis than controls in the normocholesterolemic group and nearly significant in the hypercholesterolemic group. P gingivalis was detected by polymerase chain reaction in arteries from most (94%, 16 of 17) P gingivalis–challenged pigs but not controls.

Conclusions— Recurrent P gingivalis bacteremia induces aortic and coronary lesions consistent with atherosclerosis in normocholesterolemic pigs and increases aortic and coronary atherosclerosis in hypercholesterolemic pigs.

To determine whether recurrent intravenous injections with Porphyromonas gingivalis promotes coronary artery and aortic atherosclerosis, pigs fed low- or high-fat chow were divided into P gingivalis–challenged or control groups. P gingivalis–challenged pigs developed significantly greater amount of atherosclerosis in the normocholesterolemic group and nearly significant in the hypercholesterolemic group.

Key Words: coronary and aortic atherosclerosis • Porphyromonas gingivalis • periodontitis • pig model • bacteremia

	Introduction

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Periodontitis has been associated with cardiovascular disease in many, but not all, epidemiological studies.^1–15 Mild forms of periodontal disease affect 75% of the population, and more severe forms affect 20% to 30% of adults in the United States.⁸ The mechanism by which periodontitis could contribute to cardiovascular disease is unknown but likely relates to the associated intense local and systemic inflammation and the virulence properties of the causative organisms.^16–21 Porphyromonas gingivalis has been strongly associated with adult periodontitis. This bacteria is a Gram-negative, nonmotile, obligate anaerobe that can invade and infect epithelium, endothelium, and vascular smooth muscle cells^22–29 and appears to alter endothelial function.³⁰ The capacity for invasion is in part mediated by fimbriae,^31,32 and the virulence of P gingivalis may vary among individual strains.³³ Recurrent bacteremias with P gingivalis and a mixture of other oral bacteria can occur in periodontitis patients during simple mastication.^34–36 P gingivalis has been detected by immunostaining and polymerase chain reaction (PCR) in some human carotid and coronary atheromas.^27,37 In addition, P gingivalis produces arginine-specific cysteine proteases, called gingipain Rs, that can activate both the cellular and humoral coagulation systems. These gingipain Rs activate protease-activated receptors 1 and 4 (PAR-1 and PAR-4) on platelets to a degree sufficient to support ex vivo aggregation.^38–42 Moreover, gingipain Rs can activate coagulation factors II, IX, and X by protease cleavage.^43–45 In vitro studies have shown that gingipains R and K produced by P gingivalis degrade plaque constituents and that cultured monocytes exhibit foam cell phenotypes when exposed to P gingivalis FDC 381.³¹ The combination of intense oral and systemic inflammation and frequent bacteremias with an invasive prothrombotic organism suggests that periodontitis could contribute to atherosclerosis and its thrombotic complications by several mechanisms.

The purpose of this study was to develop a useful animal model for examining the potential atherogenic effects ofP gingivalis in normocholesterolemic and hypercholesterolemic pigs with a mixed genetic background. An important facet of the rationale for choosing pigs is the fact that they develop coronary lesions that closely simulate human disease.^46–50

	Methods

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Please see supplemental Methods, available online at http://atvb.ahajournals.org and summarized in Figure 1.

View larger version (29K): [in this window] [in a new window]   Figure 1. Experimental groups and protocols and study timeline. Four weeks after sensitization with heat-killed P gingivalis, the pigs in Groups 1, 2, and 5 were injected intravenously thrice weekly with 106 to 107 CFU of the respective strains for a period of 5 months. Monthly plasma and serum (P+S) and complete blood count (CBC) samples were taken. The P gingivalis–sensitized and –unsensitized control pigs in Groups 3, 4, and 6 were injected intravenously with sterile saline thrice weekly. The high-fat diet was started week –4 in Groups 5 and 6; all other pigs received low-fat diet continuously. After completion of injections, a washout period of 2 weeks was allowed. All samples were collected again and all pigs were euthanized. Tissue samples were taken for histomorphometry, immunohistochemistry, and PCR.

	Results

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Clinical Assessment
No clinical signs of infection were noted in any pig during this study. Although the pigs in Groups 1 to 4 differed in weight on entry, there were no significant differences in body weight between the 6 groups of pigs at the end of the study (Table I, available online at http://atvb.ahajournals.org). No gross abnormalities were noted at necropsy in any organ. Specifically, no abnormalities were detected on heart valves, nor were any abscesses found. No specific analysis of lymphoid tissues or other cells was performed.

Liver Enzymes and Function Values, White Blood Cell Counts, Anti–P gingivalis Antibody Level, and Serum Cholesterol
No group had liver enzymes or other function values (AST, ALT, total bilirubin, GGTP, or alk phos) outside the reference range except for a slight elevation of GGTP in pigs from Groups 5 and 6 after receiving the high-fat diet (Table I). In addition, white blood cell counts were intermittently slightly above the reference range in all groups (Table I). Minor differences that achieved statistical significance even though within the reference range are indicated in Table I. The pigs that received intravenous P gingivalis challenges demonstrated a >100-fold increase in anti–P gingivalis antibody levels (Groups 1, 2, and 5; Table II, available online at http://atvb.ahajournals.org). There was no significant difference in the level of the anti–P gingivalis antibody level between pigs receiving the A7436 or FDC 381 strains. Control pigs that were sensitized to P gingivalis exhibited an increase in anti–P gingivalis antibody levels but to a lesser degree than that seen in the P gingivalis–challenged pigs (Group 3; Table II). Pigs fed the low-fat diet did not show significant changes in cholesterol throughout the study period (Groups 1 to 4; Table III, available online at http://atvb.ahajournals.org). Pigs fed a high-fat diet exhibited a significant increase in serum cholesterol from their respective baseline values that was not different between the P gingivalis–challenged and saline controls (Groups 5 and 6; Table III).

Morphometric Analysis of Aortas and Coronary Arteries
Low-Fat Diet
When P gingivalis–challenged pigs (Groups 1 and 2) were compared with their respective control groups individually (Groups 3 and 4) or as pooled data, the differences were significant (P=< 0.001 for the pooled data; Table 1, see also Table IV, available online at http://atvb.ahajournals.org). When compared with each other, pigs in Group 1 challenged with P gingivalis A7436 had significantly greater aortic intimal area as a percent medial area when compared with Group 2 pigs challenged with FDC 381 (P=0.0317). Otherwise, Group 1 and 2 pigs challenged with the 2 strains of P gingivalis had no significant differences in the intimal area of aortic atherosclerosis or the 3 measures of coronary atherosclerosis (P>0.05). In the P gingivalis–sensitized pigs fed a low-fat diet (Group 3), the coronary intimal area and the intimal area as a percent of medial area was significantly smaller than the unsensitized pigs (Group 4; P=0.026 and P=0.0411, respectively). The percent luminal narrowing by intima of the coronary arteries was not significantly different between Groups 3 and 4 (P=0.0649). In addition, the aortic intimal area and the intimal area as a percent medial area were not significantly different between these sensitized and unsensitized control pigs (P=0.0649 and 0.2403, respectively).

View this table: [in this window] [in a new window]   TABLE 1. Aortic and Coronary Artery Atherosclerosis Morphometry in Pigs Fed a Low-Fat Diet

High-Fat Diet
Pigs sensitized and challenged with P gingivalis (Group 5) had nearly significantly greater intimal plaque area and raised lesions of aortic atherosclerosis than the control unsensitized pigs (Group 6, P=0.053; Tables 2 and 3; see also Tables V & VI, available online at http://atvb.ahajournals.org). Also, there was a trend toward a greater amount of coronary atherosclerosis in P gingivalis–challenged hypercholesterolemic pigs (Group 5) when compared with the unsensitized hypercholesterolemic pigs (Group 6; Table 2; see also Table V).

View this table: [in this window] [in a new window]   TABLE 2. Aortic and Coronary Artery Atherosclerosis Morphometry in Pigs Fed a High-Fat Diet

View this table: [in this window] [in a new window]   TABLE 3. Percent Abdominal Aortic Surface With Raised Atherosclerotic Lesions

Detection of P gingivalis Ribosomal DNA in Aortas and Carotids by Nested PCR
P gingivalis ribosomal DNA was detected in the carotids (5 of 5) and the aortas (4 of 5) from the 5 P gingivalis-treated pigs (A7436, n=2; FDC 381, n=3) on the low-fat diet and the aortas of all 7 P gingivalis–challenged pigs on the high-fat diet. None of the aortas or carotids from saline-challenged control pigs, whether sensitized or not, had amplifiable P gingivalis ribosomal DNA.

	Discussion

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Our goal was to develop a useful animal model for examining the potential atherogenic effects of P gingivalis bacteremia mimicking that found in clinical periodontitis. Our data show that normocholesterolemic pigs challenged with recurrent P gingivalis bacteremia develop significantly larger coronary and aortic atherosclerotic lesions (Table 1; see also Table IV). The average coronary and aortic intimal areas were 5.9 times and 3 times larger than controls, respectively. These lesions were predominantly composed of smooth muscle cells consistent with early atherosclerosis (Figure 2). In addition, hypercholesterolemic pigs likewise challenged with recurrent P gingivalis bacteremia develop larger coronary and aortic atherosclerotic lesions, both having an intimal area that was 2.2 times larger than controls (Figure 2 and Table V). Relative to controls, the difference in lesion size approached significance in the hypercholesterolemic group (P=0.053). A larger sample size of pigs fed a high-fat diet would likely have achieved statistical significance. Hypercholesterolemia then was the primary atherogenic stimulus in these unsensitized control pigs fed a high fat diet and not exposed to P gingivalis. Notably, the involvement of the coronary arteries in P gingivalis–challenged animals is of particular significance as the coronary lesions seen in the pig closely simulate human disease.^46–50

View larger version (68K): [in this window] [in a new window]   Figure 2. Immunohistochemical staining of right coronary artery atherosclerosis from study pigs. A and B, Coronary artery from a P gingivalis sensitized and challenged pig (Group 1) fed low-fat diet is stained with an anti– smooth muscle cell actin antibody. Smooth muscle cells comprise the majority of cells in the lesion. The area identified by the rectangle inserted on A is shown at higher magnification in B. Arrow indicates positive staining cell (A, bar=50 µm; B, bar=25 µm, hematoxylin counterstain). C and D, Coronary arteries are shown from pigs fed a high-fat diet in the saline control, Group 6, (C) and P gingivalis–challenged, Group 5 (D). Both pigs fed a high fat diet had lesions with foam cells and fibrous caps as seen in previous studies.46–50 E, Coronary artery from a P gingivalis sensitized pig fed low-fat diet, Group 3 (hematoxylin counterstain). AP indicates atherosclerotic plaque; L, lumen. Bar in C, D, and E=1 mm.

Elegant studies with inbred heterozygous and homozygous apoE-deficient mice exhibited increased aortic atherosclerosis when challenged orally or intravenously with invasive strains of P gingivalis.^32,51–53 Our data confirm and extend these findings as well as highlight important differences. First, P gingivalis challenges increased aortic atherosclerosis in apoE-deficient mice in a hypercholesterolemic background only, whereas our normocholesterolemic pigs developed both coronary and aortic lesions with P gingivalis challenges.^32,51–53 This finding suggests that P gingivalis sensitization and bacteremia may exert an atherogenic stimulus independent of hypercholesterolemia in pigs. Second, assuming a mouse weight of 25 g, the apoE-deficient mice received 4x10⁹ CFU/kg intravenously^51,53 weekly, 8x10¹⁰ CFU po/kg weekly,⁵² or 4x10⁸ CFU po/kg weekly.³² We administered a dose of 10⁶ to 10⁷ CFU per pig or 5x10³ /kg intravenously thrice weekly assuming an average pig weight of 200 kg. The clinically relevant dose is unknown at present and probably varies greatly.^54–56 Furthermore, although oral administration is essential for determining the influence of oral inflammation in these models, the significance of the different routes of administration or access to the vasculature is unknown. Importantly, P gingivalis challenges consistently increased the amount of atherosclerosis despite these different routes of administration and dosing regimens in both species. Third, P gingivalis 16 ribosomal DNA was detected by PCR in some but not all of these mutant mice. We also detected P gingivalis in 16 of 17 (94%) arteries from P gingivalis–challenged pigs by PCR. Our samples were collected after a 2-week washout period to allow for P gingivalis clearance from blood. In this manner, any P gingivalis detected by PCR on necropsy samples would be more likely to come from tissue invasion and not persistent bacteremia. The coronary arteries were not tested for P gingivalis amplification by PCR. The data to date suggest that both the host response and the degree of virulence of the specific P gingivalis strains are two of the most important variables in these models.^{9,12,29,32,57,58}

In humans, P gingivalis colonization of the periodontal tissues is ubiquitous by the time an individual reaches adolescence and serum antibody to this pathogen remains present throughout adulthood, even persisting after tooth loss.^59,60 Periodontal disease is a chronic infection that occurs as a consequence of P gingivalis emergence in the periodontal flora in susceptible individuals and results in frequent daily low-level bacteremic showers with P gingivalis and other organisms.^36,54–56 Thus periodontitis and its associated bacteremia develop in humans who have been immunologically sensitized to organisms such as P gingivalis. We chose to sensitize the pigs in this study with strains known to cause disease in humans to model this chronic bacteremic exposure in a sensitized host. The data from the sensitized control pigs (Group 3) were obtained to provide a control for the pigs that received P gingivalis sensitization and intravenous challenges. Considered together, the control and experimental data support the hypothesis that P gingivalis sensitization and intravenous challenges induce coronary and aortic atherosclerosis. The unsensitized controls suggest that the intravenous challenges by themselves do not explain the observed differences. Importantly, prior immunization decreased periodontitis and atherosclerosis in apoE–/– mice.³² We also chose to study only strain A7436 in pigs fed a high-fat diet because the response to the 2 strains appeared similar in pigs fed a low-fat diet. We cannot exclude the possibility that pigs fed a high-fat diet would respond differently to FDC 381.

Two potential study limitations deserve note. First is that our pig model does not replicate the likely oral origin of bacteremia from inflamed human periodontal tissues.⁵² Nonetheless, as seen previously in rodents, the P gingivalis–challenged pigs developed an anti–P gingivalis antibody response as a consequence of P gingivalis challenges in association with increased atherosclerosis.^32,51,52 Second, the number of pigs was not sufficient to exclude an effect of sex in our study. Notably, recent data that was not available at the time this study was designed suggest that human males have a greater prevalence of asymptomatic carotid disease than females with comparable severity of periodontitis.^58,61

Our data support the hypothesis P gingivalis bacteremia promotes coronary and aortic atherogenesis in pigs with or without significant hypercholesterolemia. Confirmation of this hypothesis will require determining the relative contributions of the virulence properties intrinsic to P gingivalis, the degree of systemic inflammation, or other host responses to the experimental protocol. At present, the mechanism by which P gingivalis contributes to atherogenesis or comes to reside in atherosclerotic plaques is unknown. Pigs are a promising species for investigating mechanisms by which P gingivalis exerts a coronary and aortic atherogenic effect in a normocholesterolemic or hypercholesterolemic background.

	Acknowledgments

 
This work was supported by a research grant from the National Institutes of Health/National Institute of Dental and Craniofacial Research. 1-P60-DE 13079. The authors gratefully acknowledge Robin Raymer and Kent Passingham and their staff for the outstanding care given to the animals used in this study.

Received January 5, 2005; accepted April 6, 2005.

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