Murine Model of Accelerated Atherosclerosis in Mice Infected with a Periodontal Pathogen

Toward that end, our laboratory employed mice deficient in apolipoprotein E (apo E null). In these animals, spontaneous atherosclerosis develops on a normal chow diet driven primarily by hypercholesterolemia. We, and others, have shown that induction of superimposed stresses, such as hyperglycemia or hyperhomocysteinemia, accelerates atherosclerosis in this model. To test the premise that oral infection may accelerate vascular inflammation and macrovascular disease, male apo E null mice were inoculated with the human periodontal pathogen Porphyromonas gingivalis, strain 381, by oral gavage and oral/anal topical application, beginning at age 6 weeks. Control animals received vehicle (phosphate buffered saline; PBS). In parallel with accelerated alveolar bone loss in infected mice versus those animals receiving PBS, P. gingivalis-infected mice displayed a significant 40 percent increase in atherosclerotic lesion area at the aortic root versus vehicle at age 17 weeks (Figure 1). Although the majority of lesions in both groups of animals were limited to fatty streaks, it is possible that upon longer evaluation periods, acceleration of lesion area and progression of fatty streaks to more unstable plaques would occur.

Noninfected Infected

Figure 1 Oral infection with P. gingivalis accelerates early atherosclerosis in apoE-null mice. Representative photographs of the proximal aorta demonstrate increased atherosclerosis in a P. gingivalis-infected (b) versus a noninfected (a) apo E-null mouse at 17 weeks of age. Scale bar, 1 mm. Oil red O staining of cryosections at the aortic sinus from control mice revealed few small fatty streaks (c), whereas atherosclerotic lesions were greater in number and size in infected animals (d), scale bar 50 mm. Quan-titation of atherosclerotic lesion area at the aortic sinus (e) revealed that mean lesion area was significantly increased in infected versus noninfected mice (12,753 ± 1,128 versus 9,080 ± 951mm2, n = 25/group, p = 0.008). Republished with permission of Arteriosclerosis Thrombosis and Vascular Biology from: Lalla, E., et al. (2003). Oral infection with a periodontal pathogen accelerates early atherosclerosis in apolipoprotein E-null mice. Arterioscler. Thromb. Vasc. Biol. 23(8), 1405-1411. (see color insert)

Noninfected Infected

Figure 1 Oral infection with P. gingivalis accelerates early atherosclerosis in apoE-null mice. Representative photographs of the proximal aorta demonstrate increased atherosclerosis in a P. gingivalis-infected (b) versus a noninfected (a) apo E-null mouse at 17 weeks of age. Scale bar, 1 mm. Oil red O staining of cryosections at the aortic sinus from control mice revealed few small fatty streaks (c), whereas atherosclerotic lesions were greater in number and size in infected animals (d), scale bar 50 mm. Quan-titation of atherosclerotic lesion area at the aortic sinus (e) revealed that mean lesion area was significantly increased in infected versus noninfected mice (12,753 ± 1,128 versus 9,080 ± 951mm2, n = 25/group, p = 0.008). Republished with permission of Arteriosclerosis Thrombosis and Vascular Biology from: Lalla, E., et al. (2003). Oral infection with a periodontal pathogen accelerates early atherosclerosis in apolipoprotein E-null mice. Arterioscler. Thromb. Vasc. Biol. 23(8), 1405-1411. (see color insert)

The key question to address is: what are the mechanisms underlying these observations? We postulated that direct invasion of the vasculature by pathogenic bacteria accounted, at least in part, for these findings. Consistent with this premise, PCR on DNA prepared from aortic tissue at age 17 weeks (8 weeks after infection) revealed transcripts encoding P gingivalis in two of nine infected mice; in contrast, none of the PBS-treated mice demonstrated transcripts for P. gingivalis in the aorta. Direct evidence for enhanced vascular inflammation was obtained by assessment of proinflammatory/prothrombotic markers in the vasculature. By Western blotting, levels of vascular cell adhesion molecule-1 (VCAM-1) and tissue factor antigen were significantly increased in infected versus nonin-fected aortic lysates. In parallel, levels of matrix metalloproteinase-2 antigen and activity were increased in aortas retrieved from P. gingivalis-infected mice versus controls. These findings suggested that either by direct invasion of the vessel wall, or by exposure to inflammation-evoking bacterial products, or both, the vasculature of infected mice expressed increased inflammatory molecule profile and activity.

Strongly suggestive of activation of the systemic host response by infection with P. gingivalis was the finding that statistically significant increases in levels of plasma IL-6, an acute phase reactant, were evident in P. gingivalis-infected mice versus PBS-treated controls (Figure 2a). A significant correlation between the levels of IL-6 and extent of atherosclerotic lesion area was observed in these animals (Figure 2b; r = 0.37, p = 0.007). Further, a trend in plasma levels of another acute phase reactant, serum amyloid A, was observed in infected versus noninfected apo E null mice at age 17 weeks.

Critical to the premise that oral infection accelerated atherosclerosis was the observation that infection with P. gingivalis did not modify established risk factors for atherogenesis

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Figure 2 Oral infection and the systemic host response. (a) Box-and-whisker plot. At sacrifice, levels of plasma IL-6 by ELISA were significantly increased in infected apo E-null mice versus controls. Horizontal lines represent median values (noninfected: 7.3pg/mL versus infected: 10 pg/mL). Top and bottom of each box mark the first and third quartile of the values and lines extending from the end of each box mark the minimum and maximum for the group. Nonparametric analysis, p = 0.04, n = 25/group. (b) Pearson's correlation analysis revealed a statistically significant positive correlation between levels of plasma IL-6 and extent of atherosclerotic lesion area (r = 0.37, p = 0.007; n = 50). Republished with permission of Arteriosclerosis Thrombosis and Vascular Biology from: Lalla, E., et al. (2003). Oral infection with a periodontal pathogen accelerates early atherosclerosis in apolipoprotein E-null mice. Arterioscler. Thromb. Vasc. Biol. 23(8), 1405-1411.

in this model. Thus, levels and profile of total plasma cholesterol and triglyceride did not differ between infected and noninfected mice. Similarly, no differences in glucose, insulin, or creatinine were observed between the two groups.

Where do we go from here? The next series of studies must examine the contribution of each cellular component implicated in atherogenesis and progression of atherosclerosis to the acceleration of vascular disease triggered by infection with P. gingivalis. Specifically, the impact of circulating monocytes and lymphocytes in diverse processes such as engulfment/phagocytosis of bacteria and antigen presentation/processing must be studied. Resident cells of the vessel wall must also be examined, such as endothelial cells and smooth muscle cells. Interestingly, it is also possible that platelets interface with bacterial pathogens such as P. gingi-valis to provide a hospitable environment for vascular activation, perhaps by increasing expression of chemokines such as MCP-1. In addition, the potential impact of distinct oral pathogens on atherosclerosis should be addressed experimentally.

The importance of such endeavors cannot be overstated. Only by a thorough understanding of the molecular and biochemical pathways by which bacterial infection activates signaling pathways linked to cellular migration/activation can logical therapeutic targets be identified. In this context, recent studies have shown that immunization against P. gingivalis may reduce acceleration of atherosclerosis in a murine model [5].

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