hTERT Peptide Fragment GV1001 Prevents the Development of Porphyromonas gingivalis-Induced Periodontal Disease and Systemic Disorders in ApoE-Deficient Mice

Abstract

GV1001, an anticancer vaccine, exhibits other biological functions, including anti-inflammatory and antioxidant activity. It also suppresses the development of ligature-induced periodontitis in mice. Porphyromonas gingivalis (Pg), a major human oral bacterium implicated in the development of periodontitis, is associated with various systemic disorders, such as atherosclerosis and Alzheimer's disease (AD). This study aimed to explore the protective effects of GV1001 against Pg-induced periodontal disease, atherosclerosis, and AD-like conditions in Apolipoprotein (ApoE)-deficient mice. GV1001 effectively mitigated the development of Pg-induced periodontal disease, atherosclerosis, and AD-like conditions by counteracting Pg-induced local and systemic inflammation, partly by inhibiting the accumulation of Pg DNA aggregates, Pg lipopolysaccharides (LPS), and gingipains in the gingival tissue, arterial wall, and brain. GV1001 attenuated the development of atherosclerosis by inhibiting vascular inflammation, lipid deposition in the arterial wall, endothelial to mesenchymal cell transition (EndMT), the expression of Cluster of Differentiation 47 (CD47) from arterial smooth muscle cells, and the formation of foam cells in mice with Pg-induced periodontal disease. GV1001 also suppressed the accumulation of AD biomarkers in the brains of mice with periodontal disease. Overall, these findings suggest that GV1001 holds promise as a preventive agent in the development of atherosclerosis and AD-like conditions associated with periodontal disease.

Keywords: Alzheimer disease; GV1001; Porphyromonas gingivalis; atherosclerosis; periodontitis.

Figure 1

Electrophoretic image of amplified Pg DNA from mice sacrificed four weeks after completing the last inoculation (inoculation duration: five weeks). (A) PBS + PBS: Swab samples from the mice receiving gingival inoculation with PBS with systemic PBS administration; (B) PBS + GV1001: Swab samples from the mice receiving gingival inoculation with PBS with systemic GV1001 administration; (C) Pg + PBS: Swab samples from the mice receiving gingival inoculation with Pg with systemic PBS administration; (D) Pg + GV1001: Swab samples from the mice receiving gingival inoculation with Pg with systemic GV1001 administration. The lanes indicate the following: 1–6, samples taken from the gingival pocket; +, positive control (1000 CFU of purified Pg DNA); -, negative control.

Figure 2

Reversal effect of GV1001 on the enhanced expression of IL-1β, TNF-α, and IL-6 in the gingival tissue induced by Pg inoculation into the gingival pocket. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns: not statistically different. Results represent the mean ± SEM performed with n = 8–10 mice per group.

Figure 3

(A) Representative FISH staining images of Pg colony numbers and immunofluorescent images of LPS, Kgp, and RgpB aggregates in the gingival tissue. Scale bar: 100 μm; M: molar; AB: alveolar bone; G: gingival tissue. Nuclei were counterstained with DAPI (blue dots). Bright green color dots (with white arrows) are Pg DNA aggregates. Red colors are aggregates of LPS, Kgp, and RgpB. M: maxillary second molar; G: gingival tissue; P: pulp; AB: alveolar bone. White dashed lines are used to separate different tissues. Bar: 100 μm. (B) Results represent the means ± SEM. Statistical analysis was performed with one-way ANOVA from 6 samples of each group. ns: not significantly different; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

(A) Representative two- or three-dimensional μCT images of mice maxillae with PBS inoculation into the pockets and systemic PBS or GV1001 administration. (B) Representative two- or three-dimensional μCT images of mice maxillae with Pg inoculation into the pockets and systemic PBS or GV1001 administration. (C) The average distance (unit: mm) from the palatal and buccal CEJ to the ABC of the second molar. Results represent the means ± SEM performed in ten samples. **** p < 0.0001, * p < 0.1. ns: not significantly different (p > 0.05); CEJ: cement–enamel junction; ABC: alveolar bone crest. Scale bar: 0.2 mm.

Figure 5

Reversal effect of GV1001 on the increased serum levels of proinflammatory cytokines by Pg inoculation into the gingival pockets. Statistical analysis was performed with one-way ANOVA. ns: not significantly different; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Reversal effect of GV1001 on the increased expression levels of IL-1β, TNF-α, and IL-6 in the arterial wall induced by Pg inoculation in gingival pocket. The gene expression was measured with RT-qPCR. GAPDH served as a loading control. Statistical analysis was performed with one-way ANOVA. ns: not significantly different; * p < 0.05, ** p < 0.01, and **** p < 0.0001. Results represent the means ± SEM performed in 5 samples.

Figure 7

(A) Representative fluorescence in situ hybridization (FISH) and immunofluorescent staining images of the Pg LPS, Kgp, and RgpB aggregates in the arterial wall images of aortic roots of mice inoculated with Pg into the gingival pockets of ApoE-deficient mice. Blue dots are nuclei of cells stained with DAPI. Pg DNA aggregates are green with white arrows (scale bar: 20 μm), and the Pg LPS, Kgp, and RgpB aggregates are bright red color with arrows (scale bar: 100 μm). Nuclei were counterstained with DAPI (blue dots). Green color waves are non-specific staining. AW: arterial wall; L: lumen; AL: arterial leaflet. (B) Results represent the means ± SEM of five samples in each group. Statistical analysis was performed with one-way ANOVA. ns: not significantly different; ** p < 0.01, **** p < 0.0001.

Figure 8

En face analysis of mice arteries. (A) Representative photographs of mouse arteries from the en face preparation after staining with Sudan IV (8–10 mice per group). (B) Quantification of areas stained by Sudan IV using ImageJ analysis. ns: not significantly different; ** p < 0.01.

Figure 9

(A) Representative immunofluorescent staining images of CD-31 (green), FSP-1 (red), and nucleus (DAPI staining blue) in HUVECs after 48 h exposure of cells to TNF-$\alpha$ with or without GV1001. (A-1) Quantification of the fluorescent intensity using ImageJ analysis. (B) Representative immunofluorescent staining images of CD-31 (green), FSP-1 (red), and nucleus (DAPI staining blue) in HUVECs exposed to Pg LPS for 48 h. (B-2) RT-PCR assay was performed for CD31 and FSP-1 mRNA levels in HUVECs exposed to Pg LPS for 48h. CD31: endothelial cell marker; FSP-1: mesenchymal cell marker. Scale bar: 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 10

(A) Representative immunofluorescent staining images of dil-ox-LDL (Oil Red O staining) in macrophages. A bright red color indicates ox-LDL. Blue dots are nuclei stained with DAPI. (B) Quantification of the fluorescent intensity using ImageJ analysis. ns: not significantly different; **** p < 0.0001. Scale bar: 60 μm.

Figure 11

(A) Representative immunofluorescent staining images of HCASMCs treated with TNF-α and Pg LPS, both with and without GV1001. Bright red color indicates CD47. Blue dots are nuclei stained with DAPI. (B) Statistical analysis of CD47 expression in HASMCs exposed to TNF-α or Pg LPS, comparing the presence and absence of GV1001. (C) Western blot analysis illustrating CD47 levels in HCASMCs subjected to TNF-α or Pg LPS, with and without GV1001 treatment. (D) Quantification of CD47 protein levels based on Western blot results. (E) RT-qPCR analysis evaluating CD47 expression in HCASMCs exposed to TNF-α or Pg LPS, with or without concurrent GV1001 treatment. (F) Dual luciferase reporter assays for CD47 promoter activity in HCASMCs treated with TNF-α or Pg LPS, with and without GV1001. ns: not significantly different; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 50 μm.

Figure 12

Reversal effect of GV1001 on the enhanced expression levels of IL-1β, TNF-α, and IL-6 by Pg inoculation into gingival pocket in whole brain tissues. Statistical analysis was conducted through one-way analysis of variance (ANOVA), where ns indicates not significant, and ** denotes p < 0.01. The presented results represented the means ± SEM from 6–10 samples.

Figure 13

(A) Representative FISH images illustrating Pg DNA aggregates and immunofluorescent staining images depicting Pg LPS, Kgp, and RgpB aggregates in the cerebral cortex of mice with Pg-induced periodontal disease. Pg DNA aggregates are indicated by the green color with white arrows, while Pg LPS, Kgp, and RgpB aggregates are highlighted in bright red with arrows. Nuclei were counterstained with DAPI (blue dots), and neurons are visualized in green. (B) Statistical analysis was conducted using one-way ANOVA. Abbreviations: ns, not significantly different; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 40 μm.

Figure 14

(A) Representative FISH images illustrating Pg DNA aggregates and immunofluorescent staining images depicting Pg LPS, Kgp, and RgpB aggregates in the hippocampus of mice with Pg-induced periodontal disease. Pg DNA aggregates are shown with the green color with white arrows, while Pg LPS, Kgp, and RgpB aggregates are highlighted in bright red with arrows. Nuclei were counterstained with DAPI (blue dots), and neurons are visualized in green. (B) Statistical analysis was conducted using one-way ANOVA. Abbreviations: ns, not significantly different; * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar: 40 μm.

Figure 15

(A) Representative immunofluorescence staining images illustrating Aβ42 and p-Tau aggregates in the cerebral cortex and hippocampus of mouse brain. Aβ42 or p-Tau aggregates are depicted as bright red dots, nuclei are stained with DAPI (blue dots), and neurons are marked with MAP2 in green. (B) The results are presented as means ± SEM. Statistical analysis was performed using one-way ANOVA based on data obtained from 6 samples in each group. Abbreviations: ns, not significantly different; * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar: 40 μm.

Figure 16

Effect of GV1001 on the expression of Pg gingipains RgpA, RgpB and Kgp in bacterial culture. Pg 16S rRNA served as the loading control. Statistical analysis was conducted through one-way analysis of variance (ANOVA), where ns indicates not significant, * indicates p < 0.05 and **** denotes p < 0.0001. The presented results represent the means ± SEM from three samples.