Repeated Porphyromonas gingivalis W83 exposure leads to release pro-inflammatory cytokynes and angiotensin II in coronary artery endothelial cells

Abstract

The role of Porphyromonas gingivalis (P. gingivalis) or its virulence factors, including lipopolysaccharide (LPS) not only has been related with periodontitis but also with endothelial dysfunction, a key mechanism involved in the genesis of atherosclerosis and hypertension that involving systemic inflammatory markers as angiotensin II (Ang II) and cytokines. This study compares the effect of repeated and unique exposures of P. gingivalis W83 LPS and live bacteria on the production and expression of inflammatory mediators and vasoconstrictor molecules with Ang II. Human coronary artery endothelial cells (HCAEC) were stimulated with purified LPS of P. gingivalis (1.0, 3.5 or 7.0 μg/mL) or serial dilutions of live bacteria (MOI 1: 100 - 1:0,1) at a single or repeated exposure for a time of 24 h. mRNA expression levels of AGTR1, AGTR2, IL-8, IL-1β and MCP-1 were determined by RT-qPCR, and IL-6, MCP-1, IL-8, IL-1β and GM-CSF levels were measured by flow cytometry, ELISA determined Ang II levels. Live bacteria in a single dose increased mRNA levels of AGTR1, and repeated doses increased mRNA levels of IL-8 and IL-1β (p < 0.05). Repeated exposure of live-P. gingivalis induced significant production IL-6, MCP-1 and GM-CSF (p < 0.05). Moreover, these MCP-1, IL-6 and GM-CSF levels were greater than in cells treated with single exposure (p < 0.05), The expression of AGTR1 and production of Ang II induced by live-P. gingivalis W83 showed a vasomotor effect of whole bacteria in HCAEC more than LPS. In conclusion, the findings of this study suggest that repeated exposure of P. gingivalis in HCAEC induces the activation of proinflammatory and vasoconstrictor molecules that lead to endothelial dysfunction being a key mechanism of the onset and progression of arterial hypertension and atherosclerosis.

Figure 1

Viability of HCAECs after repeated treatments with live-P. gingivalis (A) and P. gingivalis-LPS (B). The HCAECs were stimulated to repeated live-P. gingivalis (MOI 1:100 - 1:0,1) and P. gingivalis-LPS (1.0, 3.5 and 7.0 µg/mL) exposures, during 24 h. Cell viability was determined according to the fluorometric detection after reduction of resazurin in the resorufin product using AlamarBlue. 1% was considered our positive control of cell death. Percentage of cell viability with respect to the control. *Represents the statistical difference with respect to the control or without stimulus. (p < 0.05). Three independent experiments were performed; the results are presented as the means ± SEM (n = 3).

Figure 2

mRNA expression levels in HCAEC stimulated with P. gingivalis-LPS or live-P. gingivalis. Monolayers of HCAEC cultured in 12-well plates were stimulated with P. gingivalis-LPS (1.0, 3.5, 7.0 µg/mL) or serial  dilutions of P. gingivalis (MOI 1:100- 1:0,1) for 24 h under repeated exposure (+++) or single exposure (+). After stimulation, (A) AGTR1, (B) AGTR2, (C) IL-8, (D) IL-1β, (E) MCP-1, mRNA levels were measured by are expressed as the means by RT-qPCR. Results are expressed as the means ± SEM (n=3). Statiscal significance is represented as *p < 0.05, and (ns) for not significant.

Figure 3

Chemokines and cytokines secreted in HCAEC stimulated with P. gingivalis-LPS or live-P. gingivalis. Monolayers of HCAEC cultured in 12-well plates were stimulated with P. gingivalis-LPS (1.0, 3.5, 7.0 µg/mL) or P. gingivalis (MOI 1:100 - 1:0,1) for 24 h under repeated exposure (+++) or single exposure (+). After stimulation, levels of the following chemokines were measured in cell culture supernatants using a cytometric bead array: (A) IL-8, (B) MCP-1, (C) IL-6, (D) GM-CSF, (E) IL-1β. Symbol (*) means p < 0.05 vs control cells; (**) means p < 0.01 vs control cells; ^†p < 0.05 compared vs cells treated by single exposure; ^††p < 0.01 compared vs cells treated by single exposure; (ns) for not significant. Three independent experiments were performed; the results are presented as the means ± SEM (n = 3).

Figure 4

Angiotensin II levels are determined in the HCAEC  cell culture supernatant stimulated to single (+) or repetitive (+++) exposures of P. gingivalis-LPS or live-P. gingivalis by the ELISA kit. The results are expressed as the means ± SEM (n=3)  with a statistical significance represented as (*)p < 0.05 vs. control, (^†)p < 0.05 compared vs cells treated by single exposure; (ns) for not significant.