IADR Abstract Archives
In-vitro differential endothelial response at repeated exposure of Porphyromonas gingivalis
Objectives: The purpose of this study was to compare the chemokine production of HCAEC stimulated with single and repeated exposure of live Porphyromonas gingivalis or its lipopolysaccharide.
Methods: Human Coronary Artery Endothelial Cells (HCAEC) were stimulated with 1 μg/ml of purified Porphyromonas gingivalis-LPS W83 or live-P. gingivalis at single or repeated doses up to 24 h. HCAECs were multiple challenges to LPS or live bacteria, thus the monolayer was stimulated for 6 h, after that, the stimulus was removed and then replaced with the next stimulus for another 6 h and finally for the last stimulation HCAEC were exposed to another 12 h. The supernatant between each exposure were collected for a total exposure time of 24 h and storage for soluble factors measuring, whilst for the second approach, the conventional stimulus was HCAECs stimulated with LPS or live bacteria at a single exposure for 1 day. Chemokine production and mRNA levels of IL-8 and MCP-1 were determined by flow cytometry and RT-qPCR respectively. A p-value <0.05 was considered statistically significant.
Results: Only repeated exposure of viable-P. gingivalis induced significant production MCP-1 (p<0.05) and a slight increase of IL-8 in HCAEC supernatants, whilst for monolayer challenged with P. gingivalis-LPS, none of both approaches affected the chemokine identification compared with control. Similar results were observed with the mRNA level.
Conclusions: The differential endothelial response is dependent on repeated exposure of viable P. gingivalis. This study supports the importance of more than one in-vitro exposure (repeated) of periodontopathogens which could lead to a better understanding of the endothelial dysfunction or pro-inflammatory activation.
Methods: Human Coronary Artery Endothelial Cells (HCAEC) were stimulated with 1 μg/ml of purified Porphyromonas gingivalis-LPS W83 or live-P. gingivalis at single or repeated doses up to 24 h. HCAECs were multiple challenges to LPS or live bacteria, thus the monolayer was stimulated for 6 h, after that, the stimulus was removed and then replaced with the next stimulus for another 6 h and finally for the last stimulation HCAEC were exposed to another 12 h. The supernatant between each exposure were collected for a total exposure time of 24 h and storage for soluble factors measuring, whilst for the second approach, the conventional stimulus was HCAECs stimulated with LPS or live bacteria at a single exposure for 1 day. Chemokine production and mRNA levels of IL-8 and MCP-1 were determined by flow cytometry and RT-qPCR respectively. A p-value <0.05 was considered statistically significant.
Results: Only repeated exposure of viable-P. gingivalis induced significant production MCP-1 (p<0.05) and a slight increase of IL-8 in HCAEC supernatants, whilst for monolayer challenged with P. gingivalis-LPS, none of both approaches affected the chemokine identification compared with control. Similar results were observed with the mRNA level.
Conclusions: The differential endothelial response is dependent on repeated exposure of viable P. gingivalis. This study supports the importance of more than one in-vitro exposure (repeated) of periodontopathogens which could lead to a better understanding of the endothelial dysfunction or pro-inflammatory activation.