Effect of nicotine and porphyromonas gingivalis lipopolysaccharide on endothelial cells in vitro

Abstract

Smoking is considered a significant risk factor for both periodontal disease and cardiovascular disease (CVD). Endothelial cells play an important role in the progression of both diseases. In the present study, we investigated in vitro the impact of nicotine on functional properties of human umbilical vein endothelial cells (HUVECs) stimulated with lipopolysaccharide (LPS) of periodontal pathogen Porphyromonas gingivalis. HUVECs were stimulated with different concentrations of nicotine (10 µM-10 mM) and/or P. gingivalis LPS. Expression levels of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, monocyte chemoattractant protein 1, and interleukin-8 were measured on both gene and protein levels. Cell proliferation/viability, apoptosis, and migration were also investigated. Nicotine at a concentration of 10 mM significantly decreased P. gingivalis LPS-induced expression of all investigated proteins after 4 h stimulation, while lower nicotine concentrations had no significant effect on protein expression with or without P. gingivalis LPS. Proliferation/viability of HUVECs was also significantly inhibited by 10-mM nicotine but not by lower concentrations. Migration of HUVECs was significantly decreased by nicotine at concentrations of 1-10 mM. Nicotine at a concentration similar to that observed in the serum of smokers had no significant effect on the functional properties of HUVECs. However, high concentrations of nicotine, similar to that observed in the oral cavity of smokers, inhibited the inflammatory response of HUVECs. This effect of nicotine might be associated with decreased gingival bleeding indices in smoking periodontitis patients.

Figure 1. Effect of nicotine and P. gingivalis LPS on the proliferation/viability of HUVECs.

HUVECs were stimulated with nicotine (10 µM-10 mM) and/or P. gingivalis LPS, and the proliferation/viability was measured after 4, 24, 48, and 72 h using the MTT assay. The y-axis represents mean ±SD values of optical densities measured at 450 nm from four wells of one representative experiment. * – significantly lower compared to control group, p<0.05.

Figure 2. Effect of nicotine and P. gingivalis LPS on the proportion of viable HUVECs.

HUVECs were stimulated with nicotine (10 µM-10 mM) and/or P. gingivalis LPS, and the proportion of viable cells was measured using a flow cytometry apoptosis assay after 4 (A), 24 (B), and 72 (C) h. Viable cells were those negative for annexin V and propidium iodide. Data are presented as mean ±SD of three independent experiments. * – significantly lower compared to control group, p<0.05.

Figure 3. Effect of nicotine on the expression of pro-inflammatory mediators in HUVECs.

HUVECs were stimulated with nicotine (10 µM-10 mM), and the expression levels of ICAM-1 (A), VCAM-1 (B), E-selectin (C), MCP-1 (D), and IL-8 (E) were measured by qPCR. GAPDH was used as endogenous control gene. Each value represents mean ±SEM of three independent assays. Non-stimulated HUVECs were used as a control ( = 1). The expression levels of pro-inflammatory mediators were not analyzed after stimulation with 10-mM nicotine for 24 and 72 h because the cells were not viable.

Figure 4. Effect of nicotine on the P. gingivalis LPS-induced mRNA expression of pro-inflammatory mediators in HUVECs.

HUVECs were stimulated by P. gingivalis LPS in the presence or absence of nicotine (10 µM–10 mM), and the expression levels of ICAM-1 (A), VCAM-1 (B), E-selectin (C), MCP-1 (D), and IL-8 (E) were measured by qPCR after 4, 24, and 72 h. GAPDH was used as endogenous control gene. Each value represents mean ±SEM of three independent assays. Non-stimulated HUVECs were used as a control ( = 1). All proteins exhibited significantly higher mRNA expression levels after stimulation with P. gingivalis LPS compared to the control group (p<0.05). The expression levels of pro-inflammatory mediators were not analyzed after stimulation with 10-mM nicotine for 24 and 72 h because the cells were not viable. * – significantly different between groups, p<0.05.

Figure 5. Effect of nicotine on the P. gingivalis LPS-induced protein expression of pro-inflammatory mediators in HUVECs.

HUVECs were stimulated by P. gingivalis LPS in the presence or absence of nicotine (10 µM–10 mM) for 4, 24, and 72 h. After stimulation, the surface expression levels of ICAM-1 (A), VCAM-1 (B), and E-selectin (C) were measured by flow cytometry, and the quantity of MCP-1 (D) and IL-8 (E) in conditioned media was measured by ELISA. Each value represents mean ±SD of three independent assays. Non-stimulated HUVECs were used as a control. The protein expression levels of pro-inflammatory mediators were not analyzed after stimulation with 10-mM nicotine for 24 and 72 h because the cells were not viable. * – significantly different between groups, p<0.05. † – significantly higher compared to controls, p<0.05.

Figure 6. Effect of nicotine and P. gingivalis LPS on HUVEC migration measured in the microchemotaxis chamber.

HUVEC migration through a polycarbonate membrane with pore size of 8 µm over 8 h was assessed in a 48-well microchemotaxis chamber. The y-axis represents mean ±SD of cells per microscope field of four different wells of one representative experiment. * – significantly different between groups, p<0.05.