Effect of Porphyromonas gingivalis lipopolysaccharide on calcification of human umbilical artery smooth muscle cells co-cultured with human periodontal ligament cells

Abstract

Periodontitis is an independent risk factor for coronary heart disease. Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) was considered to be one of the main virulence factors. In addition, vascular smooth muscle cells transform into osteoblast-like cells in an arterial calcification process under chronic inflammatory conditions. The present study aimed to determine the calcification induced by Pg-LPS in human umbilical artery smooth muscle cells (HUASMCs) co-cultured with human periodontal ligament cells (HPDLCs). An in vitro co-culture system was established using Transwell inserts. HUASMC proliferation and alkaline phosphatase (ALP) activity were measured with a Cell Counting Kit-8 and an ALP kit, respectively. Calcium nodule formation was detected using alizarin red S staining. The effects of Pg-LPS on the mRNA expression of the calcification genes of ALP, core-binding factor α1 (Runx2) and bone sialoprotein (BSP) were assessed using reverse transcription-quantitative PCR. The results indicated that Pg-LPS increased HUASMC proliferation and ALP activity. Furthermore, among all of the groups, calcium nodule formation was most extensive in co-cultured cells in the mineralization-inducing medium containing Pg-LPS. In addition, the expression of specific osteogenic genes (Runx2, ALP and BSP) significantly increased in the presence of Pg-LPS and mineralization-inducing medium, which was further enhanced in co-culture with HPDLCs. In conclusion, co-culture with HPDLCs increased the effect of Pg-LPS to stimulate the calcification of HUASMCs. It was suggested that besides the inflammation, periodontitis may promote the occurrence of vascular calcification. The study indicated that periodontal treatment of subgingival scaling to reduce and/or control Porphyromonas gingivalis may decrease the occurrence or severity of vascular calcification.

Keywords: Porphyromonas gingivalis lipopolysaccharide; calcification; co-culture; periodontitis.

Figure 1

HPDLCs and HUASMCs were characterized. (A) HPDLCs were positive for anti-vimentin staining. (B) HPDLCs were negative for anti-keratin staining. (C) HUASMCs expressed α-smooth muscle actin. The DAPI staining image is superimposed with green fluorescence, with α-SMA being fluorescent green and the nuclei blue (scale bars, 100 µm). HUASMCs, human umbilical artery smooth muscle cells; HPDLCs, human periodontal ligament cells.

Figure 2

HPDLC-HUASMC co-culture system was established with Transwell inserts. (A) HPDLCs, with micropores in the porous membrane visible. (B) HUASMCs, exhibiting a radial spindle-shaped arrangement when attached to the plate (scale bars, 20 µm). HUASMCs, human umbilical artery smooth muscle cells; HPDLCs, human periodontal ligament cells.

Figure 3

Cell viability. (A) Effect of Pg-LPS on HUASMC viability in the co-culture system. (B) Cell viability in HUASMCs treated with or without Pg-LPS in the calcified-inducing culture medium. Values are expressed as the mean ± standard deviation of three independent experiments in each group. ^*P<0.05 vs. HUASMCs (group C); ^#P<0.05. HUASMCs, human umbilical artery smooth muscle cells; PDLCs, periodontal ligament cells; Pg-LPS, Porphyromonas gingivalis lipopolysaccharide; ALP, alkaline phosphatase.

Figure 4

Induction of ALP activity. Effects of the normal medium (A) and the calcification-inducing culture medium (B) on ALP activity of HUASMCs and the HUASMCs-HPDLCs co-culture system. Values are expressed as the mean ± standard deviation of three independent experiments in each group. ^*P<0.05 vs. HUASMCs in calcification-inducing culture medium (group CC); ^#P<0.05. HUASMCs, human umbilical artery smooth muscle cells; PDLCs, periodontal ligament cells; Pg-LPS, Porphyromonas gingivalis lipopolysaccharide; ALP, alkaline phosphatase; OD, optical density.

Figure 5

Induction of osteogenic gene transcription in HUASMCs. Transcript levels for the osteogenic genes (A) Runx2, (B) ALP and (C) BSP were determined at 48 h by reverse transcription-quantitative PCR and normalized to the level of GAPDH transcript. Values are expressed as the mean ± standard deviation (n=3). The expression of Runx2, ALP and BSP was significantly higher in co-culture groups treated with Pg-LPS (1 µg/ml) and calcification-inducing medium than in the control groups. ^*P<0.05 vs. HUASMCs (group C); ^#P<0.05. HUASMCs, human umbilical artery smooth muscle cells; PDLCs, periodontal ligament cells; Pg-LPS, Porphyromonas gingivalis lipopolysaccharide; ALP, alkaline phosphatase; Runx2, core-binding factor α1; BSP, bone sialoprotein; OD, optical density.

Figure 6

Effect of Pg-LPS on calcified nodule formation of HUASMCs in different groups. (AA) HUASMCs (group C); (AB) HUASMCs + Pg-LPS (group C-P); (AC) PDLCs-HUASMCs (group CO); (AD) PDLCs-HUASMCs + Pg-LPS (group CO-P); (AE) HUASMCs in calcification-inducing culture medium (group C-C); (AF) HUASMCs + Pg-LPS in calcification-inducing culture medium (group C-CP); (AG) PDLCs-HUASMCs in calcification-inducing culture medium (group CO-C); and (AH) PDLCs-HUASMCs + Pg-LPS in calcification-inducing culture medium (group CO-CP). The calcified nodules were scattered in the C-P and CO-P groups and were almost absent in the C and CO groups. All of the groups under calcification induction or calcification induction + Pg-LPS (1 µg/ml) conditions exhibited many calcified nodules, whilst the co-cultured CO-C and CO-CP groups had slightly more calcified nodules than those cultured in DMEM medium (scale bar, 100 μm). The quantified results of calcification based on alizarin red S staining three weeks after calcification induction (B). The calcified nodule formation was significantly higher in calcification-inducing medium and co-culture groups treated with Pg-LPS (1 μg/ml) than in the control groups. Values are expressed as the mean ± standard deviation (n=3). ^#P<0.05, calcification-inducing culture medium vs. normal culture medium (i.e. group C-C vs. group C; group C-CP vs. group C-P; group CO-C vs. group CO; group CO-CP vs. group CO-P); ^*P<0.05. HUASMCs, human umbilical artery smooth muscle cells; PDLCs, periodontal ligament cells; Pg-LPS, Porphyromonas gingivalis lipopolysaccharide..