The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation

Abstract

Background: Porphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function.

Results: AoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change > |1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways.

Conclusions: This study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.

Figure 1

P. gingivalis invades AoSMCs and changes the F-actin distribution. AoSMCs were cultured on glass coverslips in multiwall plates stimulated with (B and C) or without (A) FITC-stained P. gingivalis (green arrows) for 24 h and then stained for F-actin (red arrows) and nucleus (blue arrows). The cells were mounted upside-down and visualized by scanning confocal laser microscopy. The 3D images (D-G) are shown by different combination of stained components: F-actin and nucleus (D); F-actin and FITC-stained P. gingivalis(E); nucleus and FITC-stained P. gingivalis(F); F-actin, nucleus and FITC-stained P. gingivalis(G).

Figure 2

P. gingivalis increases AoSMCs proliferation. The neutral red assay was used to analyze changes in AoSMCs growth after challenge with P. gingivalis at the concentration of 10MOI for 24 h in the absence or presence of viable P. gingivalis. The results are presented as the mean ± SEM of four separate experiments. *, p < 0.05. **, p < 0.005. n = 4.

Figure 3

The protein-protein interaction network and SPIA analysis of the 28 up-regulated genes related to positive regulation of cell proliferation and 21 down- regulated genes related to negative control of cell proliferation. The network (A) was mapped using STRING, and the confidence parameter was set as 0.15. Lines between proteins stand for possible interactions, and are color-coded based on the type of interaction. In SPIA analysis (B), X-axis indicates probability to observe differentially expressed genes on the pathway; y-axis refers to the probability to observe perturbation of genes within pathways. Each number refers to a KEGG pathway ID. Pathways above the blue line are significant at 5% after FDR correction; those above the red lines are significant at 5% after Bonferroni correction. 49 genes were analyzed by SPIA analysis. The Notch pathway is shown as a yellow dot and TGF-beta pathway is shown as a green dot.

Figure 4

The expression of selected genes and proteins in AoSMCs stimulated with P. gingivalis. Quantitative real-time PCR results demonstrate relative transcription for TGF-β1 (A), CTGF (B), SMAD3 (C), NOTCH1 (D), and HEY1 (E) of AoSMCs stimulated (P. g) or unstimulated (control) with P. gingivalis at 10 MOI for 24 h. All these results were normalized by the gene expression level of the house-keeping gene GAPDH. Representative western blot showing NOTCH1 protein expression levels of AoSMCs exposed to P. gingivalis at 8 and 10 MOI (F). Quantification of NOTCH1 protein expression levels by densitometry is shown in (G). NOTCH1 density signals were normalized to GAPDH signal values. P. gingivalis resulted in a dose-dependent (MOI 8 and 10) induction of TGF-β1 protein expression (H). *, p < 0.05; **, p < 0.005; ***, p < 0.0001. A, n = 10; B-E, n = 6; F, G, n = 3; H, n = 3-4.

Figure 5

The graph of gene-gene association among TGF-β1, Smad3 and other differentially expressed genes in AoSMCs exposed to P. gingivalis. Each dot refers to one gene and the color of dots refers to the log fold change of the gene expression level. Red dots indicate up-regulated genes. Green dots indicate down-regulated genes. The deeper is the color, the bigger fold change of the genes.

Figure 6

TGF-β and NOTCH activation in AoSMCs stimulated with P. gingivalis. Treatment of AoSMCs with viable P. gingivalis, increases the mRNA expression level of TGF-beta. TGF-beta is secreted as a latent complex which can be activated by a number of agents. The activation of TGF-beta interacts with TGFRII and this complex further phosphorylates TGFRI to activate the Smad pathway by phosphorylation at the carboxyl terminus. During this process, Smad anchor for receptor activation (SARA) can facilitate TGF-beta receptor complex to bind to R-Smads (Smad 2/3). Once the R-Smads being phosphorylated, they bind to Co-Smads to form a complex which go into nucleus and regulates the transcription of several targets genes involved in atherosclerosis. Beside this, TGF-beta and NOTCH pathway can collaborate with each other by direct interaction between Smad3 and NICD.