Treponema denticola alters cell vitality and induces HO-1 and Hsp70 expression in porcine aortic endothelial cells

Abstract

Treponema denticola is an oral spirochete that is associated with periodontal disease and detected occasionally in extraoral lesions associated with systemic disorders such as cardiovascular diseases. The effect of specific bacterial products from oral treponemes on endothelium is poorly investigated. This study analyzed the ability of components of the outer membrane of T. denticola (OMT) to induce apoptosis and heat shock proteins (HO-1 and Hsp70) in porcine aortic endothelial cells (pAECs), compared with results obtained with classical pro-inflammatory lipopolysaccharide (LPS) treatment. Cellular apoptosis was detected when pAECs were treated with either OMT or LPS, suggesting that OMT can damage endothelium integrity by reducing endothelial cell vitality. Stimulation with OMT, similarly to LPS response, increased HO-1 and Hsp-70 protein expression in a time-dependent manner, correlating with a rise in HO-1 and Hsp-70 mRNA. Collectively, these results support the hypothesis that T. denticola alters endothelial cell function. Moreover, our in vitro experiments represent a preliminary investigation to further in vivo study using a pig model to elucidate how T. denticola leaves the initial endodontic site and participates in the development of several systemic diseases.

Fig. 1

SDS-PAGE analysis of the OMT preparation, before and after treatment at 100°C for 10 min (lanes 1 and 2, respectively), lanes 3, and 4 contain whole T. denticola cells, before and after treatment at 100°C for 10 min. Filled circle indicates the position of the monomeric MSP (approximately 53 kDa); filled triangle indicates the position of the monomeric CTLP (approximately 72 kDa); number sign indicates the typical double band of multimeric CTLP (approximately 95 kDa); asterisk indicates the position of the CTLP/MSP complex. The positions of molecular size standards (low-range SDS-PAGE standards; Bio-RAD) are shown in kilodalton (kDa) on the left

Fig. 2

Effect of T. denticola outer membrane in pAECs: apoptosis levels after 24 h of exposure to OMT or LPS. a Apoptosis was assessed by Tunel assay, and cells are counterstained with propidium iodide. Cells with green fluorescence are apoptotic; b percentage of apoptotic cells. A minimum of 200 cells were evaluated for each treatment; data represent the mean ± 2 SEM of three replicates. Different letters indicate statistically significant differences (p < 0.05). C control, OMT T. denticola outer membrane, LPS lipopolysaccharide

Fig. 3

Effect of OMT (a) or LPS (b) HO-1 mRNA levels in pAECs. Data are presented as ΔCt (HPRT Ct–HO-1Ct) ±2 SEM of three replicates. C control, OMT T. denticola outer membrane, LPS lipopolysaccharide

Fig. 4

Effect of OMT (a, c) or LPS (b, d) on HO-1 protein levels in pAECs. a, b Representative Western blot of HO-1 and relative housekeeping HPRT are presented. c, d Data are expressed as arbitrary units (AUs) and represent the mean ± 2 SEM of three replicates. Different letters indicate statistically significant differences (p < 0.05). C control, OMT T. denticola outer membrane, LPS lipopolysaccharide

Fig. 5

Effect of OMT (a) or LPS (b) Hsp70 mRNA levels in pAECs. Data are presented as ΔCt (HPRT Ct–Hsp70Ct) ±2 SEM of three replicates. C control, OMT T. denticola outer membrane, LPS lipopolysaccharide

Fig. 6

Effect of OMT (a, c) or LPS (b, d) on Hsp70 protein levels in pAEC. a, b Representative Western blot of HO-1 and relative housekeeping HPRT are presented. c, d Data are expressed as arbitrary units (AUs) and represent the mean ± 2 SEM of three replicates. Different letters indicate statistically significant differences (p < 0.05). C control, OMT T. denticola outer membrane, LPS lipopolysaccharide