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.