Gingipains from Porphyromonas gingivalis promote the transformation and proliferation of vascular smooth muscle cell phenotypes

The aim of the present study was to ascertain the effect of Porphyromonas gingivalis cysteine protease gingipain on the proliferation of rat aortic smooth muscle cells (RASMCs). Gingipains were isolated and purified from the supernatant of P. gingivalis W83, which was cultured under standard anaerobic conditions; primary RASMCs were also cultured. RASMCs were exposed to 200, 100, 50, 25, 12, 6, 3, 1, and 0 mu g/mL activated gingipains and the proliferation was evaluated using a cell counting kit-8 (CCK-8) assay after 48 h. alpha-Smooth muscle actin (alpha-SMA) and osteopontin (OPN) expression were measured by immunohistochemical staining. In addition, RASMCs were stimulated with 5, 10, 20, and 40 mu M KYT-1 (arg-gingipain inhibitor) and KYT-36 (lys-gingipain inhibitor) in combination with the gingipain extracts. Different concentrations of gingipains significantly promoted the proliferation of RASMCs, except those treated with 1 mu g/mL, compared to the untreated controls. The proliferation was sustained at a concentration above 12 mu g/mL. Immunohistochemical staining showed OPN expression after gingipain stimulation. The proliferative effects of gingipains on RASMCs were blocked after treatment with 10 mu M KYT-1 or 10 mu M KYT-36 (P < 0.0001); however, the difference between KYT-1 and KYT-36 groups was not statistically significant. These results demonstrated that gingipains can promote phenotypic transformation and proliferation of RASMCs and their effects were blocked by KYT-1 and KYT-36, which help us to ascertain whether Rgp or Kgp contributes to the development of atherosclerosis.