TLR2 Mediates Bacteria-induced PAI-1 expression in Vascular Smooth Muscle Cells

Inflammation plays an important role in atherogenesis. Emerging epidemiological studies have linked periodontal infection and development of atherosclerosis. Plasminogen activator inhibitor type 1 (PAI-1), a “stress response” protein, is increased in atherosclerotic vascular smooth muscle cells (VSMC). Toll-like receptors (TLRs) signaling is associated with proinflammatory phenotypes in VSMC. Objectives: To investigate the effects of a major periodontal pathogen Actinobacillus actinomycetemcomitans(Aa) on PAI-1 expression in VSMC and the role of TLR. Methods:VSMC were explanted from wild type(WT) and TLR2 deficient(TLR2-/-) mice and confirmed with smooth muscle-actin antibody by immunofluorescent staining. Expression of PAI-1 in VSMC was assessed by immunofluroscent staining and Western blot. VSMC were exposed to Gram-negative periodontal pathogen Aa or Gram-positive oral bacterium Streptococcus sanguis (Ss) for different time points. Expression and activation of PAI-1 and mitogen-activated protein kinase (MAPK, p38 and p42/44) were determined by Western blot. Results: VSMC identity was confirmed in all explanted cells. Expression of PAI-1 was similar in WT and TLR2-/- VSMC (n=4). Time-dependent increases (fold induction=3.9±0.2, n=4, p<0.001) in the appearance of PAI-1 and urokinase plasminogen activator (uPA) complex were observed in WT VSMC exposed to Aa (107CFU/ml). Such increases were correlated with time-dependent activation of p38 and p42/44 MAPK (fold induction, p-p38 MAPK=3.5±0.2, p-p42/44 MAPK=2.8±0.1, n=4, p<0.001). By contrast, induction of the PAI-1/uPA complex was not apparent in TLR2-/- VSMC. Further, no significant increases of the PAI-1/uPA complex were identified when the cells were exposed to Ss. Conclusions: We have found that periodontal pathogen Aa, but not commensal bacterium S. sanguis significantly induced the formation of PAI-1/uPA complex in VSMC in a time-dependent manner. Such induction is correlated with activation of MAPK (p38 and p42/44) and appears to be mediated by TLR2. Our results provided an important molecular insight into the mechanisms responsible for interaction between periodontal infection and cardiovascular disease.