Osteoprotegerin protects endothelial cells against apoptotic cell death induced by Porphyromonas gingivalis cysteine proteinases

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis during the progression of periodontitis. Recent reports suggest that osteoprotegerin may also prevent arterial calcification and contribute to endothelial cell survival. To determine whether the vascular functions of osteoprotegerin are involved in periodontitis, we examined whether osteoprotegerin contributed to the survival of endothelial cells damaged by Porphyromonas gingivalis cysteine proteinases (gingipains). Gingipain proteinases cleave a broad range of host proteins, and are important virulence factors of P. gingivalis, a major causative bacterium of adult periodontitis. Human microvascular endothelial cells (HMVEC) were exposed to activated gingipain extracts from P. gingivalis 381, with and without pretreatment with osteoprotegerin. Cell viability was quantified by the tetrazolium (WST-8) reduction assay, and apoptosis was examined using Hoechst 33342 nuclear staining. After 16 h of treatment with activated gingipain extracts, HMVEC showed near-complete detachment from the tissue culture dish, and apoptosis was evident by 24 h. Pretreatment of HMVEC with osteoprotegerin reduced the extent of both cellular detachment and apoptotic cell death. Our results indicated that osteoprotegerin pretreatment protected HMVEC against detachment and apoptotic cell death induced by gingipain-active bacterial cell extracts. These results also suggest that osteoprotegerin may function as a survival factor for endothelial cells during periodontitis.     

Modulation of cytokine production by Porphyromonas gingivalis in a macrophage and epithelial cell co-culture model

Epithelial cells and macrophages play a major role in the host response to Porphyromonas gingivalis, a major etiologic agent of chronic periodontitis. Secretion of high levels of cytokines by these cells is believed to contribute to periodontal tissue destruction. To investigate the interactions between P. gingivalis and these two major cell types, we characterized the production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and regulated on activation normal T cell expressed and secreted (RANTES) by an in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 cell line) following a challenge with different strains of P. gingivalis. P. gingivalis cells stimulated the secretion of pro-inflammatory cytokines (IL-1beta and IL-6) and chemokines (IL-8 and RANTES) in the co-culture model. Responses to P. gingivalis infection were influenced by the macrophage/epithelial cell ratios of the cultures. In addition, the level of secretion of these inflammatory mediators was dependent on the bacterial strain and the multiplicity of infection (MOI) used. The use of a gingipain-deficient mutant of P. gingivalis or the addition of a cysteine protease inhibitor suggested that the level of cytokines secreted by the co-culture model was underestimated due to an extensive proteolytic degradation. This study showed that P. gingivalis can modulate the levels of inflammatory mediators, which may contribute to the progression of periodontitis.     

Induction of monocyte chemoattractant protein-1 by Porphyromonas gingivalis in human endothelial cells

The association between periodontal and cardiovascular diseases could be mediated by direct interaction of periodontal pathogens with cardiac tissue. In order to explore this possibility, the effect of the periodontal pathogen Porphyromonas gingivalis on monocyte chemoattractant protein-1 (MCP-1) production by endothelial cells was investigated. When incubated with live P. gingivalis 381, MCP-1 production by human umbilical vein endothelial cells (HUVEC) was potently increased. Compared to the type strain 381, non-adhesive/invasive strains (W50 and DPG3) did not increase MCP-1 production, which was also demonstrated at the mRNA level. Killed P. gingivalis 381 was much less effective than live bacteria for MCP-1 induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, prevented MCP-1 mRNA up-regulation by P. gingivalis 381, suggesting that internalization of P. gingivalis is necessary for MCP-1 induction. In conclusion, the secretion of high levels of MCP-1 resulting from interactions of P. gingivalis with endothelial cells could enhance atherosclerosis progression by contributing to the recruitment of monocytes.     

Arginine-specific gingipain A from Porphyromonas gingivalis induces Weibel-Palade body exocytosis and enhanced activation of vascular endothelial cells through protease-activated receptors

Gingipains, cysteine proteases derived from Porphyromonas gingivalis, are important virulence factors in periodontal diseases. We found that arginine-specific gingipain A (RgpA) increased the responsiveness of vascular endothelial cells to P. gingivalis lipopolysaccharides (LPS) and P. gingivalis whole cells to induce enhanced IL-8 production through protease-activated receptors (PARs) and phospholipase C (PLC) gamma. We therefore investigated whether RgpA-induced enhanced cell activation is mediated through exocytosis of Weibel-Palade bodies (WPBs) because they store vasoactive substances. RgpA rapidly activated PAR- and PLCgamma-dependent WPB exocytosis. In addition, angiopoietin (Ang)-2, a substance of WPB, enhanced IL-8 production by P. gingivalis LPS, suggesting that Ang-2 mediates the RgpA-induced enhanced cell responses. Thus, we propose a novel role for RgpA in induction of a proinflammatory event through PAR-mediated WPB exocytosis, which may be an important step for enhanced endothelial responses to P. gingivalis.     

Porphyromonas gingivalis influences actin degradation within epithelial cells during invasion and apoptosis

Porphyromonas gingivalis, a Gram-negative oral pathogen, has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We have previously shown that the P. gingivalis proteases arginine and lysine gingipains, are necessary and sufficient to induce host cell apoptosis. In the present study, we demonstrate that 'P. gingivalis-induced apoptosis' is mediated through degradation of actin leading to cytoskeleton collapse. Stimulation of human gingival epithelial cells with P. gingivalis strains 33277 and W50 at moi:100 induced β-actin cleavage as early as 1 h and human serum inhibited this effect. By using gingipain-deficient mutants of P. gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time-dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P. gingivalis internalization is necessary for actin cleavage. Further, we also show that lysine gingipain from P. gingivalis can cleave active caspase 3. Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in microbial host cell invasion and apoptosis.     

Porphyromonas gingivalis fimbriae induce adhesion of monocytic cell line U937 to endothelial cells

This study used the human monocytic cell line U937 to examine whether or not Porphyromonas gingivalis fimbriae could induce the adhesion of monocytes to endothelial cells. An in vitro adhesion assay was used to investigate the effects of the fimbriae on U937 cell adhesion to human umbilical vein endothelial cells (HUVEC). The fimbriae enhanced U937 cell adhesion to HUVEC in a dose-dependent manner. U937 cells adhered better to HUVEC pretreated with the fimbriae for a minimum of 2 hr than to untreated HUVEC. The enhanced adhesion was inhibited by a monoclonal antibody against P. gingivalis 381 fimbriae. Pretreatment of U937 cells with the fimbriae for 24 hr enhanced U937 cell adhesion to HUVEC approximately 4-fold. This phenomenon was inhibited by an anti-CD11b antibody, suggesting the involvement of CD11b. These results indicate that P. gingivalis fimbriae can induce monocyte adhesion to the endothelial cell surface. They also suggest that the fimbriae may be involved in the initial event for infiltration of monocytes into the periodontal tissues of individuals with adult periodontitis.     

Periodontal infections and atherosclerosis: mere associations?

PURPOSE OF REVIEW: Several lines of evidence from the last few decades suggest that periodontitis is an important risk factor for cardiovascular diseases. In this review we discuss the recent findings on the systemic effects of periodontitis, which may contribute to the pathogenesis of atherosclerosis, with a special emphasis on lipoproteins. RECENT FINDINGS: In addition to the epidemiological studies exploring the direct or indirect relationship between clinical periodontitis and cardiovascular diseases, studies utilizing serology, animal models, cell cultures, and biochemistry of lipoproteins have been published. Local infection in the periodontal pockets triggers a systemic inflammatory response releasing inflammatory mediators and awakens a strong immune response against periodontal pathogens. Elevated systemic antibody levels especially to Porphyromonas gingivalis are associated with an increased risk for atherosclerosis. Periodontitis is also accompanied by proatherogenic changes in both low and high density lipoproteins, which lead to enhanced cholesteryl ester uptake by and reduced cholesterol efflux from macrophages. Vesicles and lipopolysaccharide isolated from P. gingivalis activate macrophages to convert into foam cells. Moreover, animal studies have demonstrated that infection by P. gingivalis enhances progression of atherosclerosis. SUMMARY: Recent studies have clarified the mechanisms by which periodontitis may contribute to the development of atherosclerosis. Serological, animal, and cell culture studies provide evidence that infection by P. gingivalis may promote atherosclerosis. The influence of periodontitis on lipoprotein metabolism has emerged as a new, important factor. Recent studies provide experimental proof that periodontitis may predispose to atherosclerosis.     

Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells

Epidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice, while an isogenic fimbria-deficient (FimA-) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzyme-linked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)-1beta, IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and E-selectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL-1beta production. In addition, the major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1beta. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1beta appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.     

Arg-gingipain is responsible for the degradation of cell adhesion molecules of human gingival fibroblasts and their death induced by Porphyromonas gingivalis

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are two major cysteine proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis, which has been shown to act as major pathogen in the development and progression of periodontal diseases. These enzymes are also important for this organism to proliferate and survive in periodontal pockets. Here we show that Rgp is responsible for the disruption of fibronectin-integrin interactions in human gingival fibroblasts by P. gingivalis. Fibroblasts incubated with the culture supernatant of P. gingivalis showed a time-dependent loss of the adhesion activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that fibronectin and integrin subunits alpha2, beta1 and beta3 in the fibroblast culture largely disappeared with the treatment. The detached cells became committed to death by disruption of contacts between adhesion molecules. In contrast, the culture supernatants from the Rgp-deficient mutants produced no significant changes in either cell adhesion or viability. Prior treatment of the culture supernatant of P. gingivalis with an Rgp inhibitor, but not a Kgp inhibitor, strongly inhibited the detachment of fibroblasts followed by cell death. These results suggest that Rgp disrupts the integrin-fibronectin interactions in fibroblasts, thereby contributing to the damage of periodontal tissues in periodontal diseases caused by P. gingivalis.     

P38 mitogen-activated protein kinase pathways are involved in the hypertrophy and apoptosis of cardiomyocytes induced by Porphyromonas gingivalis conditioned medium

The surrounding medium of periodontal pathogen Porphyromonas gingivalis (P. gingivalis) increased cardiomyocyte hypertrophy and apoptosis whereas Actinobaeillus actinomycetemcomitans and Prevotella intermedia had no effects. The purpose of this study is to clarify the role of p38 pathway in P. gingivalis conditioned medium-induced H9c2 myocardial cell hypertrophy and apoptosis. DNA fragmentation, cellular morphology, nuclear condensation, p38 protein products, and mitochondrial-dependent apoptotic related proteins in cultured H9c2 myocardial cell were measured by agarose gel electrophoresis, immunofluorescence, DAPI, and western blotting following P. gingivalis conditioned medium and/or pre-administration of SB203580 (p38 inhibitor). The p38 protein products and associated activities in H9c2 cells were both upregulated by P. gingivalis conditioned medium. P. gingivalis conditioned medium increased cellular sizes, DNA fragmentation, nuclear condensation, mitochondrial Bcl2-associated death promoter (Bad), cytosolic cytochrome c (cyt c), and the activated form of caspase-9 proteins in H9c2 cells. The increased cellular sizes, DNA fragmentation, nuclear condensation, Bad, cyt c, and caspase-9 activities of H9c2 cells treated with P. gingivalis conditioned medium were all significantly reduced after pre-administration of SB203580. Our findings suggest that the activity of p38 signal pathway may be initiated by P. gingivalis conditioned medium and further activate mitochondrial-dependent apoptotic pathways leading to cell death in cultured H9c2 myocardial cells.     

NF-kappaB-dependent induction of osteoprotegerin by Porphyromonas gingivalis in endothelial cells

Porphyromonas gingivalis is a major etiological pathogen of adult periodontitis characterized by alveolar bone resorption. Vascular endothelial cells supply many inflammatory cytokines into periodontal tissue. However, whether the cells contribute to bone metabolism in periodontitis remains unknown. In this study, we investigated the effect of P. gingivalis on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) production, both of which are key regulators of bone metabolism, in human microvascular endothelial cells (HMVECs). We showed that P. gingivalis upregulated expression of OPG but not RANKL mRNA in HMVEC. P. gingivalis induced NF-kappaB activation, and the induction of OPG in HMVEC by the pathogen was blocked by the inhibitors of NF-kappaB. In addition, incubation of OPG with P. gingivalis supernatant resulted in loss of the protein. These results indicate that P. gingivalis-stimulated HMVEC secrete OPG via a NF-kappaB-dependent pathway, while the OPG is partly degraded by the bacteria. Thus, microvascular endothelial cells can act as a source of OPG and thereby may play an important role in regulating bone metabolism in periodontitis.     

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.     

Fibroblast apoptosis induced by Porphyromonas gingivalis is stimulated by a gingipain and caspase-independent pathway that involves apoptosis-inducing factor

Porphyromonas gingivalis is an oral bacterium that causes pathology in a number of dental infections that are associated with increased fibroblast cell death. Studies presented here demonstrated that P. gingivalis stimulates cell death by apoptosis rather than necrosis. Unlike previous studies apoptosis was induced independent of proteolytic activity and was also independent of caspase activity because a pancaspase inhibitor, Z-VAD-fmk, had little effect. Moreover, P. gingivalis downregulated caspase-3 mRNA levels and caspase-3 activity. The consequence of this downregulation was a significant reduction in tumour necrosis factor-alpha-induced apoptosis, which is caspase-3-dependent. Immunofluorescence and immunoblot analysis revealed P. gingivalis-induced translocation of apoptosis-inducing factor (AIF) from the cytoplasm to the nucleus. siRNA studies were undertaken and demonstrated that P. gingivalis stimulated cell death was significantly reduced when AIF was silenced (P < 0.05). Treatment of human gingival fibroblasts with H-89, a protein kinase A inhibitor that blocks AIF activation also reduced P. gingivalis-induced apoptosis (P < 0.05). These results indicate that P. gingivalis causes fibroblast apoptosis through a pathway that involves protein kinase A and AIF, is not dependent upon bacterial proteolytic activity and is also independent of the classic apoptotic pathways involving caspase-3.     

Glutamic protease distribution is limited to filamentous fungi

Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans, which has been linked to an increased risk for atherosclerosis-related events. In this study, we examined the effect of P. gingivalis infection on human macrophages with respect to foam cell formation, the hallmark of early atherogenesis, and the potential of P. gingivalis to induce its uptake by these cells. Human monocyte-derived macrophages were incubated with low density lipoprotein and infected with P. gingivalis FDC381 or its fimbriae deficient mutant, DPG3. Consistent with a role for fimbriae in this process, strain 381 significantly increased foam cell formation as compared to DPG3. Recovery of viable P. gingivalis in antibiotic protection experiments was significantly higher for strain 381 than for DPG3. By transmission electron microscopy, the wild-type strain was shown to adhere to and enter THP-1 cells. These results suggest that properties of P. gingivalis which render it capable of adhering to/invading other cell types may also be operative in macrophages and play an important role in its atherogenic potential.