Cinnamaldehyde Protects against P. gingivalis Induced Intestinal Epithelial Barrier Dysfunction in IEC-6 Cells via the PI3K/Akt-Mediated NO/Nrf2 Signaling Pathway.

Porphyromonas gingivalis (Pg), a Gram-negative oral pathogen, promotes and accelerates periodontitis-associated gut disorders. Intestinal epithelial barrier dysfunction is crucial in the pathogenesis of intestinal and systemic diseases. In this study, we sought to elucidate the protective role of cinnamaldehyde (CNM, an activator of Nrf2) against P. gingivalis (W83) and Pg-derived lipopolysaccharide (Pg-LPS) induced intestinal epithelial barrier dysfunction via antioxidative mechanisms in IEC-6 cells. IEC-6 (ATCC, CRL-1592) cells were pretreated with or without CNM (100 µM), in the presence or absence of P. gingivalis (strain W83, 109 MOI) or Pg-LPS (1, 10, and 100 µg/mL), respectively, between 0-72 h time points by adopting a co-culture method. Intestinal barrier function, cytokine secretion, and intestinal oxidative stress protein markers were analyzed. P. gingivalis or Pg-LPS significantly (p < 0.05) increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels expressing oxidative stress damage. Pg-LPS, as well as Pg alone, induces inflammatory cytokines via TLR-4 signaling. Furthermore, infection reduced Nrf2 and NAD(P)H quinone dehydrogenase 1 (NQO1). Interestingly, inducible nitric oxide synthase (iNOS) protein expression significantly (p < 0.05) increased with Pg-LPS or Pg infection, with elevated levels of nitric oxide (NO). CNM treatment suppressed both Pg- and Pg-LPS-induced intestinal oxidative stress damage by reducing ROS, MDA, and NO production. Furthermore, CNM treatment significantly upregulated the expression of tight junction proteins via increasing the phosphorylation levels of PI3K/Akt/Nrf2 suppressing inflammatory cytokines. CNM protected against Pg infection-induced intestinal epithelial barrier dysfunction by activating the PI3K/Akt-mediated Nrf2 signaling pathway in IEC-6 cells.

Rho activation drives luminal collapse and eversion in epithelial acini.

Epithelial cells lining a gland and cells grown in a soft extracellular matrix polarize with apical proteins exposed to the lumen and basal proteins in contact with the extracellular matrix. Alterations to polarity, including an apical-out polarity, occur in human cancers. Although some aberrant polarity states may result from altered protein trafficking, recent observations of an extraordinary tissue-level inside-out unfolding suggest an alternative pathway for altered polarity. Because mechanical alterations are common in human cancer, including an upregulation of RhoA-mediated actomyosin tension in acinar epithelia, we explored whether perturbing mechanical homeostasis could cause apical-out eversion. Acinar eversion was robustly induced by direct activation of RhoA in normal and tumor epithelial acini, or indirect activation of RhoA through blockage of ß1-integrins, disruption of the LINC complex, oncogenic Ras activation, or Rac1 inhibition. Furthermore, laser ablation of a portion of the untreated acinus was sufficient to induce eversion. Analyses of acini revealed high curvature and low phosphorylated myosin in the apical cell surfaces relative to the basal surfaces. A vertex-based mathematical model that balances tension at cell-cell interfaces revealed a fivefold greater basal cell surface tension relative to the apical cell surface tension. The model suggests that the difference in surface energy between the apical and basal surfaces is the driving force for acinar eversion. Our findings raise the possibility that a loss of mechanical homeostasis may cause apical-out polarity states in human cancers.

Osteoprotegerin is sensitive to actomyosin tension in human periodontal ligament fibroblasts.

Periodontal ligament fibroblasts (PdLFs) are an elongated cell type in the periodontium with matrix and bone regulatory functions which become abnormal in periodontal disease (PD). Here we found that the normally elongated and oriented PdLF nucleus becomes rounded and loses orientation in a mouse model of PD. Using in vitro micropatterning of cultured primary PdLF cell shape, we show that PdLF elongation correlates with nuclear elongation and the presence of thicker, contractile F-actin fibers. The rounded nuclei in mouse PD models in vivo are, therefore, indicative of reduced actomyosin tension. Inhibiting actomyosin contractility by inhibiting myosin light chain kinase, Rho kinase or myosin ATPase activity, in cultured PdLFs each consistently reduced messenger RNA levels of bone regulatory protein osteoprotegerin (OPG). Infection of cultured PdLFs with two different types of periodontal bacteria (Porphyromonas gingivalis and Fusobacterium nucleatum) failed to recapitulate the observed nuclear rounding in vivo, upregulated nonmuscle myosin II phosphorylation and downregulated OPG. Collectively, our results add support to the hypothesis that PdLF contractility becomes decreased and contributes to disease progression in PD.

Porphyromonas gingivalis infection alters Nrf2-phase II enzymes and nitric oxide in primary human aortic endothelial cells.

BACKGROUND: Periodontal disease (PD) is known to be associated with endothelial dysfunction in patients with coronary artery and/or cardiovascular disease. In our study, we sought to explore the virulence of P. gingivalis (Pg) affecting glycogen synthase kinase 3 beta (GSK-3ß)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/tetrahydrobiopterin (BH4 )/ nitric oxide synthase (NOS) expression in primary human aortic endothelial cells (pHAECs). METHODS: pHAECs were infected for 48 hours with Pg in vitro using the Human oxygen-Bacteria anaerobic coculture technique. Cell viability was determined, and target gene expression changes were evaluated by quantitative real-time polymerase chain reaction at the end of each incubation period. RESULTS: Pg impaired pHAEC viability 24 hours post-infection. Pg infection reduced mRNA expression levels of endothelial NOS (eNOS), Nrf2, and Phase II enzymes (heme oxygenase-1, catalase, superoxide dismutase-1) in a time-dependent manner. Significant (P <0.05) increase in the inflammatory markers (interleukin [IL]-1ß, IL-6, and tumor necrosis factor-α) were observed in the medium as well as in the infected cells. Interestingly, inducible NOS mRNA levels showed a significant (P <0.05) increase at 12 hours and 24 hours and were reduced at later time points. BH4 (cofactor of eNOS) biosynthesis enzyme dihydrofolate reductase (DHFR, salvage pathway) mRNA levels showed a significant (P <0.05) decrease, while mRNA levels of GSK-3ß were elevated. CONCLUSIONS: These results suggest that periodontal bacterial infection may cause significant changes in the endothelial GSK-3ß/BH4 /eNOS/Nrf2 pathways, which may lead to impaired vascular relaxation. Greater understanding of the factors that adversely affect endothelial cell function could contribute to the development of new therapeutic compounds to treat PD-induced vascular diseases.

Fusobacteria modulate oral carcinogenesis and promote cancer progression.

Background: Evidence suggest periodontal bacterial infection can contribute to oral cancer initiation and progression. Aim: To investigate the effects of periodontal bacteria on oral cancer cell behavior using a cell-based system and a mouse carcinogenesis model. Methods: Oral cancer cell lines were polyinfected with four periodontal bacteria. Cytokine levels and relative changes in oncogene mRNA expression were determined post-infection. Oral tumours in mice induced by 4-nitroquinoline-1-oxide (4NQO) were compared with and without administrating periodontal bacteria. Results: Polyinfected oral cancer cells had upregulated MMP1, MMP9, and IL-8. The expression of cell survival markers MYC, JAK1, and STAT3 and epithelial-mesenchymal transition markers ZEB1 and TGF-ß were also significantly elevated. Monoinfections showed F. nucleatum alone had comparable or greater effects than the four bacteria together. Fusobacterial culture supernatant, primarily LPS, was sufficient to induce IL-8 secretion, demonstrating that direct contact of live Fusobacteria with cancer cells might not be required to exert changes in cancer cell behaviour. In the 4NQO-induced oral tumour model, mice infected with bacteria developed significantly larger and more numerous lesions compared to those not infected. Conclusion: This study demonstrated that Fusobacteria could potentially enhance cancer cell invasiveness, survival, and EMT when presented in the oral tumour microenvironment. Abbreviations: 4NQO, 4-nitroquinoline-1-oxide; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial-mesenchymal transition; IL-8, interleukin-8; JAK1, Janus kinase 1; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; OSCCs, oral squamous cell carcinomas; PK, proteinase K; PMB, Polymyxin B; qRT-PCR, quantitative real-time polymerase chain reaction; STAT3, signal transducer and activator of transcription 3; TGF-ß, transforming growth factor beta; ZEB1, zinc finger E-Box binding homeobox 1.

Periodontal cell mechanotransduction.

The periodontium is a structurally and functionally complex tissue that facilitates the anchorage of teeth in jaws. The periodontium consists of various cell types including stem cells, fibroblasts and epithelial cells. Cells of the periodontium are constantly exposed to mechanical stresses generated by biological processes such as the chewing motions of teeth, by flows generated by tongue motions and by forces generated by implants. Mechanical stresses modulate the function of cells in the periodontium, and may play a significant role in the development of periodontal disease. Here, we review the literature on the effect of mechanical forces on periodontal cells in health and disease with an emphasis on molecular and cellular mechanisms.

Cerebral Oxidative Stress and Microvasculature Defects in TNF-α Expressing Transgenic and Porphyromonas gingivalis-Infected ApoE-/- Mice.

The polymicrobial dysbiotic subgingival biofilm microbes associated with periodontal disease appear to contribute to developing pathologies in distal body sites, including the brain. This study examined oxidative stress, in the form of increased protein carbonylation and oxidative protein damage, in the tumor necrosis factor-α (TNF-α) transgenic mouse that models inflammatory TNF-α excess during bacterial infection; and in the apolipoprotein knockout (ApoE-/-) mouse brains, following Porphyromonas gingivalis gingival monoinfection. Following 2,4-dinitrophenylhydrazine derivatization, carbonyl groups were detected in frontal lobe brain tissue lysates by immunoblotting and immunohistochemical analysis of fixed tissue sections from the frontotemporal lobe and the hippocampus. Immunoblot analysis confirmed the presence of variable carbonyl content and oxidative protein damage in all lysates, with TNF-α transgenic blots exhibiting increased protein carbonyl content, with consistently prominent bands at 25 kDa (p = 0.0001), 43 kDa, and 68 kDa, over wild-type mice. Compared to sham-infected ApoE-/- mouse blots, P. gingivalis-infected brain tissue blots demonstrated the greatest detectable protein carbonyl content overall, with numerous prominent bands at 25 kDa (p = 0.001) and 43 kDa (p = 0.0001) and an exclusive band to this group between 30-43 kDa* (p = 0.0001). In addition, marked immunostaining was detected exclusively in the microvasculature in P. gingivalis-infected hippocampal tissue sections, compared to sham-infected, wild-type, and TNF-α transgenic mice. This study revealed that the hippocampal microvascular structure of P. gingivalis-infected ApoE-/- mice possesses elevated oxidative stress levels, resulting in the associated tight junction proteins being susceptible to increased oxidative/proteolytic degradation, leading to a loss of functional integrity.

Apolipoprotein E Related Co-Morbidities and Alzheimer's Disease.

The primary goal of advancement in clinical services is to provide a health care system that enhances an individual's quality of life. Incidence of diabetes mellitus, cardiovascular disease, and associated dementia coupled with the advancing age of the population, have led to an increase in the worldwide challenge to the healthcare system. In order to overcome these challenges, prior knowledge of common, reliable risk factors and their effectors is essential. Oral health constitutes one such relatively unexplored but indispensable risk factor for aforementioned co-morbidities, in the form of poor oral hygiene and tooth loss during aging. Behavioral traits such as low education, smoking, poor diet, neglect of oral health, lack of exercise, and hypertension are few of the risk factors that are shared commonly among these conditions. In addition, common genetic susceptibility traits such as the apolipoprotein E gene, together with an individual's lifestyle can also influence the development of co-morbidities such as periodontitis, atherosclerosis/stroke, diabetes, and Alzheimer's disease. This review specifically addresses the susceptibility of apolipoprotein E gene allele 4 as the plausible commonality for the etiology of co-morbidities that eventually result from periodontal diseases and ultimately progress to dementia.

Periodontal pathogens invade gingiva and aortic adventitia and elicit inflammasome activation in αvß6 integrin-deficient mice.

The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin ß6(-/-) mouse model to study the causality. We investigated the ability of a polymicrobial consortium of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum to colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infected Itgß6(-/-) mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques in Itgß6(-/-) mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescence in situ hybridization demonstrated active invasion of the aortic adventitial layer by P. gingivalis. Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the mechanism of the host aortic inflammatory response induced by polymicrobial infection with well-characterized periodontal pathogens.

Myxomavirus anti-inflammatory chemokine binding protein reduces the increased plaque growth induced by chronic Porphyromonas gingivalis oral infection after balloon angioplasty aortic injury in mice.

Thrombotic occlusion of inflammatory plaque in coronary arteries causes myocardial infarction. Treatment with emergent balloon angioplasty (BA) and stent implant improves survival, but restenosis (regrowth) can occur. Periodontal bacteremia is closely associated with inflammation and native arterial atherosclerosis, with potential to increase restenosis. Two virus-derived anti-inflammatory proteins, M-T7 and Serp-1, reduce inflammation and plaque growth after BA and transplant in animal models through separate pathways. M-T7 is a broad spectrum C, CC and CXC chemokine-binding protein. Serp-1 is a serine protease inhibitor (serpin) inhibiting thrombotic and thrombolytic pathways. Serp-1 also reduces arterial inflammation and improves survival in a mouse herpes virus (MHV68) model of lethal vasculitis. In addition, Serp-1 demonstrated safety and efficacy in patients with unstable coronary disease and stent implant, reducing markers of myocardial damage. We investigate here the effects of Porphyromonas gingivalis, a periodontal pathogen, on restenosis after BA and the effects of blocking chemokine and protease pathways with M-T7 and Serp-1. ApoE-/- mice had aortic BA and oral P. gingivalis infection. Arterial plaque growth was examined at 24 weeks with and without anti-inflammatory protein treatment. Dental plaques from mice infected with P. gingivalis tested positive for infection. Neither Serp-1 nor M-T7 treatment reduced infection, but IgG antibody levels in mice treated with Serp-1 and M-T7 were reduced. P. gingivalis significantly increased monocyte invasion and arterial plaque growth after BA (P<0.025). Monocyte invasion and plaque growth were blocked by M-T7 treatment (P<0.023), whereas Serp-1 produced only a trend toward reductions. Both proteins modified expression of TLR4 and MyD88. In conclusion, aortic plaque growth in ApoE-/- mice increased after angioplasty in mice with chronic oral P. gingivalis infection. Blockade of chemokines, but not serine proteases significantly reduced arterial plaque growth, suggesting a central role for chemokine-mediated inflammation after BA in P. gingivalis infected mice.