Periodontitis is associated with incidental valvular heart disease: A nationwide population-based cohort study.

AIM: Periodontitis and valvular heart disease (VHD) are common diseases. Both diseases are related to chronic inflammation and share many common risk factors. Previous periodontal studies had focused mainly on atherosclerotic cardiovascular disease. This study aimed to determine whether periodontitis is associated with the development of VHD. MATERIALS AND METHODS: This was a retrospective nationwide cohort study using Taiwan's Longitudinal Health Insurance Database. Using ICD-9-CM coding, both the periodontitis and non-periodontitis groups were matched. RESULTS: There were 8483 cases and 4919 cases of VHD diagnosed in the periodontitis group and non-periodontitis group, respectively. The cumulative incidence of VHD was significantly higher in the periodontitis group (log-rank test, p < .001), with the incidence density of 6.44 (95% CI, 6.31-6.58) per 1000 person-years in the periodontitis group compared to 4.65 (95% CI, 4.52-4.78) in the non-periodontitis group. The relative risk for VHD was 1.39 (95% CI, 1.34-1.44). After multivariate analysis, periodontitis was independently associated with a risk for VHD (HR, 1.38; 95% CI, 1.33-1.42, p < .001). Intensive treatment of periodontitis significantly lowered the risk for VHD (HR, 0.68; 95% CI, 0.60-0.77, p < .001). CONCLUSIONS: Periodontitis was significantly associated with the development of VHD. Treatment of periodontitis reduced the risk for VHD.

Exploring MicroRNA Expression Profiles Related to the mTOR Signaling Pathway in Mouse Embryonic Fibroblast Cells Treated with Polyethylenimine.

Although the toxicology of poly(ethylenimine) (PEI) in gene expression levels has been previously investigated, little is known about the effects of PEI on the expression of microRNAs (miRNAs) that regulate gene expression at the post-transcriptional level. In this study, we explored miRNA expression profiles related to cell death mechanisms in mouse embryonic fibroblast (MEF) cells treated with PEI by applying microarray analysis. Based on the analysis of the mTOR signaling pathway, three upregulated miRNAs (mmu-miR-3090-5p, mmu-miR-346-3p, and mmu-miR-494-3p) were verified in MEF cells treated with PEI at 24 h using real-time quantitative reverse transcriptase-polymerase chain reaction. We further demonstrated that these three upregulated miRNAs resulted in the decrease of gene and protein expressions of the target gene growth factor Igf1 in MEF cells treated with PEI or transfected with three upregulated miRNA mimics. However, these three upregulated miRNAs are not all cell-specific. Finally, we demonstrated that the mTOR signaling pathway is inhibited by autophagy induction and that the cell viability decreases in MEF cells treated with PEI or transfected with these three miRNA mimics. Collectively, our data suggested that PEI may affect the regulation of miRNAs in target cells.

Autophagy-related gene expression analysis of wild-type and atg5 gene knockout mouse embryonic fibroblast cells treated with polyethylenimine.

The molecular mechanisms of autophagy in polyethylenimine (PEI)-treated cells are not well understood because of the use of nonspecific autophagy inhibitors. Here, we applied autophagy-related gene expression analysis to pinpoint the molecular mechanisms of autophagy in PEI-treated wild-type and atg5 gene knockout (atg5(-/-)) mouse embryonic fibroblast (MEF) cells. It was demonstrated that the majority of induced genes are downregulated in wild-type and atg5(-/-) MEF cells, indicating that autophagy exhibits a trend toward downregulation after treatment with PEI. In addition to regulating genes encoding autophagy machinery components, genes related to coregulation of autophagy and apoptosis were induced in wild-type and atg5(-/-) cells treated with PEI. These data indicate that autophagy and apoptosis are closely related in the PEI-induced mechanism of cell death. In the absence of autophagy, the regulation of apoptosis was enhanced in atg5(-/-) MEF cells treated with PEI, indicating that inhibition of autophagy may lead to higher levels of apoptosis. Our study may provide deeper insight into the molecular mechanisms of cell death caused by PEI.

Targeting a cysteine protease from a pathobiont alleviates experimental arthritis.

BACKGROUND: Several lines of evidence suggest that the pathobiont Porphyromonas gingivalis is involved in the development and/or progression of auto-inflammatory diseases. This bacterium produces cysteine proteases, such as gingipain RgpA, endowed with the potential to induce significant bone loss in model systems and in patients. OBJECTIVE: We sought to gain further insight into the role of this pathobiont in rheumatoid arthritis (RA) and to identify novel therapeutic targets for auto-inflammatory diseases. METHODS: We profiled the antibody response to RgPA-specific domains in patient sera. We also tested the potential protective effects of RgpA domains in an experimental arthritis model. RESULTS: Pre-immunization of rats with purified recombinant RgpA domains alleviated arthritis in the joints of the rodents and reduced bone erosion. Using a functional genomics approach at both the mRNA and protein levels, we report that the pre-immunizations reduced arthritis severity by impacting a matrix metalloprotease characteristic of articular injury, a chemokine known to be involved in recruiting inflammatory cells, and three inflammatory cytokines. Finally, we identified an amino acid motif in the RgpA catalytic domain of P. gingivalis that shares sequence homology with type II collagen. CONCLUSION: We conclude that pre-immunization against gingipain domains can reduce the severity of experimentally induced arthritis. We suggest that targeting gingipain domains by pre-immunization, or, possibly, by small-molecule inhibitors, could reduce the potential of P. gingivalis to translocate to remote tissues and instigate and/or exacerbate pathology in RA, but also in other chronic inflammatory diseases.

Lactobacillus Attenuate the Progression of Pancreatic Cancer Promoted by Porphyromonas Gingivalis in K-rasG12D Transgenic Mice.

Accumulating evidence suggests that there is a link between the host microbiome and pancreatic carcinogenesis, and that Porphyromonas gingivalis (P. gingivalis) increases the risk of developing pancreatic cancer. The aim of the current study was to clarify the role of P. gingivalis in the pathogenesis of pancreatic cancer and the potential immune modulatory effects of probiotics. The six-week-old LSL-K-rasG12D; Pdx-1-cre (KC) mice smeared P. gingivalis on the gums, causing pancreatic intraepithelial neoplasia (PanIN) after four weeks to be similar to the extent of lesions in untreated KC mice at 24 weeks. The oral inoculation of P. gingivalis of six-week-old LSL-K-rasG12D; Pdx-1-cre (KC) mice caused significantly pancreatic intraepithelial neoplasia (PanIN) after treatment four weeks is similar to the extent of lesions in untreated KC mice at 24 weeks. The pancreas weights of P. gingivalis plus probiotic-treated mice were significantly lower than the mice treated with P. gingivalis alone (P = 0.0028). The histological expressions of Snail-1, ZEB-1, collagen fibers, Galectin-3, and PD-L1 staining in the pancreas were also notably lower. In addition, probiotic administration reduced the histological expression of Smad3 and phosphorylated Smad3 in P. gingivalis treated KC mice. We demonstrated that oral exposure to P. gingivalis can accelerate the development of PanIN lesions. Probiotics are likely to have a beneficial effect by reducing cancer cell proliferation and viability, inhibiting PanIN progression, and cancer cell metastasis (Epithelial-mesenchymal transition, EMT). The transforming growth factor-ß signaling pathway may be involved in the tumor suppressive effects of probiotics.

The microRNA expression profiles in extracellular vesicles from HeLa cancer cells in response to cationic lipid- or polyethylenimine-mediated gene delivery.

Intercellular communication is known to be involved in various stages of tumour development and metastasis through the secretion of extracellular vesicles (EVs) containing messengers such as microRNAs (miRNAs). Therefore, this study explored miRNA profiles in cancer cell-derived EVs after non-viral gene delivery in order to better understand the molecular information of intercellular communication in cancer cells after gene delivery. Two commonly used non-viral vectors (Lipofectamine 2000 and jet polyethylenimine) were used for the delivery of gene fluorescent protein plasmid in HeLa cancer cells. EVs were extracted and the contents of their RNA were subjected to the next-generation sequencing. In order to illustrate the common characteristics of non-viral vectors in the cancer cells, two overlapped up-regulated miRNAs (hsa-miR-143-3p and hsa-miR-193b-3p) were confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction in the secreted EVs in response to both lipoplexes and polyplexes. The prediction of target genes and molecular pathways involved in these two miRNAs were determined, and the protein expressions related to the pathways of cell death and stress in HeLa cells were identified. Hsa-miR-143-3p and hsa-miR-193b-3p were found to be up-regulated by the use of different non-viral vectors and can thus serve as potential targets of non-viral cancer gene therapy.

The vector-related influences of autophagic microRNA delivery by Lipofectamine 2000 and polyethylenimine 25K on mouse embryonic fibroblast cells.

Despite the greater potential for clinical applications of autophagic microRNA (miRNA) delivery, the vector-related effects of such delivery on cells have not been fully explored. In this study, autophagic mmu-miR-494-3p (miR-494) in mouse embryonic fibroblast (MEF) cells was selected as a cargo miRNA, and two commonly used non-viral carriers (Lipofectamine 2000 (Lipo) and polyethylenimine 25K (PEI)), were used as delivery vectors to mechanistically elucidate its vector-related effects. The cellular uptake, nuclear localization, and quantitative miR-494 levels of the complexes of miR-494 with Lipo (miR-494 lipoplexes) were lower than those of the complexes of miR-494 with PEI (miR-494 polyplexes) in MEF cells. The indicator of autophagic activity (LC3 (microtubule-associated protein 1 light chain 3)-II/LC3-I ratio) in cells treated with miR-494 lipoplexes was higher than that in cells treated with miR-494 polyplexes. Lipo alone and PEI alone induced slight increases in the quantitative levels of miR-494 in cells, but Lipo resulted in higher gene and protein expressions of target Igf1, higher LC3-II/LC3-I ratios, and higher autophagosome formation than PEI. We also demonstrated that the delivery of miR-494 by Lipo was more involved in apoptotic caspase-3 pathways than such delivery by PEI. By applying knock-out atg5 gene in MEF cells, we found that autophagy played a protective role in cell survival and also affected cellular uptake, the quantitative level of miR-494, and target gene Igf1 regulation of delivery systems. Taken together, these results indicate that there are different degrees of responses in MEF cells for autophagic miR-494 delivery through the use of Lipo or PEI vectors that also induce autophagy in cells. Therefore, Lipo and PEI vectors cannot be treated as inert molecules, and their effects must be known and evaluated when they are used in autophagic miRNA delivery systems. Most importantly, understanding these vector-related effects on cells will be helpful in achieving optimal delivery of autophagic miRNAs.

Induction of apoptosis in macrophages by air oxidation of dioleoylphosphatidylglycerol.

Dioleoylphosphatidylglycerol (DOPG) containing unsaturated sites is the target of oxidation during preparation, storage, or in vivo use of anionic liposomes. We investigated the biological effect of air oxidation of DOPG on RAW 264.7 murine macrophage-like cells. Oxidation was induced by exposing DOPG to air for 24-72 h. The extent of air oxidation was confirmed using Matrix-Assisted Laser Desorption and Ionization with Time-of-Flight (MALDI-TOF) mass spectrometry. The product of the air oxidation of DOPG was identified as the addition of one oxygen atom to one of the symmetrical fatty moieties of DOPG at m/z 814.77. The treatment of DOPG with air oxidation produced dose-dependent cytotoxicity in macrophages. RAW 264.7 cells exposed to oxidized DOPG exhibited morphological features of apoptosis, such as chromatin condensation and cell shrinkage. Typical apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with oxidized DOPG. Flow cytometric analysis demonstrated an increase in the hypodiploid DNA population (sub-G1), indicating that DNA cleavage occurred after treatment with oxidized DOPG. In addition, we showed that pretreating RAW 264.7 cells with zVAD-fmk, a general caspase inhibitor, did not prevent apoptosis induced by oxidized DOPG, suggesting that apoptosis in macrophage cells follows a caspase-independent pathway. These results point to a need for precaution in formulating DOPG liposomes for drug delivery and therapeutic purposes.

Mechanism of cell death induced by cationic dendrimers in RAW 264.7 murine macrophage-like cells.

Cationic dendrimers possess attractive nano-sized architectures that make them suitable as targeted drug/gene delivery systems. However, very little is known about their mechanisms of cell death in cellular systems. In the current study, the apoptotic and necrotic effects of starburst polyamidoamine(PAMAM) and polypropylenimine (DAB) dendrimers in cultured RAW 264.7 murine macrophage-like cells were investigated. Cationic dendrimer treatment produced a typically dose-dependent cytotoxic effect on macrophage cells. RAW 264.7 cells exposed to cationic dendrimers exhibited morphological features of apoptosis. Apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with cationic dendrimers. Analysis from flow cytometry demonstrated an increase in hypodiploid DNA population (sub-G1) and a simultaneous decrease in diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells to cationic dendrimers. Also, cells treated with DAB dendrimer induced a higher percentage of sub-G1 population than those treated with PAMAM dendrimer at the same dose. In addition, it was shown that pre-treatment of RAW 264.7 cells with the general caspase inhibitor zVAD-fmk prevented some degree of apoptosis induced by cationic dendrimers, suggesting that apoptosis in macrophage cells involves a caspase dependent pathway. Macrophage cells were also found to be sensitive to induction of apoptosis by dendrimers, whereas NIH/3T3 cells (mouse fibroblast) and BNL CL.2 (mouse liver) cells did not undergo apoptosis. These results could be helpful for optimizing the biocompatibility of dendrimers used for targeted drug/gene delivery.

Evaluation of the stability of polymer-based plasmid DNA delivery systems after ultrasound exposure.

Under ultrasound exposure, the stability of plasmid DNA protected by polymer-based gene delivery system is an important factor for achieving optimal transfection into cells. We have evaluated the effectiveness of various polymer-based plasmid DNA delivery systems, which are interactive polymers and cationic polymers, to avoid shear degradation induced by ultrasound exposure. Alternatively, it is shown that sonication of plasmid DNA for exposure time as low as 10s resulted in total DNA fragmentation and the loss of transfection potency in NIH/3T3 cells. Among these polymer-based plasmid DNA delivery systems, only cationic polymers had the ability to provide the protection of plasmid DNA from ultrasonic degradation as indicated by the reservation in supercoiled circular (SC) and open circular (OC) forms of plasmid DNA on the agarose gel electrophoresis. The DNA stability protected by cationic polymers decreased after ultrasound exposure in 1M sodium chloride solution. Also, higher molecular weight of cationic polymers and sufficient cationic polymer/DNA weight ratios are essential to prevent DNA from degradation under ultrasound exposure in aqueous or salt solution. These results suggest that the protective mechanism by cationic polymers is due to the attractive bonding between cationic polymer and negative plasmid DNA. Whereas, DNA condensation alone provoked by the addition of polyethylene glycols was not sufficient to resist the DNA fragmentation induced by ultrasound exposure.

Protective role of autophagy in branched polyethylenimine (25K)- and poly(L-lysine) (30-70K)-induced cell death.

Polyethylenimine (PEI) and poly(L-lysine) (PLL), which are cationic polymers used for gene therapy, are known to be cytotoxic, but their molecular mechanisms of cell death are not fully understood. In this study, we provide evidence that PEI and PLL induced autophagy in HeLa cervical cancer cells. In cells overexpressed with green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) fusion protein, PEI and PLL induced fluorescent puncta formations that represent LC3 recruitment to autophagosomes. In Western blot analysis, conversions of the LC3-I to LC3-II were significant, and p62 degradation was observed in cells treated with PEI and PLL. At higher doses, the ability of endosomal escape by PEI facilitates the conversion of LC3-I to LC3-II without the use of lysosomal protease inhibitors. From the analysis of annexin V-flourescein isothiocyanate (FITC) and propidium iodide (PI) staining by flow cytometry, both apoptosis and necrosis occurred in PEI- and PLL-treated cells. Significant activated caspase-3 expression was detected in PLL- and PEI-treated cells. By applying Z-VAD apoptotic inhibition, apoptosis and autophagy may occur independently or autophagy may be in the upstream of apoptosis on PEI- and PLL-treated cells. The degree of cell death was higher in incubated HeLa cells treated with PEI or PLL plus autophagy inhibitors (3-methyladenine (3-MA) and wortmannin). Treatment with these autophagy inhibitors, however, did not inhibit LC3-II formation specifically. In addition, PEI and PLL induced higher degree of cell death in atg5(-/-) mouse embryonic fibroblast (MEF) cells than in wild-type cells. Autophagy was also induced in PEI- and PLL-treated MEFs, as evidenced by the formation of LC3-II in wild-type-but not in atg5(-/-) MEFs. These results indicate that PEI and PLL can trigger both death and survival pathways simultaneously, and autophagy played a role in cell survival in PEI- and PLL-treated cells. Our study therefore provides deeper insight into the molecular mechanisms of cell death caused by cationic polymers.

Interactions between U-937 human macrophages and poly(propyleneimine) dendrimers.

Interest in using poly(propyleneimine) (PPI) dendrimers for biomedical applications is increasing. Before using PPI dendrimers in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. However, few investigators have studied in detail the intracellular responses that cationic dendrimers induce in macrophages. Here we examined the intracellular responses-reactive oxygen species (ROS) content, mitochondria membrane potential, cell size and complexity, and cell cycle profiles-in U-937 human macrophages treated with poly(propyleneimine) dendrimers generation 2 (DAB 2.0) and 3 (DAB 3.0). Our study focused on the concentration ranges within which cell viability was greater than 90% after PPI dendrimers had been incubated for 16 h. For spontaneous ROS generation, DAB 2.0 did not consistently generate hydrogen peroxide production with increasing dosages over the entire culture period while it was capable of generating superoxide content except during the 12 h of incubation. In contrast, DAB 3.0 did not induce any hydrogen peroxide and superoxide production except for an abrupt increase of superoxide content at 60 microg/mL after 6 h of incubation. Our results showed that ROS responses in macrophages were strongly influenced by the nature of the dendrimer surface. Except at 3 h, DAB 2.0 increased mitochondrial membrane potential for every dose and culture period. In contrast, DAB 3.0 caused a significant fluctuation in mitochondrial membrane potential only at 6 h, compared with other incubation times. Exposing macrophages to PPI dendrimers caused dramatic and significant changes in macrophage cell size and complexity, and DAB 3.0 caused greater changes than DAB 2.0 did. For incubation times longer than 1 h, propidium iodide staining showed that cells treated with DAB 2.0 and 3.0 had a higher subG1 phase (indicative of apoptosis) than did untreated cells. PPI dendrimers induced different activated patterns in ROS generation and changes of mitochondrial membrane potential than did other carriers such as cationic liposomes and polyalkylcyanoacrylate. The nature of interactions between macrophages and PPI dendrimers is crucial for the design of safer and more effective delivery systems for macrophages. Our findings provide a novel insight into the cytotoxic effects at the molecular level that dendrimers cause in macrophages.

Cytotoxic properties of tyloxapol.

PURPOSE: Tyloxapol, a viscous polymer of the alkyl aryl polyether alcohol type, is classified as a nonionic surfactant and is widely used in biomedical applications. Although tyloxapol has been reported to be cytotoxic in various cell lines, there is no published information about its possible mechanisms of cell death. Hence, the objective of this study was to determine whether tyloxapol causes apoptosis or necrosis. These data could be helpful for a better understanding of the action of tyloxapol in cellular systems. METHODS: RAW 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated with tyloxapol, and the activity of dehydrogenases in those cells, an indicator of cell viability, was assessed. The cell morphology changes induced by tyloxapol treatment were detected using propidium iodide nuclear staining. The hallmarks of apoptotic cells were characterized using DNA fragmentation assays, DNA fluorescence staining, and then flow analysis. RESULTS: Tyloxapol treatment produced dose- and time-dependent cytotoxicity. Tyloxapol treatment damaged RAW 264.7 cells more than it damaged NIH/3T3 cells. All the cells exposed to tyloxapol showed some morphological features of apoptosis, such as chromatin condensation and cell shrinkage. Typical apoptotic ladders were observed in DNA extracted from tyloxapol-treated cells. Flow cytometric analysis revealed an increase in the hypodiploid DNA population (sub-G1), indicating that DNA cleavage occurred after tyloxapol treatment. In addition, we showed that pretreating cells with zVAD-fmk, a general caspase inhibitor, did not prevent tyloxapol-induced apoptosis. The cytotoxicity of tyloxapol can be reduced by adding a nontoxic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine to attenuate the interaction of tyloxapol with the cell membrane. CONCLUSIONS: Our results indicate that tyloxapol induces apoptosis in RAW 264.7 and NIH/3T3 cells. These data provide a novel insight into the cytotoxic action of tyloxapol at the molecular level.