Denise Carleto Andia

Interleukin-8 Gene Promoter Polymorphism (rs4073) May Contribute to Chronic Periodontitis

BACKGROUND The proinflammatory chemokine interleukin (IL)-8 is important in the regulation of the inflammatory response. Analyses of the single nucleotide polymorphism (SNP) reference sequence (rs) 4073 showed that the A allele upregulated IL-8 levels after stimulation with lipopolysaccharides. We investigated the association of the SNP rs4073 with chronic periodontitis. METHODS Genotyping was performed by a standard polymerase chain reaction-restriction fragment length polymorphism assay in 289 genomic DNA samples of healthy control subjects and patients with chronic periodontitis; analyses were adjusted by multivariate logistic regression modeling. A real-time polymerase chain reaction performance was used to detect levels of the IL-8 mRNA. RESULTS The analysis pointed to a statistically significant association of chronic periodontitis with the heterozygous TA genotype (P = 0.001); the results showed an increase in the frequency of the A allele in the diseased group (36% in the control group versus 48% in the periodontitis group). The higher levels of the IL-8 mRNA were found in the periodontitis group, mainly in individuals who presented the TA genotype (P = 0.03). CONCLUSION The SNP rs4073 was associated with chronic periodontitis in non-smoker Brazilian subjects because the frequency of the A allele was higher in the disease group than in the control group, and the TA genotype was associated with increased levels of IL-8 mRNA transcripts.

DNA Methylation Status of the IL8 Gene Promoter in Aggressive Periodontitis

BACKGROUND Studies evaluating the methylation status of cytokine genes may have relevance for inflammatory diseases in which the expression of some cytokines is altered, such as periodontitis. This study observes the DNA methylation status in the interleukin-8 (IL8) gene promoter in cells of the oral epithelium of subjects affected by generalized aggressive periodontitis (AgP) and compares it to those of control subjects. METHODS Genomic DNA from epithelial oral cells of 37 generalized AgP patients and 37 controls were purified and modified by sodium bisulphite. Modified DNA was submitted by methylation-specific polymerase chain reaction, electrophoresed on 10% polyacrylamide gels, and stained. RESULTS Subjects who presented generalized AgP have a higher frequency of hypomethylation of the IL8 gene promoter in oral epithelium cells than that of controls (86.5% in the generalized AgP group versus 62% in the control group; P = 0.016; chi(2) test). CONCLUSIONS A marked hypomethylated status is found in the oral epithelial cells of subjects presenting with generalized AgP, compared to controls, in the promoter region of the IL8 gene. This hypomethylated status may reflect a generalized condition of oral epithelial cells, including gingival epithelium, because gingival epithelial cells were also collected during mouthwash use.

TLR2 and TLR4 gene promoter methylation status during chronic periodontitis

AIM The objective of this study was to analyse the status of DNA methylation in the promoter region of the toll-like receptor (TLR)2 and TLR4 genes in gingival tissue samples from healthy subjects, smokers and non-smokers affected by chronic periodontitis. MATERIAL AND METHODS Genomic DNA and total RNA were purified from gingival tissue using the TRIZOL reagent protocol. Genomic DNA was then digested by methylation-sensitive restriction enzymes, amplified by polymerase chain reaction (PCR), electrophoresed on a 10% polyacrylamide gel and stained using SYBR Gold. Real-time PCR was also performed to verify the transcript levels. RESULTS The CpG dinucleotides analysed were observed to be unmethylated in the majority of DNA samples of the three groups and statistical differences were not found among groups (p>0.05). However, a trend towards methylation was observed in the TLR2 HhaI site in the samples of the periodontitis non-smoker groups. In fact, the analysis of all CpG sites together shows which complete methylation is observed in the shortest level in the samples of periodontitis non-smoker group. The analysis of transcript levels demonstrated no difference among groups (p>0.05). CONCLUSION The results demonstrated major unmethylation of the TLR4 gene promoter in all groups. However, the results for the TLR2 gene promoter are inconclusive; this gene was found as a mosaic of methylated and unmethylated DNA in the majority of samples of the three groups and we also observed a trend towards the DNA methylation of CpG sites recognized by the HhaI enzyme.

Genetic analysis of the IL8 gene polymorphism (rs4073) in generalized aggressive periodontitis

OBJECTIVES Interleukin (IL)-8 is an important chemokine for regulation of the inflammatory response. A single nucleotide polymorphism (SNP) reference sequence (rs) 4073 in the IL8 gene has been shown to regulate IL-8 levels after stimulation with lipopolysaccharide. This study investigates the transmission pattern of the IL8 rs4073 risk allele A and its association with susceptibility to aggressive periodontitis (AgP) in families and in a case-control cohort of unrelated individuals from a Brazilian population. DESIGN Genotyping was performed by standard polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) in 13 nuclear families and 184 unrelated subjects. Statistical analysis was performed using the transmission disequilibrium test (TDT) for the family dataset and Chi-square test and multivariate logistic regression modelling for the case-control dataset. RESULTS TDT analyses did not detect evidence of over transmission of IL8 rs4073 alleles in affected and unaffected family members (allele T: 52%; allele A: 48%; p=0.2252). How expected, analyses of cases and unrelated controls showed a significant and inverse association of age with AgP; however, a lack of association between genotypes, ethnic groups and generalized AgP was observed. CONCLUSIONS The SNP (rs4073) was not associated with AgP in unrelated individuals and there is no evidence of over transmission of the alleles in families with AgP, from Brazilian individuals.

Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways

Although, intracellular signaling pathways are proposed to predict the quality of cell‐surface relationship, this study addressed pre‐osteoblast behavior in response to nano hydroxyapatite (HA)‐blasted titanium (Ti) surface by exploring critical intracellular pathways and pre‐osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy (AFM) and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid‐etching (DAE), and nano hydroxyapatite‐blasted (nHA). The results revealed critical differences in surface topography, impacting the water contact angle and later the osteoblast performance. In order to evaluate the effect of those topographical characteristics on biological responses, we have seeded pre‐osteoblast cells on the Ti discs for up to 4 h and subjected the cultures to biological analysis. First, we have observed pre‐osteoblasts morphological changes resulting from the interaction with the Ti texturized surfaces whereas the cells cultured on nHA presented a more advanced spreading process when compared with the cells cultured on the other surfaces. These results argued us for analyzing the molecular machinery and thus, we have shown that nHA promoted a lower Bax/Bcl2 ratio, suggesting an interesting anti‐apoptotic effect, maybe explained by the fact that HA is a natural element present in bone composition. Thereafter, we investigated the potential effect of those surfaces on promoting pre‐osteoblast adhesion and survival signaling by performing crystal violet and immunoblotting approaches, respectively. Our results showed that nHA promoted a higher pre‐osteoblast adhesion supported by up‐modulating FAK and Src activations, both signaling transducers involved during eukaryotic cell adhesion. Also, we have shown Ras‐Erk stimulation by the all evaluated surfaces. Finally, we showed that all Ti‐texturing surfaces were able to promote osteoblast differentiation up to 10 days, when alkaline phosphatase (ALP) activity and osteogenic transcription factors were up‐modulated. Altogether, our results showed for the first time that nano hydroxyapatite‐blasted titanium surface promotes crucial intracellular signaling network responsible for cell adapting on the Ti‐surface.Biotechnol. Bioeng. 2017;114: 1888–1898.

DNA methylation levels of SOCS1 and LINE-1 in oral epithelial cells from aggressive periodontitis patients

OBJECTIVE DNA methylation has been shown to be critical in the regulation of inflammatory genes. Infections are able to trigger susceptibility to disease and it can be considered as potential epimutagenic factors in reshaping the epigenome. Therefore, what would be the DNA methylation status in cells present in an infected and inflamed oral environment? The aim was to verify the DNA methylation pattern in oral epithelium cells from aggressive periodontitis (AgP) patients in a specific gene involved in the inflammation control, as suppressor of cytokine signalling (SOCS)1 and in a broader way through long interspersed nuclear element (LINE)-1. DESIGN Genomic DNA from oral cells of 30 generalized AgP patients and 30 healthy patients were purified and modified by sodium bisulfite. DNA methylation patterns were analyzed using combined bisulfite restriction analysis (COBRA) for SOCS1 and LINE-1. RESULTS An overall scenario of demethylation was seen for both groups, whereas the healthy group presented a higher percentage of demethylation (p<0.001), also presenting the majority of total demethylated samples (83.3% versus 70.8% in the AgP group). Total LINE-1 methylation or at each specific loci presented significant differences amongst groups. CONCLUSION Epithelial cells, present in an infected and inflamed oral environment, show different DNA methylation status from those present in a healthy oral environment, regarding the SOCS1 and LINE-1. In addition, the investigation allows detecting alterations in the DNA in a non-limited manner, since the results observed might reflect a generalized condition of the oral epithelial cells, besides reflecting the condition of the gingival epithelium cells.

TIMP2 gene polymorphism as a potential tool to infer Brazilian population origin

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Advances in Genomics and Genetics 2013:3 11–15 Advances in Genomics and Genetics Dovepress

The role of triiodothyronine hormone and mechanically-stressed endothelial cell paracrine signalling synergism in gene reprogramming during hBMSC-stimulated osteogenic phenotype in vitro

We therefore investigated whether there is synergism between triiodothyronine (T3) hormone and trophic molecules released from mechanically-stressed endothelial cells (EC-enriched medium) in osteogenic phenotype by mapping classical repertory of genes. Although there are studies reporting the efficiency of T3 hormone on bone cells, it is scarce considering their effect in conjunction with other physiologically active molecules, such as those released by the active endothelial cells. To address this issue, human bone marrow-derived mesenchymal stem cells (hBMSCs) were treated with EC-enriched medium subjected to shear-stress up to 72 h in vitro, in conjunction or not with T3 hormone. Although our results found an important synergism considering these parameters on modulating key bone-related gene markers, such as on the alkaline phosphatase (ALP) behavior (at both mRNA and protein content), contributing for osteoblast differentiation, important genes such as OSTERIX and RUNX2 were significantly down-expressed, while a over-expression of RANKL was found when the conjunction effect of T3 and endothelial paracrine signaling was considered. In addition, T3 hormone over expressed both OCT4 and NANOG genes in a DNA epigenetic-independent manner. However, we observed a dynamic reprogramming of DNMT1, DNMT3A, DNMT3B and TET1, important DNA-related epigenetic markers. Specifically, T3 hormone alone up-modulated TET2 transcripts profile. Complimentarily, expression of microRNA (miRs) processing-related genes also was modulated, as well as miR-10b, miR-22, miR-21, miR-143 and miR-145 transcriptional related profiles. Altogether, our results suggested a positive effect of mechanically-stressed endothelial cells-induced paracrine signaling on T3 hormone-obtaining osteogenic phenotype, contributing to understanding the paradoxal effect of T3 hormone on the bone physiology.

Laminar shear stress-provoked cytoskeletal changes are mediated by epigenetic reprogramming of TIMP1 in human primary smooth muscle cells: DA SILVA et al.

Whereas endothelial responses to shear stress are well‐characterized, the cell physiological effects of shear stress in smooth muscle cells (SMCs) remain largely obscure. As SMCs are directly challenged by shear stress after endothelial denuding injury following procedures such as angioplasty or endarterectomy, characterization of these responses represents an important scientific question. Hence we decided to contrast cytoskeletal reorganization, epigenetic reprogramming, signaling transduction, and changes in miRNA (miRs) profiles in primary human aortic smooth muscle cells (AoSMCs) between unstressed cells and cells exposed to shear stress. We observed that shear stress‐provoked reorganization of the actin cytoskeleton in an apparently Cofilin‐dependent fashion and which related to altered integrin signaling, apparently caused by remodeling of the extracellular matrix. The latter appeared a downstream effect of increased expression of matrix metalloproteinases and downregulation of tissue metalloproteinase inhibitor 1 (TIMP1) protein levels. In turn, these effects related to shear stress‐provoked changes in expression and nuclear localization of the epigenetic regulators demethylases TET1, TET2, DNMT1, DNMT3A and DNMT3B, HDAC6, and SIRT1. Accordingly, TIMP1 promotor CpG hypomethylation was a prominent effect, and resulted in a significant increase in TIMP1 transcription, which may also have related increased expression of miRs involved in modulating TIMP1 translation. Thus epigenetic‐reprogramming of TIMP1 emerges as critical element in smooth muscle responses to mechanical signals and as epigenetic machinery is amendable to pharmacological manipulation, this pathway may have important clinical consequences.

Resveratrol Reverts Epigenetic and Transcription Changes Caused by Smoke Inhalation on Bone-Related Genes in Rats

We investigated the effects of cigarette smoke (CS) and resveratrol intake on the modulation of bone repair-related genes through epigenetic mechanisms at the global and gene-specific levels, after 30 days of calvarial defects were created, in rats. The samples were assigned to three groups as follows: no CS, CS, and CS/resveratrol. After evaluation of global (5 hmC) changes and epigenetic and transcription regulation at gene-specific levels, CS group showed increased 5 hmC and Tets transcripts with demethylation at Rankl and Trap promoters (p ≤ 0.01), linked to their increased gene expression (p ≤ 0.001). These modifications were reverted in the CS/resveratrol group. Opposite patterns were observed among CS and CS/resveratrol for epigenetic enzyme transcripts with higher levels of Dnmts in the CS/resveratrol (p ≤ 0.01). No CS and CS/resveratrol demonstrated similar gene expression levels for all Tets and bone-related genes. Resveratrol reverts epigenetic and transcription changes caused by CS at both global and gene-specific levels in bone-related and epigenetic machinery genes, emphasizing the resveratrol as biological modulator for CS in rats.