Insulin-like growth factor 1 and transforming growth factor-β stimulate cystine/glutamate exchange activity in dental pulp cells

Introduction

The growth factors insulin-like growth factor (IGF-1) and transforming growth factor-β (TGF-β) are protective to dental pulp cells in culture against the toxicity of the composite materials Durafill VS and Flow Line (Henry Schein Inc, New York, NY). Because the toxicity of these materials is mediated by oxidative stress, it seemed possible that the protective effects of IGF-1 and TGF-β were through the enhancement of an endogenous antioxidant mechanism.

Methods

We used cultured dental pulp cells to determine the mechanism of the protective effects of IGF-1 and TGF-β, focusing on the glutathione system and the role of cystine/glutamate exchange (system xc-).

Results

We found that the toxicity of Durafill VS and Flow Line was attenuated by the addition of glutathione monoethylester, suggesting a specific role for the cellular antioxidant glutathione. Supporting this hypothesis, we found that IGF-1 and TGF-β were protective against the toxicity of the glutathione synthesis inhibitor buthionine sulfoximine. Because levels of cellular cystine are the limiting factor in the production of glutathione, we tested the effects of IGF-1 and TGF-β on cystine uptake. Both growth factors stimulated system xc-mediated cystine uptake. Furthermore, they attenuated the glutathione depletion induced by Durafill VS and Flow Line.

Conclusions

The results suggest that IGF-1 and TGF-β are protective through the stimulation of system xc-mediated cystine uptake, leading to maintenance of cellular glutathione. This novel action of growth factors on dental pulp cells has implications not only for preventing toxicity of dental materials but also for the general function of these cells.     

Evaluation of milk fat globule-epidermal growth factor–factor VIII and IL-1β levels in gingival crevicular fluid and saliva in periodontal disease and health

Odontology - The aim of this study is to determine the levels of MFG-E8 and interleukin (IL)-1β in saliva and gingival crevicular fluid (GCF) associated with periodontal health and disease....     

Smad7 blocks transforming growth factor-β1-induced gingival fibroblast-myofibroblast transition via inhibitory regulation of Smad2 and connective tissue growth factor

Background: Transforming growth factor‐β1 (TGF‐β1), its downstream signaling mediators (Smad proteins), and specific targets, including connective tissue growth factor (CTGF), play important roles in tissue remodeling and fibrosis via myofibroblast activation. We investigated the effect of overexpression of Smad7, a TGF‐β1 signaling inhibitor, on transition of gingival fibroblast to myofibroblast. Moreover, we analyzed the participation of CTGF on TGF‐β1–mediated myofibroblast transformation. Methods: To study the inhibitory effect of Smad7 on TGF‐β1/CTGF‐mediating gingival fibroblast transition into myofibroblasts, we stably overexpressed Smad7 in normal gingival fibroblasts and in myofibroblasts from hereditary gingival fibromatosis (HGF). Myofibroblasts were characterized by the expression of the specific marker isoform α of the smooth muscle actin (α‐SMA) by Western blot, flow cytometry, and immunofluorescence. Enzyme‐linked immunosorbent assay for type I collagen was performed to measure myofibroblast activity. CTGF's role on myofibroblast transformation was examined by enzyme‐linked immunosorbent assay and small interference RNA. Results: TGF‐β1 induced the expression of α‐SMA and CTGF, and small interference RNA–mediating CTGF silencing prevented fibroblast‐myofibroblast switch induced by TGF‐β1. In Smad7‐overexpressing fibroblasts, ablation of TGF‐β1–induced Smad2 phosphorylation marked decreased α‐SMA, CTGF, and type I collagen expression. Similarly, HGF transfectants overexpressing Smad7 demonstrated low levels of α‐SMA and phospho‐Smad2 and significant reduction on CTGF and type I collagen production. Conclusions: CTGF is critical for TGF‐β1–induced gingival fibroblast‐myofibroblast transition, and Smad7 overexpression is effective in the blockage of myofibroblast transformation and activation, suggesting that treatments targeting myofibroblasts by Smad7 overexpression may be clinically effective in gingival fibrotic diseases, such as HGF.     

Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor β

Rawal SY, Dabbous MKh, Tipton DA. Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor β. J Periodont Res 2012; 47: 320–329. © 2011 John Wiley & Sons A/S Background and Objective: Marijuana (Cannabis sativa) use may be associated with gingival enlargement, resembling that caused by phenytoin. Cannabidiol (CBD), a nonpsychotropic Cannabis derivative, is structurally similar to phenytoin. While there are many reports on effects of phenytoin on human gingival fibroblasts, there is no information on effects of Cannabis components on these cells. The objective of this study was to determine effects of CBD on human gingival fibroblast fibrogenic and matrix‐degrading activities. Material and Methods: Fibroblasts were incubated with CBD in serum‐free medium for 1–6 d. The effect of CBD on cell viability was determined by measuring activity of a mitochondrial enzyme. The fibrogenic molecule transforming growth factor β and the extracellular matrix molecule fibronectin were measured by ELISA. Pro‐MMP‐1 and total MMP‐2 were measured by ELISA. Activity of MMP‐2 was determined via a colorimetric assay in which a detection enzyme is activated by active MMP‐2. Data were analysed using ANOVA and Scheffe’s F procedure for post hoc comparisons. Results: Cannabidiol had little or no significant effect on cell viability. Low CBD concentrations increased transforming growth factor β production by as much as 40% (p < 0.001), while higher concentrations decreased it by as much as 40% (p < 0.0001). Cannabidiol increased fibronectin production by as much as approximately 100% (p < 0.001). Lower CBD concentrations increased MMP production, but the highest concentrations decreased production of both MMPs (p < 0.05) and decreased MMP‐2 activity (p < 0.02). Conclusion: The data suggest that the CBD may promote fibrotic gingival enlargement by increasing gingival fibroblast production of transforming growth factor β and fibronectin, while decreasing MMP production and activity.     

Role of vascular endothelial growth factor in reconstructive surgery after surgical excision of malignant tumor

As a key mediator of normal physiological angiogenesis, vascular endothelial growth factor(VEGF) has been regarded as an emancipator to plastic surgeon, and yet a misfortune to oncology surgeon, due to its singular biological effect. Therefore in some clinical cases, especially for some malignant tumor patients having endured radical surgery and being craving for a reconstructive surgery, VEGF plays a role full of paradoxes. To make a clinical balance, we should find a point to inhibit tumor cell from utilizing VEGF and make a permission to normal tissues to employ it.     

Age-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in osteopetrotic (op/op) mice

ObjectiveExpression of osteoclasts in osteopetrotic (op/op) mice is substantially reduced by the absence of functional macrophage colony-stimulating factor (M-CSF). However, it has been reported that osteoclasts do gradually appear in the bones of op/op mice and spontaneously correct the osteopetrosis.DesignAge-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in op/op mice were examined.ResultsThe number of femoral osteoclasts, and the serum levels of VEGF, both gradually increased in op/op mice after birth and reached a peak in 120- and 60-day-old mice, respectively. However, the serum levels of RANKL showed an inverse relationship to osteoclast number.ConclusionsThese findings suggest that the appearance of osteoclasts may be influenced by the serum levels of VEGF and that the serum levels of RANKL may be influenced by the appearance of osteoclasts.     

Plasma rich in growth factors promote gingival tissue regeneration by stimulating fibroblast proliferation and migration and by blocking transforming growth factor-β1-induced myodifferentiation

Background: Periodontitis involves inflammation and infection of the ligaments and bones that support the teeth. Gingival fibroblasts are the most abundant cells in periodontal tissue, and they play a role in maintaining the structural integrity of the tissue. Plasma rich in growth factors contain a pool of proteins and growth factors that promote wound healing and tissue regeneration. In the present study, we evaluate the potential of different formulations obtained with this approach to stimulate several biologic processes involved in wound healing, including fibroblast proliferation, migration, adhesion, and the autocrine release of some angiogenic factors and extracellular matrix components. Furthermore, the ability of this technology to prevent and inhibit transforming growth factor β1‐induced myodifferentiation was also determined. Methods: Cell proliferation was evaluated through a colorimetric assay, cell migration was performed on culture inserts, and cell adhesion was studied through a fluorescence‐based method. Enzyme‐linked immunosorbent assay was used to determine some of the biomolecules released by gingival fibroblasts. Smooth muscle actin expression was assessed through immunofluorescence microscopy. Results: Results showed that plasma rich in growth factors significantly increased gingival fibroblast proliferation, migration, and cell adhesion on type I collagen matrix. In addition, it stimulated the autocrine expression of vascular endothelial growth factor, hepatocyte growth factor, and hyaluronic acid. The myofibroblast phenotype, which is characterized by expressing α‐smooth muscle actin, was inhibited and reverted by treating with this technology. Conclusion: These findings suggest that plasma rich in growth factors is capable of promoting regeneration of gingival connective tissue by stimulating some of the main processes involved in wound regeneration.     

Detection of mutation-specific epidermal growth factor receptor (E746–A750del) and lack of detection of human papillomavirus in oral squamous cell carcinoma

The clinical impact of epidermal growth factor receptor (EGFR) (E746–A750del) mutation and human papillomavirus (HPV) in oral squamous cell carcinoma (OSCC) is unclear. EGFR (E746–A750del) expression was analyzed in OSCC specimens (n = 161) by immunohistochemistry. The expression results were correlated with clinical characteristics and impact on survival. Using INNO-LiPA Extra, high-risk HPV types were genotyped and analyzed in 211 OSCC specimens. Positive EGFR (E746–A750del) expression (n = 40/161, 25%) was not associated with any clinicopathological characteristics, prognostic factors, social habits (smoking, alcohol consumption), or tumour-specific survival. HPV16 DNA was detected in three out of 211 samples (HPV16-positive: n = 3/211, 1.4%). This study shows that mutation-specific EGFR (E746–A750del) expression and HPV do not appear to be relevant to the survival of patients with OSCC.     

Genetic polymorphism of tumor necrosis factor alpha (TNF-α) and tumor necrosis factor beta (TNF-β) genes and risk of oral pre-cancer and cancer in North Indian population

Objective

There are inconclusive data connecting single-nucleotide polymorphisms (SNPs) of TNF-α (rs361525) and TNF-β (rs909253) to potential malignant oral disorder (PMOD) such as lichen planus and oral fibrosis. Here, we have investigated the risk of oral squamous cell carcinoma as well as oral pre-cancerous lesions in North Indian population with the polymorphism of the TNFα/ β genes.

Material and methods

A total 500 patients with oral pre-cancer and OSCC and 500 healthy volunteers were genotypes for the TNF-α (-238) G/A (rs361525) and TNF-β (252) A/G (rs909253) gene polymorphism. Genotypes were identified by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). Genotype frequencies were evaluated by Chi-square test.

Results

Compared to the GG genotype, the GA genotype of TNF-α (G238A) polymorphism (rs361525) has been found to significantly increase the risk of oral disease (OR = 1.99) and especially the risk of lichen planus and OSCC (OR = 2.805 and 5.790, respectively). Similarly, the risk of oral disease was also more in the heterozygote (AG) than the common allele homozygote (AA) of TNF-β (A252G) polymorphism (rs909253) (OR = 1.483).

Conclusion

We conclude that the SNPs rs361525 and rs909253 were significantly associated with oral pre-cancer and OSCC.

     

Are intrauterine growth restriction and preterm birth associated with dental caries?

OBJECTIVES: To assess the association between two intrauterine growth restriction (IUGR) surrogates - IUGR [small for gestational age birth (SGA) and fetal growth restriction (FGR)] and preterm birth with dental caries. METHODS: Data from the Third National Health and Nutritional Examination Survey (1988-1994) were used, including 2- to 5.9-year-old singletons (n = 3189). Dental caries was defined as presence of any teeth with dental caries (treated or untreated) and also as presence of at least two teeth with dental caries. Exposure variables were preterm birth (<37 gestational weeks), FGR, and SGA. Covariates included were poverty, race/ethnicity, age, sex, sucrose intake, environmental tobacco smoking, dental visits, education of head of household, breastfeeding, and use of baby bottle. Separate statistical analyses were conducted for IUGR and for preterm birth through the estimation of prevalence ratio (PR), taking complex sampling design into consideration and adjusting for confounders. Sensitivity analysis was conducted including and excluding 2-year-old children and also with the two definitions of dental caries. RESULTS: In general, the inclusion of 2-year-old children and the case definition of presence of any teeth with dental caries biased the results toward the null, but with no major changes in the results. In bivariate analysis, SGA and FGR birth were both negatively but not significantly associated with dental caries while a significant positive association was found for preterm birth. Sensitivity analysis showed that the PR for preterm in bivariate analysis varied from 1.65 (95% CI 1.14-2.40) to 1.84 (95% CI 1.19-2.83). After adjusting for confounders, the PR for preterm birth varied from 1.38 (95% CI 1.00-1.89) to 1.64 (95% CI 1.22-2.20). After adjustment, the PR for SGA varied from 0.79 (95% CI 0.56-101) to 0.66 (95% CI 0.33-0.96). For children from 3 to 5.9 years old, the adjusted PR for FGR using the category 'none' as reference were mild (PR 1.10; 95% CI 0.76-1.58), moderate (PR 0.66; 95% CI 0.26-167), and severe (PR 0.59; 95% CI 0.36-0.99). These values for FGR were very similar for the other models using other classifications of case definition or inclusion of 2-year-old children. CONCLUSIONS: Preterm birth was found to be positively associated with dental caries while there is an indication that SGA and FGR are negatively associated with dental caries. Although the negative association is counterintuitive, it is possible that increased antibiotic use and delayed tooth eruption may explain the negative association between IUGR and dental caries.     

Human papilloma virus: An etiological and prognostic factor for oral cancer?

The increasing prevalence of human papilloma virus (HPV)‐positive oral tumors can be considered an epidemic. Although the incidence of HPV cervical cancer is decreasing, the incidence of oral cavity and oropharyngeal cancers associated with HPV is increasing. The presence of certain HPV genotypes could be a predictor of future oral cancer lesions, although lesions associated with HPV could be less aggressive and exhibit a higher survival rate. In the present study, we review the most important biologic, clinic, epidemiologic, and prognostic factors associated with HPV infection and oral cancer.     

Mechanism of long-chain noncoding RNA PCGEM1 in the regulation of the invasion and metastasis of oral squamous carcinoma cells via transforming growth factor β2/Smad2 signaling pathway

目的 探讨长链非编码RNA（lncRNA）PCGEM1通过转化生长因子β2（TGF β2）/Smad2信号通路调控口腔鳞状细胞癌（OSCC）侵袭和转移的机制。方法 将60例OSCC患者癌组织及距离癌组织超过2 cm处的正常组织纳入研究,实时定量聚合酶链反应（qRT-PCR）检测miR-148a、lncRNA PCGEM1在OSCC组织及正常组织、人正常口腔黏膜上皮细胞（OMEC）及人源OSCC细胞株KB、BcaCD885、SCC-4、CAL27、SCC-15中的表达情况;分析lncRNA PCGEM1和miR-148a表达与患者临床病理信息之间的关系。构建lncRNA PCGEM1沉默细胞系KB-siPCGEM1及阴性对照（KB-NC）,并以KB作为空白对照组,采用MTT、Transwell和划痕实验检测lncRNA PCGEM1对KB细胞增殖、侵袭和迁移能力的影响;使用生物信息学网站starBase预测lncRNA PCGEM1可以互补结合的微小RNA（miRNA）,再根据www.microRNA.org网站预测相应miRNA可靶向结合的基因;免疫印迹（Western blotting）检测TGF β2/Smad2信号通路蛋白表达情况。结果 qRT-PCR结果显示,OSCC组织中lncRNA PCGEM1、miR-148a的表达水平高于正常组织（P<0.05）;lncRNA PCGEM1和miR-148a在不同TNM分期、淋巴结转移和组织分化程度的患者癌组织中表达差异均有统计学意义（P<0.05）;与空白对照组和KB-NC组相比,KB-siPCGEM1组细胞OD_{492 nm}值下降,细胞侵袭数量及迁移率降低,差异均具有统计学意义（P<0.05）;生物信息学预测结果显示,lncRNA PCGEM1可与miR-148a互补结合,miR-148a与TGFβ2存在靶向结合位点;qRT-PCR和Western blotting检测结果显示,KB-siPCGEM1组中miR148a、TGFβ2及p-Smad 2的表达明显低于空白对照组与KB-NC组（P<0.05）,空白对照组和KB-NC组的差异无统计学意义（P>0.05）。结论 lncRNA PCGEM1在OSCC中高表达,高表达lncRNA PCGEM1可能通过上调miR-148a水平,强化TGF β2/Smad2信号通路,从而促进OSCC的进展。