Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E(2) by hypoxia-inducible factor-1α up-regulation in human periodontal ligament cells

Kim Y‐S, Shin S‐I, Kang K‐L, Herr Y, Bae W‐J, Kim E‐C. Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E₂ by hypoxia‐inducible factor‐1α up‐regulation in human periodontal ligament cells. J Periodont Res 2012; 47: 719–728. © 2012 John Wiley & Sons A/S Background and Objective: Although hypoxia‐inducible factor 1α (HIF‐1α) is up‐regulated in the periodontal pockets of periodontitis patients, the expression and precise molecular mechanisms of HIF‐1α remain unknown in human periodontal ligament cells (PDLCs). The aim of this study was to explore the effects, as well as the signaling pathway, of nicotine and lipopolysaccharide (LPS) on the expression of HIF‐1α and on the production of its target genes, including cyclooxygenase‐2 (COX‐2)‐derived prostaglandin E₂ (PGE₂), MMP‐2 and MMP‐9 in PDLCs. Material and Methods: The expression of COX‐2 and HIF‐1α proteins was evaluated using western blotting. The production of PGE₂ and MMPs was evaluated using enzyme immunoassays and zymography, respectively. Results: LPS and nicotine synergistically induced the production of PGE₂, MMP‐2 and MMP‐9, and increased the expression of MMP‐2, MMP‐9, COX‐2 and HIF‐1α proteins. Inhibition of HIF‐1α activity by chetomin or knockdown of HIF1α gene expression by small interfering RNA markedly attenuated the production of LPS‐ and nicotine‐stimulated PGE₂ and MMPs, as well as the expression of COX‐2 and HIF‐1α. Furthermore, pretreatment with inhibitors of COX‐2, p38, extracellular signal‐regulated kinase, Jun N‐terminal kinase, protein kinase C, phosphatidylinositol 3‐kinase and nuclear factor‐kappaB decreased the expression of nicotine‐ and LPS‐induced HIF‐1α and COX‐2, as well as the activity of PGE₂ and MMPs. Conclusion: These data demonstrate novel mechanisms by which nicotine and LPS promote periodontal tissue destruction, and provide further evidence that HIF‐1α is a potential target in periodontal disease associated with smoking and dental plaque.     

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor by periodontal fibroblasts

Agis H, Watzek G, Gruber R. Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor by periodontal fibroblasts. J Periodont Res 2012; 47: 165–173. © 2011 John Wiley & Sons A/S Background and Objective: Pharmacological inhibitors of prolyl hydroxylases (PHDs) can induce a proangiogenic response that favors wound healing and bone regeneration. However, the response of periodontal cells to PHD inhibitors is unknown. Material and Methods: To determine the effects of PHD inhibitors on periodontal cells, we exposed human fibroblasts from the gingiva and the periodontal ligament to dimethyloxallyl glycine, desferrioxamine, l ‐mimosine and CoCl₂. Viability, proliferation, and protein synthesis were assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), [³H]thymidine, and [³H]leucine incorporation, respectively. The levels of Ki67, hypoxia‐inducible factor 1α (HIF‐1α), p27, phosphorylated c‐Jun N‐terminal kinase (JNK) and phosphorylated p38 were determined by immunohistochemistry and western blotting. Vascular endothelial growth factor (VEGF) mRNA levels were measured by quantitative PCR. Protein levels of VEGF and interleukin (IL)‐6 were evaluated by immunoassays. Results: We found that PHD inhibitors, while leaving cell viability unchanged, reduced proliferation and protein synthesis. This was paralleled by decreased Ki67 levels and increased p27 levels, suggesting that PHD inhibitors provoke growth arrest. Independently from this response, PHD inhibitors stabilized HIF‐1α and increased the production of VEGF. This increase of VEGF was observed in the presence of proinflammatory IL‐1 and pharmacological inhibitors of JNK and p38 signaling. Moreover, PHD inhibitors did not modulate expression of IL‐6 and the phosphorylation of JNK and p38. Conclusion: These results suggest that PHD inhibitors enhance the production of VEGF in periodontal fibroblasts, even in the presence of proinflammatory IL‐1. The data further suggest that PHD inhibitors do not provoke a significant proinflammatory or anti‐inflammatory response in this in vitro setting.     

Up-regulation of vascular endothelial growth factor in synovial fibroblasts from human temporomandibular joint by hypoxia

INTRODUCTION: Enhanced expression of vascular endothelial growth factor (VEGF) has been described in patients with internal derangement (ID). Herein, we examined the expression of VEGF in synovial fibroblasts from temporomandibular joint (TMJ) under hypoxia and investigated the regulation of hypoxia-inducible factor-1alpha (HIF-1alpha) involved in the expression of VEGF. METHODS: Synovial fibroblasts were prepared from human TMJ. These cells were incubated under hypoxia or normoxia for the indicated time periods. VEGF levels in cultured supernatant were measured by an ELISA. VEGF mRNA isoforms and stability were assessed using RT-PCR and Northern blot analysis respectively. HIF-1alpha accumulation was evaluated by Western blotting and immunofluorescence. RESULTS: VEGF were significantly induced by hypoxia in synovial fibroblasts. In response to hypoxia, VEGF121 and VEGF165 mRNA were both remarkably increased, while there was no change in VEGF mRNA stability. The accumulation and nuclear translocation of HIF-1alpha occurred under hypoxia. CONCLUSIONS: Hypoxia may mainly induce the expression of VEGF121 and VEGF165 in synovial fibroblasts to promote inflamed angiogenesis of TMJ. HIF-1alpha, which is clearly activated in response to hypoxia, may control the expression of VEGF in synovial fibroblasts from TMJ.     

Lymphatic growth factors are expressed in human gingiva and upregulated in gingival fibroblasts after stimulation

1 Background

The lymphatic growth factors vascular endothelial growth factor (VEGF)‐C and ‐D are important for maintenance and growth of lymphatic vessels (lymphangiogenesis), but their localization in human gingiva is unknown. This study investigated the expression of VEGF‐C and ‐D in human gingiva and isolated human gingival fibroblasts (HGFs). In addition, the localization of their main receptor VEGFR‐3 was explored.

2 Methods

Non‐inflamed gingiva from six donors was used for immunohistochemistry or isolation of HGFs. HGFs were stimulated with either E.coli lipopolysaccharide (LPS) or IL‐6/soluble IL‐6 receptor (sIL‐6R) complex for 1, 6, and 24 hours. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was used to quantify the relative changes in gene expression of VEGF‐A, ‐C, and ‐D and enzyme‐linked immunosorbent assay (ELISA) for quantification of protein levels.

3 Results

VEGF‐C, ‐D and VEGFR‐3 were seen in keratinocytes, blood vessels and in scattered single cells in gingiva. VEGFR‐3 was also found in lymphatic vessels and VEGF‐C in cells with fibroblastic appearance. Gene analysis showed no expression of VEGF‐D in the HGFs, but showed constitutive expression of VEGF‐C and ‐A. Stimulation of HGFs with LPS or IL‐6/sIL‐6R complex was followed by gene upregulation of VEGF‐C and ‐A and increased protein levels in cell culture supernatant (P ≤0.05).

4 Conclusions

The localization of VEGF‐C, ‐D, and VEGFR‐3 expression imply that signaling via VEGFR‐3 is linked to vascular homeostasis and keratinocyte function under normal conditions in gingiva. Inflammatory stimulation of HGFs upregulates VEGF‐C and ‐A expression and may contribute to angiogenesis and lymphangiogenesis.     

Interferon-γ stimulates CD14, TLR2 and TLR4 mRNA expression in gingival fibroblasts increasing responsiveness to bacterial challenge

ObjectiveTo investigate the potential effects of IFN-03A5 on the responsiveness of human gingival fibroblasts to bacterial challenge.DesignmRNA and protein expression of CD14, TLR2 and TLR4 in human gingival fibroblasts was detected by quantitative polymerase chain reaction (Q-PCR) and flow cytometry. The effect of preincubation with IFN-03A5 on subsequent bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. Bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot.ResultsHuman gingival fibroblasts express CD14, TLR2 and TLR4 mRNAs. IFN-03A5, but not IL-103B2, induced mRNA expression of all three receptors and the expression of membrane bound CD14 protein. Pre-incubation of fibroblasts with IFN-03A5 and subsequent stimulation with Escherichia coli LPS or Porphyromonas gingivalis LPS led to increased production of IL-6 and IL-8. LPS-induced pro-inflammatory cytokine production was abrogated by a blocking antibody to CD14. Both E. coli LPS and P. gingivalis LPS induced IκBα degradation in human gingival fibroblasts.ConclusionOur data indicate that IFN-03A5 primes human gingival fibroblasts, through the upregulation of CD14 expression, which results in increased responsiveness to bacterial LPS challenge, as determined by pro-inflammatory cytokine production.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

The role of psychologic stress-induced hypoxia-inducible factor-1α in rat experimental periodontitis

Background: Studies have shown that psychologic stress plays a significant role in the outcome of many diseases. The present study is designed to investigate the effect of stress on experimental ligature‐induced periodontal disease in rats by means of a variable moderate chronic stress model. Methods: Sixty‐six age‐matched male Wistar rats of specific pathogen‐free grade were randomly divided into four groups: 1) normal control group, naive rats; 2) experimental periodontitis group, received only silk ligatures at the gingival margins of the second maxillary molar; 3) stress‐stimulation group, treated only with experimental stress conditions; and 4) experimental periodontitis plus stress‐stimulation group (e.g., experimental groups also exposed to stress). Stress was imposed by means of restraint stress, cold‐water immersion stress, and cat shock stress, which were all applied randomly. The rats were sacrificed at weeks 1, 4, 6, and 8 of the experiment. Attachment losses (AL) were measured by a specially made periodontal probe. The histopathologic changes of periodontia stained with hematoxylin and eosin were observed under a microscope. The expression of hypoxia‐inducible factor‐1α used to evaluate tissue hypoxic degree in periodontal tissues was tested by immunohistochemistry. Results: Our results show that there was no significant difference of AL among the normal control and the stress‐stimulation groups (P >0.05); AL of the periodontitis plus stress‐stimulation group was significantly higher than that of the experimental periodontitis group at weeks 4, 6, and 8 (P <0.01), and the hypoxia‐inducible factor‐1α expression scores of the periodontitis plus stress‐stimulation group were significantly higher than those of the experimental periodontitis group at weeks 4, 6, and 8 (P = 0.0477). Conclusions: Stress‐stimulation may aggravate periodontitis by decreased tissue oxygenation in rats. We conclude that there is a correlation of periodontitis severity with psychologic stress and periodontal tissue hypoxia.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

Involvement of P2X(7) purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts

Montreekachon P, Chotjumlong P, Bolscher JGM, Nazmi K, Reutrakul V, Krisanaprakornkit S. Involvement of P2X ₇ purinergic receptor and MEK1/2 in interleukin‐8 up‐regulation by LL‐37 in human gingival fibroblasts. J Periodont Res 2011; 46: 327–337.© 2011 John Wiley & Sons A/S Background and Objective: The antimicrobial peptide LL‐37, derived from human neutrophils, can directly chemoattract leukocytes and up‐regulate the expression of several immune‐related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL‐37 on interleukin‐8 (IL‐8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL‐8 induction. Material and Methods: Cultured fibroblasts were treated with different concentrations of LL‐37 or interleukin‐1β (IL‐1β), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT‐PCR and real‐time PCR were conducted to analyze the expression of IL‐8 mRNA, and the IL‐8 levels in cell‐free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL‐37. Results: Nontoxic concentrations of LL‐37 (up to 10 μm ) and IL‐1β significantly up‐regulated the expression of IL‐8 mRNA in a dose‐dependent manner (p < 0.05). The IL‐8 protein levels were consistently significantly elevated in conditioned media of LL‐37‐treated HGFs (p < 0.05). IL‐8 up‐regulation by LL‐37 was completely abrogated by 20 μm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X₇ receptor) and the neutralizing antibody against P2X₇ blocked IL‐8 up‐regulation in a dose‐dependent manner, consistent with expression of the P2X₇ receptor in HGFs. Conclusion: These findings indicate that LL‐37 induces IL‐8 expression via the P2X₇ receptor and the MEK1/2‐dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL‐37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues.