Expression of circadian core clock genes in fibroblasts of human gingiva and periodontal ligament is modulated by L-Mimosine and hypoxia in monolayer and spheroid cultures

Janjić, Klara; Kurzmann, Christoph; Moritz, Andreas; Agis, Hermann

doi:10.1016/j.archoralbio.2017.03.007

Cited by 27 publications

(27 citation statements)

References 25 publications

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“…However, the atmosphere in the normoxic and hypoxic groups in the system does not only differ with regard to the oxygen levels but also with regard to the CO 2 levels. The results indicate that cells survive these conditions and that a similar hypoxia-like response in the HMA groups and the hypoxia group is initiated within 24 h. Similar approaches have been successfully used in previous studies even for longer culture times (Steinbach et al 2004, Gruber et al 2008, Janjic et al 2017. Altogether, these results support the feasibility of the system.…”

Section: Discussionsupporting

confidence: 79%

“…, Janjic et al . ). Hypoxic conditions were induced by the BD GasPak EZ Pouch system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).…”

Section: Methodsmentioning

confidence: 97%

“…Spheroids were cultured under normoxic and hypoxic conditions. To induce hypoxia, a previously published in vitro approach was implemented with minor modifications (Steinbach et al 2004, Gruber et al 2008, Janjic et al 2017. Hypoxic conditions were induced by the BD GasPak EZ Pouch system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).…”

Section: Spheroid Formationmentioning

confidence: 99%

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Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents

et al. 2017

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Aim To evaluate the impact of hypoxia and hypoxia mimetic agents (HMA) on the formation and activity of spheroids by dental pulp cells (DPC). Methodology DPC on agarose-coated plates were treated with hypoxia and the HMA dimethyloxallyl glycine (DMOG), desferrioxamine (DFO) and L-mimosine (L-MIM). Images of spheroids were taken directly after seeding and at 6 h and 24 h. Spheroid sizes were quantified by area measurement with ImageJ software. Viability was assessed with Live-Dead staining, MTT and resazurin-based toxicity assay. Production of VEGF, IL-8 and SDF-1 was evaluated using immunoassays. Data were analysed using KruskalWallis test and post hoc Mann-Whitney U-test. Results DPC formed spheroids in the presence of hypoxia, HMA and combined treatment with hypoxia and HMA. No pronounced difference in spheroid size was found in the groups treated with hypoxia, DMOG, DFO, L-MIM and the combination of hypoxia and the HMA relative to their normoxic controls (P > 0.05). Spheroids appeared vital in Live-Dead and MTT staining and the resazurin-based toxicity assay. Evaluation of protein production with immunoassays revealed significantly enhanced levels of VEGF and IL-8 (P < 0.05), but there was no significant effect on SDF-1 production (P > 0.05). Treatment with a combination of hypoxia and HMA did not further boost VEGF and IL-8 production (P > 0.05). Conclusions Pre-conditioning with hypoxia and HMA increased the pro-angiogenic capacity of spheroids whilst not interfering with their formation. Preclinical studies will reveal whether pre-conditioning of spheroids with hypoxia and HMA can effectively improve the efficiency of cell transplantation approaches for regenerative endodontics.

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Section: Discussionsupporting

confidence: 79%

“…, Janjic et al . ). Hypoxic conditions were induced by the BD GasPak EZ Pouch system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).…”

Section: Methodsmentioning

confidence: 97%

Section: Spheroid Formationmentioning

confidence: 99%

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Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents

et al. 2017

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“…The conditions were based on preliminary unpublished experiments and previous in vitro studies . To establish hypoxic conditions we used an established protocol with minor modifications . The culture plates were placed into BD GasPak EZ Pouches (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).…”

Section: Methodsmentioning

confidence: 99%

“…27,28 To establish hypoxic conditions we used an established protocol with minor modifications. [29][30][31] The culture plates were placed into BD GasPak EZ Pouches (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The manufacturer confirms that the oxygen concentration rapidly decreases to a level of <1%.…”

Section: Monolayer Culturementioning

confidence: 99%

Angiopoietin‐like 4 production upon treatment with hypoxia and L‐mimosine in periodontal fibroblasts

Janjić

Schellner

Engenhart

et al. 2019

J of Periodontal Research

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Background and objective A key factor in the modulation of angiogenesis as well as in bone resorption is angiopoietin‐like 4. However, the role of angiopoietin‐like 4 in periodontal tissue is unknown. Here, we hypothesized that hypoxia and the hypoxia mimetic agent L‐mimosine can induce the production of angiopoietin‐like 4 in periodontal fibroblasts. Methods Human periodontal ligament fibroblasts (PDLF) were cultured in monolayer and spheroid cultures. The cultures were incubated in the presence of hypoxia or L‐mimosine. Angiopoietin‐like 4 mRNA and protein levels were measured by qPCR and ELISA, respectively. Also, the impact of Lipopolysaccharides of E. coli and P. gingivalis, interleukin (IL)‐1β and tumor necrosis factor (TNF)α was evaluated. Furthermore, we tested dependency on hypoxia‐inducible factor (HIF)‐1 activity by Western blotting for HIF‐1 and inhibitor studies with echinomycin. Potential autocrine effects were assessed by exposure of PDLF to recombinant angiopoietin‐like 4 in full length, C‐terminal and N‐terminal fragments. The impact on viability, DNA synthesis, alkaline phosphatase, and matrix mineralization was evaluated. Results Both hypoxia and L‐mimosine elevated angiopoietin‐like 4 mRNA and protein levels in monolayer cultures of PDLF. HIF‐1 was elevated after both hypoxia and L‐mimosine treatment. LPS, IL‐1β, and TNFα did not modulate angiopoietin‐like 4 levels significantly. Addition of echinomycin in the cultures inhibited the production of angiopoietin‐like 4. In spheroid cultures of PDLF, the increase did not reach the level of significance at mRNA and protein levels. Angiopoietin‐like 4 in full length, C‐terminal, and N‐terminal fragments did not modulate viability, DNA synthesis, alkaline phosphatase, and matrix mineralization. Conclusion Overall, we found that hypoxia and the hypoxia mimetic agent L‐mimosine can stimulate angiopoietin‐like 4 production in monolayer cultures of PDLF. This increase depends on HIF‐1 activity. Future studies will reveal how the modulation of angiopoietin‐like 4 in the periodontium contributes to periodontal disease and regeneration.

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Angiogenin production in response to hypoxia and l‐mimosine in periodontal fibroblasts

Janjić

Bauer

Edelmayer

et al. 2019

Journal of Periodontology

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Background: A major mediator of angiogenesis is angiogenin, which is expressed in the early phase of healing in oral tissue engineering strategies. It is unclear how angiogenin is regulated in the periodontal tissue. The objective of this study was to reveal the regulation of angiogenin in response to hypoxia and the hypoxia mimetic agent L-mimosine in periodontal fibroblasts.Methods: Human fibroblasts of the periodontal ligament (PDLF) and the gingiva (GF) in monolayer and spheroid cultures were exposed to hypoxia or L-mimosine. The production of angiogenin was evaluated at mRNA and protein levels with reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Echinomycin, an inhibitor of hypoxia-inducible factor (HIF)-1 activity, was used to test the involvement of HIF-1. Results:Our data show that hypoxia and L-mimosine can increase angiogenin mRNA and protein levels in PDLF monolayer cultures. In GF monolayer cultures, we found an increase of angiogenin at the mRNA level in response to hypoxia. The increase of angiogenin can be blocked by inhibition of HIF-1 signaling via echinomycin. In PDLF and GF spheroid cultures, the impact of hypoxia and L-mimosine did not reach the level of significance. Conclusion:Hypoxia and the hypoxia mimetic agent L-mimosine can increase the production of angiogenin via HIF-1 signaling in PDLF monolayer cultures but not in spheroid cultures. GF were less sensitive to the impact of hypoxia and L-mimosine. Overall, these results suggest a link between hypoxia, HIF-1 signaling and angiogenin in the periodontium. K E Y W O R D Sbone biology, cell biology, fibroblast(s), gene regulation, growth factors, wound healing 674

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Expression of circadian core clock genes in fibroblasts of human gingiva and periodontal ligament is modulated by L-Mimosine and hypoxia in monolayer and spheroid cultures

Cited by 27 publications

References 25 publications

Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents

Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents

Angiopoietin‐like 4 production upon treatment with hypoxia and L‐mimosine in periodontal fibroblasts

Angiogenin production in response to hypoxia and l‐mimosine in periodontal fibroblasts

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