Department of Conservative Dentistry and Periodontology, University Clinic of Dentistry, Medical University of Vienna, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
J Periodontal Res. 2019 Oct;54(5):489-498. doi: 10.1111/jre.12649. Epub 2019 Mar 20.
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.
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.
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.
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.
血管生成和骨吸收的调节的一个关键因素是血管生成素样 4（angiopoietin-like 4，ANGPTL4）。然而，ANGPTL4 在牙周组织中的作用尚不清楚。在这里，我们假设低氧和低氧模拟剂 L-千里光宁能够诱导牙周成纤维细胞产生 ANGPTL4。
将人牙周韧带成纤维细胞（periodontal ligament fibroblasts，PDLF）在单层和球体培养物中进行培养。在存在低氧或 L-千里光宁的情况下孵育这些培养物。通过 qPCR 和 ELISA 分别测量 ANGPTL4 mRNA 和蛋白水平。还评估了大肠杆菌脂多糖（lipopolysaccharides of E. coli，LPS）和牙龈卟啉单胞菌（P. gingivalis）、白细胞介素（interleukin，IL）-1β 和肿瘤坏死因子（tumor necrosis factor，TNF）α 的影响。此外，通过 Western blot 检测 HIF-1 和使用 echinomycin 进行抑制剂研究来测试对缺氧诱导因子（hypoxia-inducible factor，HIF）-1 活性的依赖性。通过用全长、C 末端和 N 末端片段的重组 ANGPTL4 暴露于 PDLF 来评估潜在的自分泌作用。评估对细胞活力、DNA 合成、碱性磷酸酶和基质矿化的影响。
低氧和 L-千里光宁均能提高 PDLF 单层培养物中 ANGPTL4 的 mRNA 和蛋白水平。HIF-1 在低氧和 L-千里光宁处理后均升高。LPS、IL-1β 和 TNFα 对 ANGPTL4 水平的调节作用不显著。在培养物中添加 echinomycin 可抑制 ANGPTL4 的产生。在 PDLF 的球体培养物中，mRNA 和蛋白水平的增加均未达到显著水平。全长、C 末端和 N 末端片段的 ANGPTL4 均未调节细胞活力、DNA 合成、碱性磷酸酶和基质矿化。
总的来说，我们发现低氧和低氧模拟剂 L-千里光宁可以刺激 PDLF 单层培养物中 ANGPTL4 的产生。这种增加依赖于 HIF-1 活性。未来的研究将揭示牙周组织中 ANGPTL4 的调节如何促进牙周病和再生。
