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

Abstract

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.

Increased expression of angiopoietin‐like protein 4 regulates matrix metalloproteinase‐13 expression in Porphyromonas gingivalis lipopolysaccharides‐stimulated gingival fibroblasts and ligature‐induced experimental periodontitis

“…Previous studies have shown that ANGPTL4 is produced during inflammation. Although ANGPTL4 increases due to hypoxia in periodontal ligament fibroblasts, 14 the involvement and role of ANGPTL4 in periodontitis have not been elucidated. Ligature‐induced experimental periodontitis was used in a rat model of periodontitis.…”

Vascular Changes and Hypoxia in Periodontal Disease as a Link to Systemic Complications

“…It has been shown that angiopoietin-like 4, an important signaling factor in modulation of angiogenesis, inflammation, lipid metabolism, and bone resorption, is induced by HIF-1 in human periodontal ligament fibroblasts and it can also be modulated by P. gingivalis LPS, IL-1 and TNF-α [103]. Although the function of angiopoietin-like-4 in periodontal tissues is not yet fully known, its mediation of angiogenesis and bone regeneration in inflammation, especially under hypoxic conditions, indicates that it may stand out as a good therapeutic agent candidate for periodontal diseases.…”

Clinical Potential of Hypoxia Inducible Factors Prolyl Hydroxylase Inhibitors in Treating Nonanemic Diseases

“…United States), DS-1093 (Daiichi Sankyo, Inc., Chuo City, Tokyo, Japan, no longer investigated for anemia) and Zyan1 (Cadila Healthcare Ltd., Ahmedabad, Gujarat, India) are still in preclinical, phase 1 or 2 clinical trials for renal anemia or are being evaluated for other indications. Other HIF-PHIs such as dimethyloxalylglycine ( Trichonas et al, 2013 ; Xie et al, 2014 ), L-mimosine (L-mim) ( Trimmel et al, 2015 ; Janjic et al, 2019 ), MK8617 ( Debenham et al, 2016 ; Li et al, 2019 ) and an FIH selective inhibitor N-oxalyl-D-phenylalanine ( Meng et al, 2018 ) are still in preclinical research. Since the action and mechanism of HIF-PHIs in CKD anemia are well summarized in reviews published before ( Forristal and Levesque, 2014 ; Gupta and Wish, 2017 ; Haase, 2017 ; Locatelli et al, 2017 ; Del Vecchio and Locatelli, 2018 ; Sanghani and Haase, 2019 ), this article focuses on milestones in the development of these clinical available HIF-PHIs and their potential in treating non-anemic diseases.…”