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

BACKGROUND: Angiopoietin-like protein 4 (ANGPTL4) is produced in chronic or acute inflammation. Although ANGPTL4 increases in the periodontal ligament fibroblasts during hypoxia, the involvement and role of ANGPTL4 in periodontitis have not been elucidated. OBJECTIVE: In this study, we investigated whether ligature-induced experimental periodontitis and/or Porphyromonas gingivalis lipopolysaccharides (Pg-LPS) would upregulate ANGPTL4 expression and whether ANGPTL4 would somehow involve in the expression of matrix metalloproteinases (MMPs) which are key molecules in the process of periodontal tissue destruction. METHODS: Experimental periodontitis was induced in 6-week-old male Sprague-Dawley rats by placing a nylon suture around the neck of the maxillary second molar. Two weeks after the induction of periodontitis, the periodontal tissue was excised and analyzed by histological/immunohistochemical staining and gene expression analyses. Human gingival fibroblasts (hGFs) were stimulated with Pg-LPS. The gene expression of ANGPTLs and receptors involved in ANGPTL4 recognition were observed. We also confirmed the changes in gene expression of MMPs upon stimulation with human ANGPTL4. Furthermore, we downregulated ANGPTL4 expression by short interfering RNA in hGFs and investigated the effect of Pg-LPS on MMP production. RESULTS: Induction of periodontitis significantly increased the expression of ANGPTL4 in the gingiva. Pg-LPS significantly increased the gene and protein expression of ANGPTL4 in hGFs but not the gene expression of other ANGPTLs or ANGPTL receptors. Recombinant human ANGPTL4 significantly increased MMP13 gene expression in hGFs. We also confirmed that MMP13 expression was increased in the gingiva during experimental periodontitis. Pg-LPS induced MMP13 gene expression in hGFs. These results suggest the pivotal role of ANGPTL4 in periodontitis. CONCLUSION: Periodontitis increases ANGPTL4 expression in the gingiva, further suggesting that increased ANGPTL4 may be a factor involved in enhancing MMP13 expression.

ANGPTL4 regulates the osteogenic differentiation of periodontal ligament stem cells.

The molecular mechanism of mechanical force regulating the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) has not been clearly elucidated. In this study, two mRNA-seqs, GSE106887 and GSE109167, which contained several samples of PDLSCs under mechanical force, were downloaded from Gene Expression Omnibus. Differential expression analysis was firstly taken between GSE106887 and GSE109167, then the common 84 up-regulated genes and 26 down-regulated genes were selected. Function enrichment analysis was used to identify the key genes and pathways in PDLSCs subjected to the tension and compression force. PDLSCs were isolated from human periodontal ligament tissues. The effects of ANGPTL4 knockdown with shRNA on the osteogenic differentiation of PDLSCs were studied in vitro. Then, the orthodontic tooth movement (OTM) rat model was used to study the expression of HIF-1α and ANGPTL4 in alveolar bone remodeling in vivo. ANGPTL4 and the HIF-1 pathway were identified in PDLSCs subjected to the tension and compression force. alizarin red staining, alcian blue staining, and oil red O staining verified that PDLSCs had the ability of osteogenic, chondrogenic, and adipogenic differentiation, respectively. Verification experiment revealed that the expression of ANGPTL4 in PDLSCs significantly increased when cultured under osteogenic medium in vitro. While ANGPTL4 was knocked down by shRNA, the levels of ALPL, RUNX2, and OCN decreased significantly, as well as the protein levels of COL1A1, ALPL, RUNX2, and OCN. During the OTM, the expression of HIF-1α and ANGPTL4 in periodontal ligament cells increased on the tension and compression sides. We concluded the positive relationship between ANGPTL4 and osteogenic differentiation of PDLSCs.

Angiopoietin-like 4 promotes osteosarcoma cell proliferation and migration and stimulates osteoclastogenesis.

BACKGROUND: Osteosarcoma is the most common primary bone cancer in children and young adults. It is highly aggressive and patients that present with metastasis have a poor prognosis. Angiopoietin-like 4 (ANGPTL4) drives the progression and metastasis of many solid tumours, but has not been described in osteosarcoma tissue. ANGPTL4 also enhances osteoclast activity, which is required for osteosarcoma growth in bone. We therefore investigated the expression and function of ANGPTL4 in human osteosarcoma tissue and cell lines. METHODS: Expression of ANGPTL4 in osteosarcoma tissue microarrays was determined by immunohistochemistry. Hypoxic secretion of ANGPTL4 was tested by ELISA and Western blot. Regulation of ANGPTL4 by hypoxia-inducible factor (HIF) was investigated using isoform specific HIF siRNA (HIF-1α, HIF-2α). Effects of ANGPTL4 on cell proliferation, migration (scratch wound assay), colony formation and osteoblastogenesis were assessed using exogenous ANGPTL4 or cells stably transfected with ANGPTL4. Osteoclastogenic differentiation of CD14+ monocytes was assessed by staining for tartrate-resistant acid phosphatase (TRAP), bone resorption was assessed by lacunar resorption of dentine. RESULTS: ANGPTL4 was immunohistochemically detectable in 76/109 cases. ANGPTL4 was induced by hypoxia in 6 osteosarcoma cell lines, under the control of the HIF-1α transcription factor. MG-63 cells transfected with an ANGPTL4 over-expression plasmid exhibited increased proliferation and migration capacity and promoted osteoclastogenesis and osteoclast-mediated bone resorption. Individually the full-length form of ANGPTL4 could increase MG-63 cell proliferation, whereas N-terminal ANGPTL4 mediated the other pro-tumourigenic phenotypes. CONCLUSIONS: This study describes a role(s) for ANGPTL4 in osteosarcoma and identifies ANGPTL4 as a treatment target that could potentially reduce tumour progression, inhibit angiogenesis, reduce bone destruction and prevent metastatic events.

HIF-1α regulates osteoclastogenesis and alveolar bone resorption in periodontitis via ANGPTL4.

OBJECTIVE: The mechanism of alveolar bone resorption caused by periodontitis is not fully understood. We sought to investigate whether microenvironmental changes of local hypoxia are involved in these processes. METHODS: In this study, periodontitis models of control mice and knockout of Hypoxia Induced Factor 1α (HIF-1α) harboring Cathepsin K (CTSK) Cre mice were constructed to study the effect of osteoclasts affected by hypoxic environment on alveolar bone resorption. RAW264.7 cells were subsequently induced by CoCl2 to observe the effects of HIF-1α and Angiopoietin-like Protein 4 (ANGPTL4) on osteoblast differentiation and fusion. RESULTS: The degree of alveolar bone resorption in the periodontitis tissues was lesser in mice with conditional knockout of HIF-1α in osteoclasts than in wild-type mice. We also observed that HIF-1α conditional knockout mice had fewer osteoclasts on the alveolar bone surface than control mice. HIF-1α increases the expression of ANGPTL4 and promotes the differentiation of RAW264.7 cells into osteoblasts and cell fusion under chemically simulated hypoxic conditions. CONCLUSION: HIF-1α regulates osteoclastogenesis and participates in bone resorption in periodontitis through ANGPTL4.

L-mimosine and hypoxia enhance angiopoietin-like 4 production involving hypoxia-inducible factor-1alpha: Insights from monolayer and spheroid cultures of dental pulp-derived cells and tooth slice cultures.

OBJECTIVE: Angiopoietin-like 4 (Angptl4) is an angiogenesis modulating signaling factor and as such involved in blood vessel formation but also in hard tissue resorption. Here we hypothesized that the hypoxia mimetic agent L-mimosine (L-MIM) and hypoxia stimulate the production of Angptl4 in the dental pulp. MATERIAL AND METHODS: Monolayer and spheroid cultures of primary human dental pulp-derived cells (DPC) were treated with L-MIM or hypoxia. Furthermore, tooth slice cultures were performed. The production of Angptl4 was assessed at mRNA and protein levels using reverse transcription qPCR and immunoassays, respectively. To assess the involvement of hypoxia inducible factor (HIF)-1α (HIF-1signaling, inhibitor studies with echinomycin and Western Blot analysis for HIF-1α were performed in DPC monolayer cultures.(HIF-1 RESULTS: L-MIM and hypoxia increased production of Angptl4 at mRNA and protein levels in monolayer cultures of DPC. The increase of Angptl4 was paralleled by an increase of HIF-1α and inhibited by echinomycin. Angptl4 protein levels were also elevated in spheroid cultures. In tooth slice cultures, the pulp tissue expressed and released Angptl4 under normoxic and hypoxic conditions and in the presence of L-MIM. There was a trend for an increase in Angptl4 mRNA levels and a trend for a decrease in the protein levels of the supernatants. CONCLUSIONS: Our results suggest that the hypoxia mimetic agent L-MIM and hypoxia can increase Angptl4 production in DPC involving HIF-1α. However, the increase in the cell culture supernatants does not translate in an increased release in tooth slice organ cultures.