title: The bisphosphonate zoledronic acid regulates key angiogenesis-related genes in primary human gingival fibroblasts.
abstract: BACKGROUND
  Osteonecrosis of the jaws is recognised as a serious complication for patients receiving bisphosphonates. The anti-angiogenic effects of bisphosphonates have been implicated in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this study was to determine the effects of zoledronic acid on cultured human gingival fibroblasts in relation to the modulation of genes associated with angiogenic regulation.
  
  
  METHODS
  Primary cultures of fibroblasts were developed from gingival tissues excised during crown-lengthening surgery from three patients. Cells were cultured with and without 30μM zoledronic acid for 6, 12 and 24h and cellular proliferation and migration investigated using CellTiter-Blue and scratch wound assays, respectively. Gene expression was determined using semi-quantitative PCR array technology that allowed the analysis of 84 pathway-focused genes known to be important in the regulation of angiogenesis.
  
  
  RESULTS
  Zoledronic acid increased the proliferation of the gingival fibroblasts in a dose dependent manner with 12 and 24h of exposure. Scratch wounding of the human gingival fibroblasts and treatment with increasing doses and time exposure to zoledronic acid (ZA) inhibited their migration. Statistically significant increases in gene expression were found for RHOB, VEGFA, CD55 and BMP2 (p≤0.05) in response to 30μM zoledronic acid. CCL2 and IL6 genes were significantly downregulated (p≤0.05).
  
  
  CONCLUSIONS
  The regulation of the prenylated protein RHOB in this study was consistent with the known effects of zoledronic acid on the mevalonate pathway. The down regulation of CCL2 and IL6 and the upregulation of CD55 may be associated with suppression of inflammation. An increase in VEGFA and BMP2 gene expression suggests that fibroblasts respond to zoledronic acid by producing a proangiogenic environment.
authors.affiliation: Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
authors.forename: E J
authors.initials: EJ
authors.lastname: Ohlrich
authors.affiliation: Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand. Electronic address: dawn.coates@otago.ac.nz.
authors.forename: D E
authors.initials: DE
authors.lastname: Coates
authors.affiliation: Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
authors.forename: M P
authors.initials: MP
authors.lastname: Cullinan
authors.affiliation: Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
authors.forename: T J
authors.initials: TJ
authors.lastname: Milne
authors.affiliation: Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
authors.forename: S
authors.initials: S
authors.lastname: Zafar
authors.affiliation: Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
authors.forename: W D
authors.initials: WD
authors.lastname: Duncan
authors.affiliation: Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand.
authors.forename: G J
authors.initials: GJ
authors.lastname: Seymour
mesh_terms: D050071:Bone Density Conservation Agents / Q000494:pharmacology*; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D018958:CD55 Antigens / Q000378:metabolism; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D004164:Diphosphonates / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005881:Gingiva / Q000166:cytology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D015850:Interleukin-6 / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000077211:Zoledronic Acid; D020743:rhoB GTP-Binding Protein / Q000378:metabolism
chemical_list: C525718:BMP2 protein, human; D050071:Bone Density Conservation Agents; D055396:Bone Morphogenetic Protein 2; C111835:CCL2 protein, human; D018958:CD55 Antigens; D018932:Chemokine CCL2; D004164:Diphosphonates; D007093:Imidazoles; D015850:Interleukin-6; D042461:Vascular Endothelial Growth Factor A; D000077211:Zoledronic Acid; D020743:rhoB GTP-Binding Protein
keywords: Angiogenic; Gene expression; Gingival fibroblasts; Inflammation; Mevalonate pathway