title: N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide promotes vascular endothelial cell angiogenesis and migration in the absence of serum and FGF-2.
abstract: OBJECTIVE
  To investigate the effect of N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide (BPC) on angiogenesis in human umbilical vein endothelial cells (HUVECs).
  
  
  METHODS
  Capillary-like tube formation on matrigel and cell migration analyses were performed in the absence of serum and fibroblast growth factor (FGF-2). Reactive oxygen species (ROS) were measured using a fluorescent probe, 2', 7'- dichlorodihydrofluorescein (DCHF). The nitric oxide (NO) production of HUVECs was examined using a NO detection kit. Morphological observation under a phase contrast microscope, a viability assay using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium (MTT) and a lactate dehydrogenase (LDH) activity analysis by a detection kit were performed to evaluate the toxicity of BPC on HUVECs in the presence of serum and FGF-2. The level of hypoxia-inducible factor 1α (HIF-1α) and the release of vascular endothelial growth factor (VEGF) were measured by Western blot and ELISA, respectively.
  
  
  RESULTS
  In the absence of serum and FGF-2, cells treated with BPC (5-20 μmol/L) rapidly aligned with one another and formed tube-like structures within 12 h. In the presence of serum and FGF-2, cells treated with BPC for 24, 48 and 72 h had no changes in morphology, viability or LDH release compared with the control group. Cell migration in the BPC-treated group was significantly increased compared with the control group. During this process, NO production and ROS level were elevated dramatically, and the levels of HIF-1α and VEGF were increased dependent on the generation of ROS.
  
  
  CONCLUSIONS
  BPC most effectively promoted angiogenesis and migration in HUVECs in the absence of FGF-2 and serum.
authors.affiliation: Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Ji-nan, China.
authors.forename: Hai-yan
authors.initials: HY
authors.lastname: Zhang
authors.forename: Le
authors.initials: L
authors.lastname: Su
authors.forename: Bin
authors.initials: B
authors.lastname: Huang
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Bao-xiang
authors.initials: BX
authors.lastname: Zhao
authors.forename: Shang-li
authors.initials: SL
authors.lastname: Zhang
authors.forename: Jun-ying
authors.initials: JY
authors.lastname: Miao
mesh_terms: D001593:Benzyl Compounds / Q000008:administration & dosage / Q000494:pharmacology*; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism; D011720:Pyrazoles / Q000008:administration & dosage / Q000494:pharmacology*; D017382:Reactive Oxygen Species; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D001593:Benzyl Compounds; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C558480:N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide; D011720:Pyrazoles; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D009569:Nitric Oxide; D007770:L-Lactate Dehydrogenase