N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide promotes vascular endothelial cell angiogenesis and migration in the absence of serum and FGF-2

Abstract

Aim: 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.

Conclusion: BPC most effectively promoted angiogenesis and migration in HUVECs in the absence of FGF-2 and serum.

Figure 1

The structure of N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide (BPC).

Figure 2

Effects of BPC on endothelial cells in the absence and presence of serum and FGF-2. (A) Cell morphological micrographs were obtained under a phase contrast microscope at 24, 48 and 72 h (×200). In the control group (Ctrl), cells were cultured in basal M199 medium (without serum and FGF-2) with DMSO [<0.1% (v/v)]. In the experimental groups, cells were treated with 5 (d-f), 10 (g-i), 20 (j-l), or 40 (m-o) μmol/L BPC. (B) Cell morphological micrographs obtained under a phase contrast microscope at 24, 48 and 72 h (×200). In the control group (Ctrl), cells were cultured in M199 medium with DMSO [<0.1% (v/v)]. In the experimental groups, cells were treated with 5 (d-f), 10 (g-i), or 20 (j-l) μmol/L BPC. (C) Cell viability was determined using MTT assay at 24, 48 and 72 h in the presence of serum and FGF-2 (n=3). (D) LDH assay was performed on cells treated as described in the text for 48 h in the presence of serum and FGF-2 (n=3).

Figure 3

BPC induced endothelial cell differentiation into capillary-like structures in vitro. (A) BPC promoted vascular structure formation in an in vitro Matrigel assay without FGF-2 and serum (×200). HUVECs were seeded without BPC (a-c) and with BPC (d-l) for 2, 4, and 8 h. (B) Quantitative assessment of the extent of tube formation (^bP<0.05, ^cP<0.01 vs Ctrl, n=5).

Figure 4

BPC promotes migration of endothelial cells in vitro. BPC induced HUVEC migration in the absence of FGF-2 and serum (200×). (A) Representative photomicrographs of migration. Cells migrated in FGF-2 and serum-free medium at 0, 12, and 24 h in the absence (a-c) and presence of BPC (d-l). (B) Quantitative assessment of migration distance (^bP<0.05, ^cP<0.01 vs Ctrl, n=4).

Figure 5

BPC induces angiogenesis through increase in ROS and NO. (A) An intracellular ROS assay was performed on the cells treated with BPC 5–20 μmol/L for 3, 6, and 12 h (×200). Relative DCF fluorescence reflected the intensity of ROS in Ctrl and BPC groups. (B) Quantitative assessment of ROS levels using relative fluorescence intensity of DCF per cell in the scan. ^bP<0.05, ^cP<0.01 vs Ctrl. (C) NAC inhibited angiogenesis induced by BPC (×200). (D)The change of NO production in cells treated with BPC 5–20 μmol/L for 3, 6, and 12 h. ^bP<0.05, ^cP<0.01 vs Ctrl, n = 3. (E) NO increase was not affected by NAC 10 mmol/L. Ctrl cells were cultured in M199 medium with DMSO. BPC cells were treated with 10 μmol/L BPC (^cP<0.01 vs ctrl, n=3).

Figure 6

BPC enhanced HIF-1α levels and VEGF release dependent on the generation of ROS. (A) BPC-enhanced HIF-1α expression was inhibited by NAC 10 mmol/L at 6 h. (B) Bar graph plots of relative HIF-1α levels (^bP<0.05, ^cP<0.01 vs Ctrl. ^fP<0.01 vs 10 μmol/L BPC; n=3). (C) BPC enhanced VEGF release. In the control group (Ctrl), cells were cultured in M199 medium with DMSO [<0.1% (v/v)]. In the experimental groups, cells were treated with BPC at 5, 10, or 20 μmol/L. (^bP<0.05, ^cP<0.01 vs Ctrl. n=3). (D) BPC-enhanced VEGF release was inhibited by NAC 10 mmol/L. Ctrl cells were cultured in M199 medium with DMSO [<0.1% (v/v)]. BPC cells were treated with 10 μmol/L BPC (^bP<0.05 vs Ctrl. ^eP<0.05 vs 10 μmol/L BPC; n=3).