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Gold nanoparticles inhibit VEGF165-induced migration and tube formation of endothelial cells via the Akt pathway

BioMed Research International (2014) 2014
DOI: 10.1155/2014/418624
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Abstract

The early stages of angiogenesis can be divided into three steps: endothelial cell proliferation, migration, and tube formation. Vascular endothelial growth factor (VEGF) is considered the most important proangiogenic factor; in particular, VEGF165 plays a critical role in angiogenesis. Here, we evaluated whether gold nanoparticles (AuNPs) could inhibit the VEGF165-induced human umbilical vein endothelial cell (HUVEC) migration and tube formation. AuNPs and VEGF165 were coincubated overnight at 4°C, after which the effects on cell migration and tube formation were assessed. Cell migration was assessed using a modified wound-healing assay and a transwell chamber assay; tube formation was assessed using a capillary-like tube formation assay and a chick chorioallantoic membrane (CAM) assay. We additionally detected the cell surface morphology and ultrastructure using atomic force microscopy (AFM). Furthermore, Akt phosphorylation downstream of VEGFR-2/PI3K in HUVECs was determined in a Western blot analysis. Our study demonstrated that AuNPs significantly inhibited VEGF165-induced HUVEC migration and tube formation by affecting the cell surface ultrastructure, cytoskeleton and might have inhibited angiogenesis via the Akt pathway. © 2014 Yunlong Pan et al.

Figures

  • Figure 1: UV-Vis spectrum and TEM image of the AuNPs. (a) Purified AuNPs were analyzed using UV-Vis absorption spectroscopy. (b)The sizes of the AuNPs in a 15 nm diameter range.
  • Figure 2: The effects of AuNPs on VEGF 165 -induced HUVEC migration. (a) Confluent HUVEC monolayers were wounded with pipette tips (upper panel) and treated with VEGF 165 (20 ng/mL) in the presence or absence of AuNPs (125 or 250 nM). After 24 h incubation (lower panel), the wound closures were imaged to analyze the abilities of the cells to migrate into and fill the wounded areas. HUVECs incubated in 0.5% serumwere used as controls. HUVECs treated with VEGF 165 migrated efficiently from the woundmargin relative to the controls. AuNPs significantly inhibited VEGF 165 -induced cell migration. (b) The average migration of cells treated with 0.5% serum (control) and VEGF 165 (20 ng/mL) in the presence or absence of AuNPs (125 or 250 nM). Data are presented as the means ± SEM based on three independent experiments. #𝑃 < 0.05 compared with the control group; ∗𝑃 < 0.05 compared with the VEGF165 group.
  • Figure 3: The effects of AuNPs on HUVEC migration in a Transwell assay. (a) Sample Transwell assay images showing the inhibitory effects of AuNPs on VEGF 165 -induced HUVEC migration after staining the transmigrated cells with crystal violet (magnification, 10x). VEGF 165 -treated (20 ng/mL) cells transmigrated through theMatrigel-coated Boyden chamber within 12 h, compared with controls, whereas pretreatment with AuNPs (125 or 250 nM) significantly reduced cell migration. (b) Quantification of the Transwell assay results: crystal violet OD values represent the amounts of transmigrated cells. Data are presented as the means ± SEM based on three independent experiments. # 𝑃 < 0.05 compared with the control group; ∗𝑃 < 0.05 compared with the VEGF165 group.
  • Figure 4: Effects of the AuNPs on VEGF 165 -induced HUVEC tube formation. (a) Sample images showing the inhibitory effect of AuNPs on VEGF 165 -induced HUVEC tube formation (magnification, 10x). HUVECs (1 × 105 cells) were inoculated on the Matrigel surface and treated with VEGF 165 (20 ng/mL) in the presence or absence of AuNPs (125 or 250 nM). (b) Tube formation was quantified by counting the connected cells in randomly selected fields at 100x magnification. Data are presented as the means ± SEM based on three independent experiments. #𝑃 < 0.05 compared with the control group; ∗𝑃 < 0.05 compared with the VEGF165 group.
  • Figure 5:The effects of AuNPs on angiogenesis in the CAM. Fertilized eggs were treated with VEGF 165 in the presence or absence of AuNPs for 48 h and subsequently harvested and photographed. (a) Control; (b) VEGF 165 (80 nM/egg); (c) VEGF 165 + AuNPs (1000 nM/egg); (d) VEGF 165 + AuNPs (1500 nM/egg); (e) angiogenesis was quantified by counting the number of arteriole branches in each photograph. Data are presented as themeans± SEMbased on three independent experiments. #𝑃 < 0.05 comparedwith the control group; ∗𝑃 < 0.05 compared with the VEGF 165 group.
  • Figure 6: Changes in the cellular ultrastructure as detected by AFM. HUVECs were seeded onto slides and treated with VEGF 165 (20 ng/mL) in the presence or absence of AuNPs (250 nM) for 24 h. The morphological changes of the HUVECs were assessed using AFM as described previously.
  • Figure 7: AuNPs inhibit VEGF 165 -induced HUVEC migration mediated by Akt pathway. (a) Serum-starved HUVECs were treated with VEGF 165 (20 ng/mL) in the presence or absence of gold nanoparticles. The effects of AuNPs on phosphorylated Akt and pan Akt were determined using Western blot analysis. (b) Confluent HUVEC monolayers were wounded with pipette tips (upper panel) and treated with VEGF 165 (20 ng/mL) in the Ly294002 (10𝜇M). After 24 h incubation (lower panel), the wound closures were imaged to analyze the abilities of the cells to migrate into and fill the wounded areas. HUVECs incubated in 0.5% serum were used as controls. (c)The average migration of cells treated with 0.5% serum (control) and VEGF 165 (20 ng/mL) in the Ly294002 (10𝜇M). Data are presented as the means ± SEM based on three independent experiments. #𝑃 < 0.05 compared with the control group; ∗𝑃 < 0.05 compared with the VEGF165 group.
  • Table 1: Statistical analyses of the particle sizes and the cell surface Rp-v, Rq, and Ra (mean ± SD; nm).

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Pan, Y., Wu, Q., Qin, L., Cai, J., & Du, B. (2014). Gold nanoparticles inhibit VEGF165-induced migration and tube formation of endothelial cells via the Akt pathway. BioMed Research International, 2014. https://doi.org/10.1155/2014/418624

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