Gold Nanoparticles Inhibit VEGF165-Induced Migration and Tube Formation of Endothelial Cells via the Akt Pathway

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.


Introduction
It is a well-known fact that angiogenesis plays important role in cancer and other diseases and vascular endothelial growth factor (VEGF) is a crucial regulatory molecule during angiogenesis [1,2]. The early stages of angiogenesis can be divided into three steps: endothelial cell proliferation, migration, and tube formation [3]. As an isoform of VEGF, VEGF-A regulates the key steps of the angiogenic process, particularly endothelial cell proliferation and migration [4,5]. This cytokine promotes endothelial cell migration by interacting with its receptor VEGFR-2 and activating the downstream VEGFR-2/PI3K/Akt/eNOS axis in endothelial cells [5,6]. VEGF-A has two main isoforms: the heparinbinding growth factor VEGF 165 and the non-heparin-binding growth factor VEGF 121 ; these isoforms are the most important proangiogenic factors during angiogenesis [4].
In recent years, several anti-VEGF strategies have been explored in clinical settings. The most clinically advanced inhibitors were the recombinant humanized monoclonal antibodies against VEGF, including bevacizumab [7]; however, the clinical applications of these antibodies are greatly limited because of the high cost and incidence of side effects [8]. Currently, research regarding the use of nanomaterials to inhibit angiogenesis is emerging rapidly [9]. Given their small particle size, good biocompatibility, and low toxicity, gold nanoparticles are considered a good prospect for biological applications [10,11].
Several reporters have demonstrated that gold nanoparticles (AuNPs) exhibited potentially antiangiogenic effects by interacting with the heparin-binding domain of VEGF 165 [12]. Our previous study showed that AuNPs inhibited VEGF 165 -induced HUVEC proliferation and contributed to reduced levels of tumor angiogenesis [13].
However, the effects of AuNPs on endothelial migration and tube formation remained unknown.
The present study was conducted to demonstrate the effects of AuNPs on endothelial migration and tube formation. In this study, we found that AuNPs suppressed VEGF 165induced endothelial cell migration and tube formation and might have inhibited these processes through the Akt signaling pathway. Furthermore, we showed that AuNPs altered the cell membrane surface ultrastructure, a phenomenon that might be associated with angiogenesis. The results suggested that AuNPs reduced angiogenesis by repressing endothelial cell migration and tube formation.

Preparation and Characterization of AuNPs.
In a typical experiment [14], 0.01 mol/L of chloroauric acid (5 mL) and 1% sodium citrate (10 mL) were added to 50 mL of an aqueous solution that had been heated and stirred until reaching the boiling point; heating and vigorous stirring were maintained until the solution reached a wine red color. The newly formed AuNPs were filtered through a 0.22 m filter, used in the experiments, and ultimately characterized via UV-Vis absorption spectroscopy and transmission electron microscopy.

HUVEC Isolation and
Culture. The HUVECs were harvested from human umbilical veins via 0.25% trypsin (Gibco, NY, USA) digestion. After washing in D-Hanks solution, the umbilical veins were perfused with a 0.25% trypsin solution for 10 min at 37 ∘ C, after which the solution was collected and centrifuged for 10 min at 1000 rpm/min. The cells were passaged via trypsin digestion and cultured in M199 media (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 2 mM glutamine (Gibco, USA), and 1% penicillin/streptomycin (Sigma, USA) in an incubator at 37 ∘ C and 5% CO 2 .
The HUVECs were treated with VEGF 165 or AuNPtreated VEGF 165 . AuNPs (125 or 250 nM) were prepared in sterile distilled water, diluted to the required concentrations in cell culture medium, and incubated with VEGF 165 (20 ng/mL) overnight at 4 ∘ C before the experiments. Cells in the control group were cultured in basal medium.

Wound-Healing
Assay. An in vitro wound-healing assay was performed to measure unidirectional HUVEC migration [15]. The HUVECs were seeded in 6-well plates (1 × 10 5 cells/well), incubated in M199 with 10% FBS for 24 h, and subsequently washed twice with PBS and incubated in M199 with 1% FBS. After the cells grew to confluence, a straight line was scratched across the culture with a 10-200 L micropipettor tip. The cells were treated with VEGF 165 (20 ng/mL) in the presence or absence of AuNPs (125 or 250 nM) and were incubated at 37 ∘ C and 5% CO 2 . Images of the cells were obtained with an inverted phase contrast microscope (Olympus, Tokyo, Japan) at 0 h and 24 h. The wound width was determined with ImageJ software.

Transwell Monolayer Permeability
Assay. HUVEC migration was evaluated with a Transwell system (Corning Costar, MA, USA) that comprised 8 m polycarbonate filter inserts in 24-well plates [16]. Briefly, serum-starved cells were trypsin-harvested in M199 with 0.5% FBS. Next, 600 L of medium containing 1% FBS and VEGF 165 (20 ng/mL) with/without AuNPs was added to the lower chambers, while HUVECs (1 × 10 5 ) were plated in the upper chambers. After 12 h incubation, the cells on the bottom of the Transwell membrane were fixed with 4% paraformaldehyde at 37 ∘ C for 20 min and stained with 1% crystal violet at 37 ∘ C for 10 min; the nonmigrating cells in the upper chamber were removed with blunt-end swabs. The membranes were washed three times with PBS and photographed under a fluorescence microscope (Olympus; Tokyo, Japan). Finally, the crystal violet was dissolved in 33% acetic acid, and the absorbance was measured at 600 nm. The amount of cell migration was determined as the ratio of the OD values of the treatment relative to the control. Each treatment was repeated in 4 independent chambers.

Capillary-Like Tube Formation
Assay. The formation of HUVECs into capillary-like structures on Matrigel (BD Biosciences, Bedford, MA, USA) was evaluated as previously described [9,16]. HUVECs were pretreated with VEGF 165 (20 ng/mL) in the presence or absence of AuNPs (125 or 250 nM) in starvation medium at 37 ∘ C in a humidified atmosphere of 5% CO 2 for 24 h. At least 30 min before the experiment, 96-well plates were coated with Matrigel. Next, trypsin-harvested HUVECs were seeded onto the plated Matrigel (1 × 10 4 cells per well) in serum-free M199 medium and incubated at 37 ∘ C. Images of the formation of capillary-like structures were obtained after 6 and 12 h with a computer-assisted microscope (Olympus, Tokyo, Japan) at 100x magnification. Tubular structures were quantified by manually counting the numbers of connected cells in randomly selected fields at 100x magnification; the total tube number in the control group was designated as 100%.

Chick Chorioallantoic Membrane (CAM) Assay.
The effects of the test compounds on angiogenesis were investigated with the CAM assay as described previously [9]. Briefly, groups of 10 fertilized chicken eggs were incubated at 37 ∘ C and 80% humidity. On the sixth day of incubation, a square window was opened in each shell. Filter paper disks saturated with VEGF 165 , AuNP-treated VEGF 165 , or PBS were placed on the areas between preexisting vessels, after which the embryos were incubated for an additional 48 h. After the second incubation, the CAM arterious branches in each treatment group were photographed and counted using a Nikon digital camera system (Chiyoda-ku, Tokyo, Japan). The antiangiogenic effect of the AuNPs was indicated by relative numbers of arterious branches. The assay was performed three times to ensure reproducibility.

2.7.
Single-Cell AFM Measurement. 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, after which the cells were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, and air-dried at room temperature. An AFM (Autoprobe CP Research, Veeco, USA) was used in the contact mode to obtain topographic images in air [17]. The silicon nitride tips (UL20B; Park Scientific Instruments) used in all AFM measurements had been ultravioletirradiated in air for 15 min to remove any organic contaminants prior to use. The tip curvature radius was <10 nm, and the cantilever length, width, and thickness were 115, 30, and 3.5 mm, respectively, with an oscillation frequency of 255 kHz and a force constant of 0.01 N/m (manufacturer's information). The prepared sample was placed on the AFM XY-scanning station, and more than 10 cells were measured. The acquired images were processed only using the software provided with the equipment (Image Processing Software Version 2.1, IP 2.1) to eliminate low-frequency background noise in the scanning direction (flattened order: 0-1).

Western Blot Analysis.
Akt and phospho-Akt antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The GAPDH antibody was obtained from Kangchen (Shanghai, China). Goat anti-mouse IgG and anti-rabbit IgG were obtained from Calbiotech (San Diego, CA, USA). HUVECs were plated into a 6-well plate (3 × 10 5 cells/well). After adhering, the cells were starved and incubated in serum-free medium for 24 h and then treated with VEGF 165 (20 ng/mL) in the presence or absence of AuNPs (125-250 nM) for another 24 h. Whole-cell lysates were collected and boiled for 10 min in 2x SDS sample buffer, subjected to 10% SDS-PAGE, and transferred to PVDF membranes (Amersham Life Sciences). The blots were blocked in blocking buffer (5% nonfat dry milk/1% Tween-20 in TBS) for 1 h at room temperature and then incubated with the primary antibodies (anti-Akt, 1 : 5000 dilution; anti-phospho-Akt, 1 : 500; and anti-GAPDH, 1 : 10,000) in blocking buffer for 2 h at room temperature. The bands were then visualized using horseradish peroxidase-conjugated secondary antibodies (1 : 2000) and ECL (Pierce Biotech, Rockford, IL, USA). The experiments were repeated three times.

Statistical Analysis.
All data were presented as the means ± SEM from at least three independent experiments. Statistical analyses were performed with SPSS 17.0 software. Comparisons between the groups were performed with the two independent samples' -test; those between multiple groups were tested with one-way ANOVA, and those between any groups were tested with SNK. A probability value of < 0.05 was considered statistically significant.

Characterization of AuNPs.
The AuNPs were characterized using UV-Vis spectrophotometry and TEM. The AuNPs were red wine-colored and exhibited a peak in the 520 nm region of the UV-Vis spectrum (Figure 1(a)). Next, TEM was used to define the size distribution of the AuNPs. The monodisperse AuNPs were found to be spherical with a mean diameter of 15 nm (Figure 1(b)). Purified AuNPs were used for further studies.

Effects of the AuNPs on VEGF 165 -Induced HUVEC Migration.
It is a well-known fact that endothelial migration is the key process in angiogenesis and VEGF 165 plays crucial role in cell migration. To determine whether AuNPs could inhibit VEGF 165 -induced endothelial cell migration, wound-healing and Transwell monolayer permeability assays were employed to explore the changes in migration following treatment with VEGF 165 that had been preincubated with or without AuNPs.
As shown in Figure 2(a), the endothelial cell migration distance was 307.7 ± 18.9 m after VEGF 165 treatment for 24 h; this represented a significant increase relative to that of the control group (152.3 ± 6.1 m, < 0.01; Figure 2(b)). However, when HUVECs were treated with VEGF 165 that had been preincubated with 125 nM or 250 nM AuNPs, the cell migration distance was significantly reduced to 241.6 ± 11.5 m or 188.3±4.0 m, respectively ( < 0.01). These data show that AuNPs reduced endothelial cell migration distance in concentration-dependent manner.
We further used a Transwell system to examine the effects of AuNPs on VEGF 165 -induced endothelial cell migration. Compared with the nonstimulated control, a large number of HUVECs migrated to the lower side of the filter after stimulation with VEGF 165 for 12 h, whereas AuNPs significantly prevented this migration in a concentrationdependent manner (Figure 3(a)). As shown in Figure 3(b), after VEGF 165 treatment, the crystal violet OD values (representative of the amounts of migrated cells) increased from 0.19 ± 0.01 to 0.48 ± 0.03; however, treatment with VEGF 165 after preincubation with 125 nM or 250 nM AuNPs reduced the crystal violet OD values to 0.38 ± 0.02 or 0.27 ± 0.02, respectively ( < 0.05; Figure 3(b)). These results confirm that AuNPs reduce crystal violet OD values in concentrationdependent manner. The above results suggest that AuNPs repress the VEGF 165 -induced cell migration. 165 -Induced HUVEC Tube Formation. Another important step in the angiogenic process is tubule formation. We used a three-dimensional Matrigel assay to examine the potential effects of AuNPs on VEGF 165induced tube formation. When HUVECs were placed on the Matrigel, robust, elongated tube-like structures formed after incubation in the presence of VEGF 165 (Figure 4(a)). The number of formed tubules was calculated using invertedphase contrast microscopy, which directly revealed the ability of the HUVECs to form tubular structures. As expected, VEGF 165 clearly promoted tube formation, whereas the AuNPs significantly inhibited VEGF 165 -dependent tube formation. As shown in Figure 4(b), the tube-formation rate after VEGF 165 treatment was approximately 142 ± 12.1% (relative to the control); however, tube formation in the presence of 125 nM or 250 nM AuNPs was reduced to 87 ± 7.4% or 43 ± 4.3% of the control, respectively ( < 0.05; Figure 4(b)). These results demonstrated the potential of AuNPs to inhibit VEGF 165 -induced tube formation. The quantitative data are summarized in Figure 5(e). The branched blood vessel formation rate in the VEGF 165 -treated group was approximately 137 ± 8.7% (relative to the control group); however, after preincubation with 1000 nM or 1500 nM AuNPs, the rate decreased to 61 ± 3.1% or 39 ± 2.8%, respectively ( < 0.05). These results demonstrated that angiogenic development of the CAM arterial endpoint was stimulated by VEGF 165 and inhibited by AuNPs.

Changes in the Cellular Ultrastructure as Detected by AFM.
Atomic force microscopy is a useful tool for the collection of cellular surface information because it can detect the cellular topography, ultrastructure, and morphological changes [17,18]. In this study, AFM was used to observe a variety of changes in the HUVEC surface morphology and ultrastructure in the different treatment groups. More than 5 cells per group were scanned and measured by AFM, although only representative images are presented. As shown in Figure 6, the nonstimulated HUVECs had fusiform morphology with smooth surfaces and few particles. VEGF 165 -treated HUVECs adhered closely to the substrate; these cells were more active and had a pleomorphic appearance with obvious pseudopodia (indicated by the black arrows in Figure 6), larger particles, and rough surfaces. In contrast, the HUVECs that had been incubated with AuNP-treated VEGF 165 had a similar appearance to those of the control group, with fewer pseudopods and particles and a smoother cell surface. Furthermore, VEGF 165 -treated HUVECs exhibited considerable cellular plasmodesmata under AFM, whereas those incubated with AuNP-treated VEGF 165 exhibited comparatively and significantly thinner plasmodesmata. This finding suggested that AuNPs could significantly block the VEGF 165 -induced cellular extension or migration.
Additionally, the particle size distributions and surface roughness in each cellular ultrastructure image (3 m × 3 m) were analyzed using software (Image Processing 2.1, IP 2.1). The statistical analyses of the particle sizes and the peak to valley roughness (Rp-v), the root-mean-square roughness (Rq), and the average roughness (Ra) of the cell surface are shown in Table 1. The results indicated that VEGF 165 induced larger particle formation ( < 0.01) and increased surface roughness ( < 0.05) in the HUVECs, whereas AuNPs suppressed these changes and led to smaller particle formation ( < 0.01) and smoother surfaces ( < 0.05).
VEGFR2 plays a major role in VEGF-dependent angiogenesis. Our previous study [13] showed that AuNPs significantly inhibited phospho-VEGFR2 induced by VEGF 165 . To investigate the mechanism associated with the AuNPmediated inhibitory effects on VEGF 165 -induced migration and tube formation, we used Western blotting to examine the activation of Akt, which is an important downstream component of the VEGFR-2/PI3K signaling pathway in HUVECs. As shown in Figure 7, Akt phosphorylation was significantly increased in the HUVECs after VEGF 165 treatment but significantly reduced in a dose-dependent manner after treatment with VEGF 165 that had been preincubated with AuNPs. Next, a specific inhibitor of PI3K/Akt pathway, LY294002, was used to inhibit Akt phosphorylation in wound-healing assay to test whether inhibition of Akt phosphorylation has effect on VEGF 165 -induced migration. As shown in Figure 7(   to 178.9 ± 19.8 m ( < 0.05). The results suggested that the AuNPs significantly inhibited VEGF 165 -induced Akt phosphorylation in the HUVECs, thus resulting in the inhibition of VEGF 165 -induced HUVEC migration.

Discussion
Angiogenesis, the growth of new capillaries from preexisting vessels, occurs as a result of dynamic endothelial cell functions such as migration and tube formation that are essential to the organized formation of vessel sprouts [3,19]. We, therefore, evaluated these two critical angiogenic processes in this study. First, we examined whether AuNPs had any effect on VEGF 165 -induced HUVEC migration; the results of a wound-healing assay indicated a significant area of the uncovered wound after AuNPs treatment relative to that of the controls, and a Transwell monolayer permeability assay showed that the number of HUVECs that migrated to the lower side of the filter decreased after AuNPs treatment. Second, we observed the effects of AuNPs on VEGF 165 -induced HUVEC tube formation; the results of a Matrigel assay demonstrated that AuNPs inhibited or delayed the formation of HUVEC tube-like structures, and the results of a chick chorioallantoic membrane assay also revealed that AuNPs suppressed new capillary formation. The present report, therefore, demonstrates the inhibitory effects of AuNPs on VEGF 165 -induced HUVEC migration and tubule formation.
During neovascularization, vascular endothelial cell proliferation and migration occur initially, followed by organization into a network of tube-like structures and the formation of new capillaries [20]. During these complex processes, VEGF is considered the most important proangiogenic factor; in particular, VEGF 165 and VEGF 121 play major regulatory roles in the functions of vascular endothelial cells [4,21]. A previous study reported that AuNPs could bind to VEGF 165 through its heparin-binding domain but could not bind to VEGF 121 , which does not contain a heparinbinding domain [12,13,22]. Consequently, VEGF 165 was used to stimulate HUVECs in all of the present experiments.
Reportedly, VEGFR-2 (Flk-1) is the main receptor through which VEGF mediates its biological effects in endothelial cells [23,24]. VEGF 165 can act through VEGFR-2 to mediate a signaling cascade that regulates all of the key steps of the angiogenic process, including endothelial cell division, proliferation, migration, and tube formation [25]. Several studies have suggested that the processes involved in the formation of new blood vessels, including proliferation, migration, and tube formation, could be suppressed by blocking VEGF signaling [15,19,25,26]. The PI3K/Akt pathway is considered very important among the VEGF/VEGFR-2 downstream signaling pathways [27]. This pathway can regulate endothelial cell migration via the VEGFR-2/PI3K/Akt/eNOS axis [5,6]. Akt (protein kinase B), a serine/threonine-specific protein kinase, regulates endothelial nitric oxide synthase activation to stimulate vasodilation, vascular remodeling, and angiogenesis [9,20,28]. Akt plays a critical role in endothelial cell migration and can be activated through the specific binding of VEGF 165 to VEGFR-2, which then triggers the downstream VEGFR-2/PI3K/Akt signaling pathway. In this study, we used Western blotting to examine Akt phosphorylation and found that AuNPs significantly reduced VEGF 165 -induced Akt phosphorylation. In summary, it seems possible that AuNPs could inhibit VEGF 165 -induced HUVEC migration and tube formation by blocking the Akt signaling pathway subsequent to highaffinity binding to the heparin-binding domain of VEGF 165 , thus preventing the VEGF 165 -VEGFR-2 interaction. As a result, VEGF 165 was inactivated by AuNPs, and the VEGFmediated HUVEC stimulation on HUVECs was weakened, thus inhibiting the migratory and tube forming abilities. Changes in cell morphology and structure are closely related to a cell's functional status; for example, endothelial cell morphological changes can be observed during proliferation, migration, or even tube formation [17]. What changes in cell morphology and structure can be observed in activated HUVECs? Therefore, it is very important to study the morphology and ultrastructure of HUVECs to investigate migration or tube formation. We observed the cell membrane surface ultrastructures using atomic force microscopy (AFM), which allowed the cellular changes after VEGF 165 treatment in the presence or absence of AuNPs to be observed visually. The AFM images revealed increased HUVEC activity in response to VEGF 165 treatment and cells with pleomorphic appearance, obvious pseudopodia, larger particles, and increased cell membrane surface roughness; in contrast, the HUVECs appeared to be less active after AuNP treatment. These results indicated that AuNPs could significantly block VEGF 165 -induced cellular proliferation or migration. AFM is a powerful method that we can with  which image cell surface ultrastructures under physiological conditions [29]. As a nondestructive surface imaging tool, AFM can obtain nanometric-scale images of the cell surface and can provide very important morphological and cell membrane details [18,30]. Changes in cell morphology and pseudopod formation indicate the level of cell activity, and the cell surface particles indicate metabolic activity and functional protein secretion [31]. Endothelial cell migration and morphological changes are essential for angiogenesis [6]. Therefore, the changes in cellular morphology and surface ultrastructure that were detected in this study might intuitively reflect the migratory and tube forming abilities of the HUVECs and could thus provide new insights into the angiogenic process in these cells.
In summary, our study focused on the inhibitory effects of AuNPs on HUVEC migration, CAM, and tube formation, the key angiogenic characteristics of endothelial cells. Moreover, we used AFM to observe changes in cellular micromorphology. The early stages of neovascularization can be divided into three well-known steps: endothelial cell proliferation, migration, and tube formation; these are regulated by VEGF and are also the main targets of antiangiogenesis [15,25]. We previously reported that AuNPs could inhibit VEGF 165induced HUVEC proliferation by binding to the sulfur and amine groups present in the heparin-binding domains of VEGF 165 [13]. In this study, we observed that AuNPs could also inhibit two other angiogenic steps.

Conclusions
In conclusion, our study demonstrated that AuNPs could significantly inhibit VEGF 165 -induced HUVEC migration and tube formation both in vitro and in vivo and provided the first nanoscale images of the AuNP-mediated inhibition of HUVEC cell migration and tube formation. We speculate that AuNPs might inhibit these processes in Akt signaling pathway-mediated processes because the particles bind to VEGF 165 and thus indirectly reduce VEGFR-2 activation. These findings have provided new insights into the antiangiogenic effects of AuNPs.