Aspalatone Prevents VEGF-Induced Lipid Peroxidation, Migration, Tube Formation, and Dysfunction of Human Aortic Endothelial Cells

Abstract

Although aspalatone (acetylsalicylic acid maltol ester) is recognized as an antithrombotic agent with antioxidative and antiplatelet potential; its efficacy in preventing endothelial dysfunction is not known. In this study, we examined the antiangiogenic, antioxidative, and anti-inflammatory effect of aspalatone in human aortic endothelial cells (HAECs). Specifically, the effect of aspalatone on VEGF-induced HAECs growth, migration, tube formation, and levels of lipid peroxidation-derived malondialdehyde (MDA) was examined. Our results indicate that the treatment of HAECs with aspalatone decreased VEGF-induced cell migration, tube formation, and levels of MDA. Aspalatone also inhibited VEGF-induced decrease in the expression of eNOS and increase in the expression of iNOS, ICAM-1, and VCAM-1. Aspalatone also prevented the VEGF-induced adhesion of monocytes to endothelial cells. Furthermore, aspalatone also prevented VEGF-induced release of inflammatory markers such as Angiopoietin-2, Leptin, EGF, G-CSF, HB-EGF, and HGF in HAECs. Thus, our results suggest that aspalatone could be used to prevent endothelial dysfunction, an important process in the pathophysiology of cardiovascular diseases.

Figure 1

Effect of aspalatone on HAECs viability: HAECs (3000 cells/well) were treated with indicated concentrations of aspalatone without or with VEGF (10 ng/mL). After 24 h, cell viability was determined by MTT assay. Values are mean ± SD (n = 5). ^∗p < 0.05 when compared with control and ^#p < 0.005 when compared with VEGF- treated group.

Figure 2

Aspalatone inhibits VEGF-induced migration of HAECs. (a) In vitro wound healing assay showing effect of aspalatone on VEGF-induced migration in HAECs. HAECs were grown in 12-well tissue culture dishes and after the cells became confluent, the monolayer was scratched with a sterile pipette tip. The media was replaced with VEGF (10 ng/mL) without or with aspalatone (50 μM) and the cells were incubated for the indicated time periods. Photographs were taken at 0 h and 18 h when the VEGF containing monolayer wound was completely filled. (b) Histogram showing migration rate calculated by formula (Width_0 hr−Width_18 hr)/Width_0 hr × 100. Representative photographs from three independent experiments are shown. Magnification 10x. Scale bar: 400 μm.

Figure 3

Aspalatone inhibits VEGF-induced tube formation of HAECs. (a) In vitro angiogenesis assay was performed in the absence or presence of different concentrations of aspalatone (10 μM, 20 μM, 50 μM, and 100 μM) by using in vitro angiogenesis assay kit from Millipore. (b) Histograms showing length of capillaries treated with different concentration of aspalatone. Bars represent mean ± SD (n = 6). Representative photographs from three independent experiments are shown. Magnification 4x. ^∗p < 0.05 when compared with control group. Scale bar: 1000 μm.

Figure 4

Aspalatone inhibits VEGF-induced adhesion of monocytes to HAECs. (a) HAECs (3000 cells/well) in 96-well plates were treated with aspalatone (50 μM) overnight followed by addition of THP-1 cells and VEGF (10 ng/mL) for another 18 h. Cell adhesion was determined by MTT absorbance recorded at 570 nm using a plate reader. Bars represent mean ± SD (n = 5). ^∗p < 0.05 when compared with control and ^#p < 0.005 when compared with VEGF-treated group. HAECs treated without or with (b) VEGF (10 ng/mL) or (c) LPS (1 μg/mL) in the absence and presence of aspalatone (50 μM) for 18 h. Equal amounts of cell extracts were subjected to Western blot analysis using specific antibodies against VCAM-1, ICAM-1, eNOS, iNOS, and GAPDH in HAECs.

Figure 5

Aspalatone modulates VEGF-induced expression of inflammatory cytokines and growth factors in HAECs. Culture media of HAECs treated with VEGF (10 ng/mL) in the absence or presence of aspalatone (50 μM and 100 μM) were analyzed for inflammatory cytokines by using a Human Angiogenesis/Growth Factor Magnetic bead panel kit from Millipore following manufacturer's instructions using a Milliplex Analyzer System. Bars represent mean ± SD (n = 4). ^∗p < 0.05 and ^∗∗p < 0.001 when compared with control and ^#p < 0.01 and ^##p < 0.001 when compared with VEGF-treated group.

Figure 6

Aspalatone inhibits VEGF-induced reactive oxygen species production in HAECs. (a) Flow cytometric analysis showing CM-H₂DCFDA fluorescence in HAECs treated with VEGF (10 ng/mL) without or with aspalatone (50 μM) for 18 h (red: VEGF alone treated, pink: VEGF + aspalatone, green: control, and blue: aspalatone). Unfilled histograms with solid black lines were unstained controls. (b) Bars showing fold change in CM-H₂DCFDA Mean Fluorescence Intensity (MFI) in HAECs treated with aspalatone (50 μM) in the absence or presence of VEGF (10 ng/mL) for 18 h. Bars represent mean ± SD (n = 3). ^∗p < 0.05 when compared with control and ^#p < 0.001 when compared with VEGF-treated group.

Figure 7

Aspalatone prevents VEGF-induced lipid peroxidation-derived malondialdehyde levels in HAECs. HAECs treated without or with 50 μM aspalatone in the absence and presence of VEGF (10 ng/mL) for 18 h. The MDA levels were measured by using a kit from Oxis International Inc. MDA values (μM) were normalized to total cellular protein content and presented as fold change compared to control. Bars represent mean ± SD (n = 3). ^∗p < 0.05 when compared with control and ^#p < 0.001 when compared with VEGF-treated group.