Cilengitide Inhibits Neovascularization in a Rabbit Abdominal Aortic Plaque Model by Impairing the VEGF Signaling

Abstract

Background: Cilengitide is a selective α _v β ₃ and α _v β ₅ integrin inhibitor. We sought to investigate the effect of cilengitide on the neovascularization of abdominal aortic plaques in rabbits and explore its underlying antiangiogenic mechanism on human umbilical vein endothelial cells (HUVECs).

Materials and methods: For the in vivo experiment, the abdominal aortic plaque model of rabbits was established and injected with different doses of cilengitide or saline for 14 consecutive days. Conventional ultrasound (CUS) and contrast-enhanced ultrasound (CEUS) were applied to measure the vascular structure and blood flow parameters. CD31 immunofluorescence staining was performed for examining neovascularization. Relative expressions of vascular endothelial growth factor (VEGF) and integrin of the plaque were determined. For in vitro experiments, HUVECs were tested for proliferation, migration, apoptosis, and tube formation in the presence of different doses of cilengitide. Relative expressions of VEGF, integrin, and Ras/ERK/AKT signaling pathways were determined for the exploration of underlying mechanism.

Results: CEUS showed modestly increased size and eccentricity index (EI) of plaques in the control group. Different degrees of reduced size and EI of plaques were observed in two cilengitide treatment groups. The expressions of VEGF and integrin in the plaque were inhibited after 14 days of cilengitide treatment. The neovascularization and apoptosis of the abdominal aorta were also significantly alleviated by cilengitide treatment. For in vitro experiments, cilengitide treatment was found to inhibit the proliferation, migration, and tube formation of HUVECs. However, cilengitide did not induce the apoptosis of HUVECs. A higher dose of cilengitide inhibited the mRNA expression of VEGF-A, β ₃, and β ₅, but not α _V. Lastly, cilengitide treatment significantly inhibited the Ras/ERK/AKT pathway in the HUVECs. Conclusions. This study showed that cilengitide effectively inhibited the growth of plaque size by inhibiting the angiogenesis of the abdominal aortic plaques and blocking the VEGF-mediated angiogenic effect on HUVECs.

Figure 1

Schematic diagram of the total vessel area (TVA) and the eccentricity index (EI). (a) The grey ellipse in the lower two vessels represents TVA. (b) The components and calculation formula for EI.

Figure 2

Visualization of representative abdominal aortic plaques and contrast agent perfusion in different imaging modes in the T2 group. (a) Plaque on CUS. (b) The boundary of the plaque is shown more distinctly on CEUS. (c) The clear appearance of contrast agent microbubbles in the plaque.

Figure 3

Ultrasound images of a representative plaque in the T2 group in different imaging modes and at different periods. (a) Pretreatment on CUS. (b) Pretreatment on CEUS. (c) Posttreatment on CUS. (d) Posttreatment on CEUS.

Figure 4

Representative image for H&E and Movat staining. (a) The representative image for H&E staining. (b) The representative image for Movat staining.

Figure 5

Cilengitide inhibits neovascularization within the plaque of the abdominal aorta. (a) The representative image for CD31-positive staining. The white arrow indicates the CD31-positive microvessels in the plaque. Scale bar, 100 μm. (b) Quantitative results of CD31-positive cells. n = 3 for the control, T0, and T1 groups; n = 4 for the T2 group. ^##p < 0.01 vs. the T0 group.

Figure 6

Cilengitide attenuates the apoptosis of abdominal aorta. (a) The representative image for TUNEL staining. Scale bar, 100 μm. (b) Quantitative results of TUNEL-positive cells. n = 3 for the control, T0, and T1 groups; n = 4 for the T2 group. ^∗∗p < 0.01 vs. the control group; ^#p < 0.05 vs. the T0 group; ^##p < 0.01 vs. the T0 group.

Figure 7

Cilengitide inhibits the VEGF expression in rabbit abdominal aortic plaque. (a, b) The representative image and quantification of VEGF-A in the plaque of the abdominal aorta. (c) The mRNA expression of VEGF in the plaque of the abdominal aorta. (d, e) The content of VEGF-A and VEGF-R2 was evaluated by ELISA in the plaque of the abdominal aorta. (f–h) Relative mRNA of α_v, β₃, and β₅ in the plaque of the abdominal aorta. (i–l) The representative image and quantification of MMP-2, MMP-9, and MCP-1 expression in the plaque of the abdominal aorta. n = 3 for the control, T0, and T1 groups; n = 4 for the T2 group. ^∗∗p < 0.01 vs. the control group; ^#p < 0.05 vs. the T0 group; ^##p < 0.01 vs. the T0 group.

Figure 8

The effect of cilengitide on the proliferation, migration, apoptosis, and tube formation of HUVECs. (a) The quantification of cell proliferation was evaluated by CCK-8 assay for HUVECs under treatment with different concentrations of cilengitide (0-10 μM) for 24 hours. (b, c) The representative image and quantification of cell migration evaluation of HUVECs under treatment with different concentrations of cilengitide (0-10 μM) for 24 hours. Scale bar, 100 μm. (d, e) The representative image and quantification of apoptosis were evaluated by flow cytometry analysis for HUVECs under treatment with different concentrations of cilengitide (0-10 μM) for 24 hours. (f) The representative image of tube formation assay of HUVECs under treatment with different concentrations of cilengitide (0-10 μM) for 8 hours. (g–i) Quantification of the length of tubes, tube area, and the number of capillary-like structures for the tube formation assay of HUVECs. n = 5 for each group. Scale bar, 100 μm. ^∗p < 0.05 vs. 0 μM; ^∗∗p < 0.01 vs. 0 μM.

Figure 9

Cilengitide treatment inhibits the mRNA expression of VEGF, α_v, β₃, and β₅ of HUVECs. (a) Cilengitide inhibits the mRNA expression of VEGF under doses of 10 μM. (b) Cilengitide did not affect the mRNA expression of α_v. (c) Cilengitide inhibits the mRNA expression of β₃ under doses of 10 μM. (d) Cilengitide inhibits the mRNA expression of β₅ under the doses of 0.1 μM, 1 μM, and 10 μM. n = 5 for each group. ^∗p < 0.05 vs. 0 μM; ^∗∗p < 0.01 vs. 0 μM.

Figure 10

Cilengitide treatment inhibits the expression of VEGF and its downstream Ras/ERK/AKT signaling pathway of HUVECs. (a) Representative image of western blots for the protein expression of VEGF, Ras, p-ERK/EKR, and p-AKT/AKT under treatment with 0 μM, 1 μM, and 10 μM cilengitide. (b–e) Quantification of the protein expression levels. n = 5 for each group. ^∗p < 0.05 vs. 0 μM; ^∗∗p < 0.01 vs. 0 μM.

Figure 11

Schematic illustration of the effect of cilengitide on the HUVECs. Recognizing the ligands with conserved arginine-glycine-aspartic acid (RGD) motifs, integrin α_vβ₃ mediates the various activities of cell adhesion. Cilengitide has a high affinity for integrin α_vβ₃ and inhibits relevant intracellular signaling. It inhibits the proliferation, migration, and tube formation of HUVECs but not affects apoptosis.