Involvement of integrin-linked kinase in capillary/tube-like network formation of human vascular endothelial cells

Abstract

Angiogenesis is a complex process involving an ECM and vascular endothelial cells (EC), and is regulated by various angiogenic factors including VEGF. The ability to form a capillary/tube-like network is a specialized function of EC. Therefore, in vitro angiogenesis was assessed by a capillary/tube-like network formation assay. There are three angiogenic parameters: capillary length, number of capillaries, and relative capillary area per field. We evaluated capillary length per field in the assay. VEGF promoted capillary/tube-like network formation of EC in a type I collagen gel matrix in vitro. Moreover, we demonstrated the involvement of ILK in a VEGF signaling pathway mediating capillary/tube-like network formation of EC using dominant-negative, kinase deficient ILK. This is a straightforward assay to monitor responses of human vascular endothelial cells.

Fig. 1. Formation of a capillary/tube-like network of human vascular endothelial cells stimulated with VEGF.

HUVEC were stimulated with 30 ng/ml of VEGF and cultured in a type I collagen gel-coated plate for eight hours at 37°C. The microscopic image was recorded by a CCD video camera and analyzed using image analysis software. a: Microscope image, b: Processed image.

Fig. 2. VEGF induced capillary/tube-like network formation of human vascular endothelial cells in a concentration-dependent fashion.

HUVEC (1.5 × 104 cells/well) were incubated in a type I collagen gel-coated plate with the indicated concentration of VEGF at 37°C for eight hours. The microscopic image was recorded by a CCD video camera and analyzed using image analysis software. The results represent the mean ± SD values.

Fig. 3. Inhibition of ILK kinase activity by ILK-KD or LY294002 in HUVEC.

The cells were stimulated with 30 ng/ml VEGF for 5 minutes, and then ILK activity was measured by ILK kinase assay. Anti-ILK blot was prepared from the same immunoprecipitates used for the kinase assay (top panel). Bars represent the mean ± SD values. EV, empty vector; KD, ILK-KD.

Fig. 4. Effect of a dominant negative, kinase-deficient ILK on VEGF-induced capillary/tube-like network formation of human vascular endothelial cells.

HUVEC were transiently transfected with the indicated plasmids (0.5 μg/ml) 18 hours prior to VEGF stimulation. The cells were incubated in a type I collagen-coated plate for eight hours at 37°C with or without 30 ng/ml VEGF. Bars indicate as the mean ± SD values. The statistical significance is given for the difference between the empty vector-transfected groups plus and minus VEGF stimulation, and for the difference between VEGF-stimulated cells transfected with an empty vector or with a vector encoding ILK-KD, respectively. EV; empty vector, KD; ILK-KD.

Fig. 5. Effect of a LY294002 on VEGF-induced capillary/tube-like network formation of human vascular endothelial cells.

LY294002 was added 30 minutes before VEGF stimulation. The cells were incubated in a type I collagen-coated plate for eight hours at 37°C with or without 30 ng/ml VEGF. Bars indicate as the mean ± SD values.

Fig. 6. Effect of ILK-KD on HUVEC proliferation stimulated with VEGF.

HUVEC were transiently transfected with the indicated vectors and incubated for 18 hours. The cells were cultured in the absence or presence of VEGF for 48 hours. Viable cells were measured by WST assay. The results represent the mean ± SD values.