Endothelin-1 induces an angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo

Abstract

The endothelial cell-derived endothelin-1 (ET-1) is a potent mitogen for endothelial cells, vascular smooth muscle cells, and tumor cells. In this study, we analyzed the role of ET-1 on human umbilical vein endothelial cell (HUVEC) phenotype related to different stages of angiogenesis. ET-1 promoted HUVEC proliferation, migration, and invasion in a dose-dependent manner. The ET(B) receptor (ET(B)R) antagonist, BQ 788, blocked the angiogenic effects induced by ET-1, whereas the ET(A)R antagonist was less effective. ET-1 stimulated matrix metalloproteinase-2 mRNA expression and metalloproteinase-2 production, as determined by reverse transcriptase-polymerase chain reaction and gelatin zymography. Furthermore ET-1 was able to enhance HUVEC differentiation into cord vascular-like structures on Matrigel. When tested in combination with vascular endothelial growth factor (VEGF), ET-1 enhanced VEGF-induced angiogenic-related effects on endothelial cells in vitro. Finally, using the Matrigel plug neovascularization assay in vivo, ET-1 in combination with VEGF stimulated an angiogenic response comparable to that elicited by basic fibroblast growth factor. These findings demonstrated that ET-1 induces angiogenic responses in cultured endothelial cells through ET(B)R and that stimulates neovascularization in vivo in concert with VEGF. ET-1 and its receptors acting as angiogenic regulators might represent new targets for anti-angiogenic therapy.

Figure 1.

Proliferative effect of ET-1 and VEGF on HUVEC. Endothelial cells (1 × 10 cells/ml) were plated in 0.5% FCS medium. Different doses of ET-1 were added to endothelial cells. ET receptor antagonists (1 μmol/L) were incubated 15 minutes before the addition of ET-1 (10 nmol/L). VEGF (1 ng/ml) was added in the absence or presence of ET-1 (10 nmol/L). In all cases, [³H]thymidine incorporation was analyzed 24 hours after the addition of agonists. Data are means of results from three experiments, each performed in sextuplicate. Bars, SD. a: P ≤ 0.001 compared to control. b: P ≤ 0.001 compared to ET-1 10 nmol/L. c:P ≤ 0.01 compared to ET-1 and VEGF.

Figure 2.

Effect of ET-1 and VEGF on HUVEC migration. Endothelial cells (5 × 10 cells/ml) pretreated for 15 minutes with 1 μmol/L of the ET_AR (BQ 123) or ET_BR antagonist (BQ 788) were placed in the upper compartment of a 48-well Boyden chamber. VEGF (1 ng/ml), ET-1 (10 nmol/L), and ET_BR agonists (ET-3 and S6c, 10 nmol/L) were added in lower wells. Cells migrated through the filter were counted after 4 hours. Data are expressed as the number of migrated cells in 10 high-power fields and are the means of results from three experiments each performed in triplicate. Bars, SD. a: P ≤ 0.02 compared to control. b: P ≤ 0.001 compared to control. c: P ≤ 0.02 compared to ET-1. d: P ≤ 0.01 compared to VEGF.

Figure 3.

Effect of ET-1 and VEGF on HUVEC invasion. HUVEC (5 × 10 cells/ml) were seeded on Matrigel layer in a Boyden chamber assay. Endothelial cells were preincubated for 15 minutes with ET_A (BQ 123) or ET_B (BQ 788) receptor antagonists (1 μmol/L) before the addition of 100 nmol/L ET-1. VEGF (1 ng/ml) in the absence or presence of ET-1 (100 nmol/L) was added in the lower wells. Cells migrating through the filter were counted after 6 hours. Data are expressed as the number of migrated cells in 10 high-power fields and are means of results from three experiments each performed in triplicate. Bars, SD. a: P ≤ 0.001compared to control. b: P ≤ 0.001 compared to ET-1. c: P ≤ 0.01 compared to VEGF.

Figure 4.

Effect of ET-1 and VEGF on MMP-2 production in HUVEC. Endothelial cells were cultured for 24 hours in 0.5% FCS in the absence or in presence of 100 nmol/L ET-1 or 1 ng/ml VEGF. A: After incubation, the conditioned medium of control (lane 1), 100 nmol/L ET-1 (lane 2), and 1 ng/ml VEGF (lane 3) -treated cells was analyzed by gelatin-zymography, showing that HUVEC released the 72-kd form of MMP-2 into their supernatants. B: MMP-2 mRNA detected by RT-PCR in HUVEC. PCR products for MMP-2 and GAPDH of control (lane 1), 100 nmol/L ET-1 (lane 2), and 1 ng/ml VEGF (lane 3) were shown as visualized by ethidium bromide. C: The relative expression of MMP-2 mRNA (open bars) or protein (hatched bars) in HUVEC. Protein production was determined by densitometric scanning of zymography of cells treated with ET-1 or VEGF. mRNA levels were determined by densitometric scanning of ethidium-bromide bands normalized to those of GAPDH. Data are the mean ± SD of three independent experiments and are expressed as the percent of control. **, P < 0.001 compared to control.

Figure 4.

Effect of ET-1 and VEGF on MMP-2 production in HUVEC. Endothelial cells were cultured for 24 hours in 0.5% FCS in the absence or in presence of 100 nmol/L ET-1 or 1 ng/ml VEGF. A: After incubation, the conditioned medium of control (lane 1), 100 nmol/L ET-1 (lane 2), and 1 ng/ml VEGF (lane 3) -treated cells was analyzed by gelatin-zymography, showing that HUVEC released the 72-kd form of MMP-2 into their supernatants. B: MMP-2 mRNA detected by RT-PCR in HUVEC. PCR products for MMP-2 and GAPDH of control (lane 1), 100 nmol/L ET-1 (lane 2), and 1 ng/ml VEGF (lane 3) were shown as visualized by ethidium bromide. C: The relative expression of MMP-2 mRNA (open bars) or protein (hatched bars) in HUVEC. Protein production was determined by densitometric scanning of zymography of cells treated with ET-1 or VEGF. mRNA levels were determined by densitometric scanning of ethidium-bromide bands normalized to those of GAPDH. Data are the mean ± SD of three independent experiments and are expressed as the percent of control. **, P < 0.001 compared to control.

Figure 5.

Morphogenic activity of ET-1 and VEGF. HUVEC (3.5 × 10 cells/well) were seeded onto Matrigel-precoated wells and cultured in low-serum conditions (0.5% FCS) in absence (control) (A) or in presence of 10 nmol/L ET-1 (B), 1ng/ml VEGF (C), or ET-1 (10 nmol/L) + VEGF (1ng/ml) (D). Photographs were taken 24 hours later. 0riginal magnification, ×200.

Figure 6.

Histological analysis of the angiogenic response induced by ET-1 and VEGF in the Matrigel pellets. A: The histology of Matrigel pellets containing vehicle presented only few infiltrating single cells. B: The addition of both ET-1 (100 nmol/L) and VEGF (300 ng/ml) to the Matrigel resulted in the induction of cellularity and in the formation of cords, tubules, and blood-containing vessels. Original magnification, ×300; counterstained with hematoxylin.