Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo

Abstract

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.

Figure 1.

Synergistic induction of HUVEC tubulogenesis by the combination of HGF and VEGF. HUVECs were cultured in 3-D collagen gels in BM alone (A), or BM supplemented with VEGF (200 ng/ml) (B), HGF (200 ng/ml) (C), or a combination of VEGF and HGF (200 ng/ml each) (D). Photographs were taken at 72 hours. Size marker, 100 μm. Note the interconnected network of HUVECs in the combined HGF- and VEGF-treated group.

Figure 2.

Morphology of HUVECs at 4, 24, and 48 hours in 3-D collagen gels when cultured in BM and VEGF (200 ng/ml) and HGF (200 ng/ml). A–C: Representative photomicrographs taken at 4 (A), 24 (B), and 48 (C) hours using Hoffman modulation optics. D: Representative transmission electron micrograph showing lumen-like structure (arrow). Size marker shown at bottom, 3 μm.

Figure 3.

HGF dose-dependently induced HUVEC tube formation in 3-D collagen gels in the presence of a fixed concentration of VEGF. HUVECs were cultured in 3-D collagen gels in BM supplemented with VEGF (400 ng/ml) in the presence of the indicated concentrations of HGF for 72 hours. Average tube length was obtained by measuring as described in Materials and Methods and is expressed as the mean ± SEM from three independent experiments. *, P < 0.05 versus VEGF treatment.

Figure 4.

The combination of HGF and VEGF inhibits HUVEC death (at 48 hours) in 3-D collagen gels. A: HUVECs were cultured in 3-D gels in BM alone or BM supplemented with HGF (200 ng/ml), VEGF (200 ng/ml), or the combination of HGF and VEGF (200 ng/ml each) for 24 hours. Cell viability was determined as described in Materials and Methods (means ± SD, n = 9). B and C: Photomicrographs of acridine orange-stained nuclei taken using a confocal microscope showing representative pyknotic (B) and normal (C) nuclei. D and E: Effects of HGF and VEGF on the expression of A1 (D) and bcl-2 (E). HUVECs were cultured in 3-D collagen gels in BM supplemented with HGF (200 ng/ml), VEGF (200 ng/ml), or the combination of HGF and VEGF (200 ng/ml each). Total RNA were extracted at 6 hours and 24 hours and duplicate samples from two independent experiments analyzed by quantitative RT-PCR (Taqman) as described in Materials and Methods. Results are expressed as the ratio of the indicated mRNA to the level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase in the same sample. The legend for A, D, and E, is shown in the inset to A.

Figure 5.

Induction of HUVEC tubulogenesis in NDFB/HUVEC co-cultures in the presence of VEGF. a: NDFBs alone. b: HUVECs alone. c: 1:1 mixture of NDFBs/HUVECs. d and e: Same field of a mixture of NDFB co-cultured with Di-I-Ac-LDL-prelabeled HUVECs under phase (d) and fluorescent (e) optics. All cells were cultured in 3-D collagen gels in BM supplemented with VEGF (200 ng/ml) for 72 hours. Note that NDFBs remained as intact, single cells (a) whereas the majority of the HUVECs appeared apoptotic or dead (b) in 3-D collagen gels. Tubular structures were formed in NDFB/HUVEC co-culture (c). Size marker, 100 μm. Corresponding Di-I-Ac-LDL-labeled HUVECs comprise the interconnected tube-like structures (d and e). Large arrow, endothelial cell labeled with Di-I-Ac-LDL; arrowhead, fibroblast, which is elongated but not fluorescent.

Figure 6.

Neutralizing antibody against HGF and HGF mutants NK1 and NK2 blocked NDFB CM-induced HUVEC tube formation in 3-D collagen gels. A: CM from NDFBs were obtained and used as the culture medium for HUVEC in 3-D collagen gels supplemented with VEGF (200 ng/ml) as described in Materials and Methods. Anti-HGF antibodies were given at time 0 at concentrations of 1 μg/ml or 10 μg/ml. Results are expressed as mean ± SEM from three independent experiments. #, P < 0.05 versus CM groups with supplemental VEGF (200 ng/ml). *, P < 0.05 versus CM without VEGF. B: HUVECs were co-cultured with NDFBs in 3-D collagen gels for 72 hours in BM containing 200 ng/ml VEGF in the presence of PBS (control), NK1, or NK2 (1 μg/ml). Data are shown as the mean tube length ± SEM of three independent experiments. *, P < 0.05 compared to control (NDFBs: HUVEC co-cultures incubated with 200 ng/ml VEGF).

Figure 7.

. Representative photomicrographs of the effects of VEGF and HGF alone and in combination on angiogenesis in vivo. A–D: Representative flat-mount photomicrographs of rat corneas 6 days after implantation of hydron pellets A: Control, excipient alone. B: HGF (200 ng/ml). VEGF (200 ng/ml) (C) and VEGF (200 ng/ml) and HGF (200 ng/ml) in combination (D). E: Summary data of the in vivo angiogenic response to control, VEGF-, HGF-, and VEGF and HGF-treated groups. Data are expressed as mean ± SE, n = 5 animals/group. *, Significantly different from control; **, significantly different from VEGF alone. ⁺, Significantly different from HGF alone (Mann-Whitney test for nonparametric values).