Using gene expression profiling to identify the molecular basis of the synergistic actions of hepatocyte growth factor and vascular endothelial growth factor in human endothelial cells

Abstract

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are two potent endothelial mitogens with demonstrated angiogenic activities in animal models of therapeutic angiogenesis. Several recent studies suggest that these growth factors may act synergistically, although the mechanism of this interaction is not understood. Changes in the gene expression profile of human umbilical vein endothelial cells treated with HGF, VEGF or the combination of the two were analyzed with high-density oligonucleotide arrays, representing approximately 22000 genes. Notably, the genes significantly up- and downregulated by VEGF versus HGF exhibited very little overlap, indicating distinct signal transduction pathways. The combination of HGF and VEGF markedly increased the number of significantly up- and downregulated genes. At 4 h, the combination of the two growth factors induced a number of chemokine and cytokines and their receptors (IL-8, IL-6, IL-11, CCR6, CXCR1,CXC1 and IL17RC), numerous genes involved in growth factor signal transduction (egr-1, fosB, grb10, grb14,MAP2K3,MAP3K8, MAPKAP2,MPK3, DUSP4 and DUSP6), as well as a number of other growth factors (PDGFA, BMP2, Hb-EGF, FGF16, heuregulin beta 1, c-kit ligand, angiopoietin 2 and angiopoietin 4 and VEGFC). In addition, the VEGF receptors neuropilin-1 and flt-1 were also upregulated. At 24 h, a clear 'cell cycle' signature is noted, with the upregulated expression of various cell cycle control proteins and gene involved in the regulation of mitosis and mitotic spindle assembly. The receptor for HGF, c-met, is also upregulated. These data are consistent with the hypothesis that the combination of HGF and VEGF results in the cooperative upregulation of a number of different molecular pathways leading to a more robust proliferative response, that is, growth factor(s), receptors, molecules involved in growth factor signal transduction, as well as, at later time points, upregulation of the necessary cellular proteins required for cells to escape cell cycle arrest and enter the cell cycle.

Figures

Figure 1

Venn diagram representation of those genes significantly upregulated (a) or downregulated (b) by HGF (solid circle), VEGF (dashed circle) or the combination of the two growth factors (circle with dashes and dots) at 4 h (top) and 24 h (bottom).

Figure 2

Dot plot representation of the expression of 566 probes upregulated by more than 1.5-fold, P<0.1 at 4 h, in the presence of the combination of HGF and VEGF at 4 h. The same probesets were compared to the expression data for HGF alone (b) and VEGF alone (a) at 4 h. Blue denotes not significantly changed from basal and red, significantly upregulated over basal expression.

Figure 3

Agglomerative cluster of genes significantly upregulated or downregulated by HGF, VEGF, or the combination of the two growth factors at 4 h and 24 h. Black indicates ‘not detected' or did not reach statistical significance (P>0.1); green represents downregulated, and red, upregulated.