Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome

Abstract

Angiogenic factors are critical to the initiation of angiogenesis and maintenance of the vascular network. Here we use human genetics as an approach to identify an angiogenic factor, VG5Q, and further define two genetic defects of VG5Q in patients with the vascular disease Klippel-Trenaunay syndrome (KTS). One mutation is chromosomal translocation t(5;11), which increases VG5Q transcription. The second is mutation E133K identified in five KTS patients, but not in 200 matched controls. VG5Q protein acts as a potent angiogenic factor in promoting angiogenesis, and suppression of VG5Q expression inhibits vessel formation. E133K is a functional mutation that substantially enhances the angiogenic effect of VG5Q. VG5Q shows strong expression in blood vessels and is secreted as vessel formation is initiated. VG5Q can bind to endothelial cells and promote cell proliferation, suggesting that it may act in an autocrine fashion. We also demonstrate a direct interaction of VG5Q with another secreted angiogenic factor, TWEAK (also known as TNFSF12). These results define VG5Q as an angiogenic factor, establish VG5Q as a susceptibility gene for KTS, and show that increased angiogenesis is a molecular pathogenic mechanism of KTS.

Figure 1

Positional cloning of the VG5Q gene. a, Definition of chromosome breakpoints involved in translocation t(5;11)(q13.3;q15.1) associated with KTS. Chr 5/Chr 11, normal chromosomes; Der 5/Der 11, derivative chromosomes. b, The amino acid sequences of human VG5Q protein. The FHA domain (435–508) and a G-patch domain (619–663) are indicated. The estimated pI of VG5Q is 5.2. c, d, Identification of VG5Q mutation E133K in five independent KTS patients. c, DNA sequence analysis (G to A substitution at codon 133). d, Schematic representation of mutation E113K (substitution of a glutamic acid residue by a lysine residue).

Figure 2

Expression profile of VG5Q and dynamic redistribution and secretion of VG5Q protein during angiogenesis. A, B, VG5Q expression in HMVECs and HUVECs determined by northern (A) and western blot analyses (B). C, Tissue expression pattern of VG5Q determined by northern blot analysis. D, Expression of VG5Q in different cell lines determined by RT–PCR. Tubulin, internal control. E, Immunostaining of mouse kidney for VG5Q protein expression. The nucleus was stained with DAPI (blue signal). CD31, endothelial cell marker; α-actin, VSMC marker. F, HMVECs were cultured on plastic (a, b, g) and matrigel (c–f, h). VG5Q protein, red; nucleus, blue (DAPI). c, d, f, One hour on matrigel; e, 4 h on matrigel; h, 24 h on matrigel. G, Competitive ELISA analysis to show that VG5Q is secreted. Relative HRP activity, absorbance reading of the wells. An asterisk indicates statistical significance (P < 0.05). H, Detection of VG5Q in the media by metabolic labelling and immunoprecipitation, indicating that cells secrete VG5Q into the media. Control, HUVECs transfected with pcDNA3.1; plastic and matrigel, HUVECs transfected with pcDNA3.1-VG5Q and plated on either plastic or matrigel plates, respectively. The 84-kDa band indicated by an arrow represents VG5Q. The nature of the small 45-kDa band is unknown, but may be a cleaved VG5Q product. Faint signal was also observed in the media from cells cultured on plastic dishes. This may suggest weak secretion of VG5Q under this condition.

Figure 3

VG5Q is an angiogenic factor and both VG5Q E133K and KTS translocation t(5;11) are functional mutations. A, Angiogenesis promoted by VG5Q protein was determined by CAM assays. EK, VG5Q with mutation E133K. B, Effect of siRNA against VG5Q on endothelial tube formation. Scramble, control siRNA. Suppression of VG5Q expression by siRNAs was determined by RT–PCR (ribosomal RNA as control) and western blot analyses (tubulin as control). C, Binding of VG5Q to endothelial cells by cell adhesion assays. D, The t(5;11)(q13.3;p15.1) translocation associated with KTS increases expression of VG5Q. Results represent mean of triplicate cultures ± standard deviation from three independent experiments. EC, endothelial cell.

Figure 4

VG5Q interacts with TWEAK. a, Pull-down assays using GST–TWEAK. Input, ³⁵S-labelled luciferase (Luc, about 61 kDa) and VG5Q (about 84 kDa). Right panel, binding of VG5Q with GST–TWEAK, but not with GST; middle panel, no interaction between GST–TWEAK with luciferase. b, Co-immunoprecipitation of TWEAK with VG5Q from HVSMC protein extract by a rabbit anti-VG5Q antibody. Bound materials were analysed by western blot using a goat anti-TWEAK antibody. NRS, normal rabbit serum as a negative control. 50-kDa band, IgG cross-reaction. c, Co-localization of VG5Q and TWEAK in HUVECs. VG5Q, red signal; TWEAK, green; nuclei, blue (DAPI).