VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo

Abstract

Background: The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms.

Methods: The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b.

Results: The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts.

Conclusion: Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

Figure 1

Alternative splicing of VEGF-A generating VEGF_xxx and VEGF_xxxb isoforms. (A) VEGF-A contains eight exons with receptor binding found in exons 3 and 4, heparin and neuropilin binding in exons 6 and 7 and 8a. (B) Alternative splicing of the C-terminal end using proximal splice site generating VEGF_xxx isoforms or distal splice site generating VEGF_xxxb isoforms. This splicing leads to an alterative last six amino acids (CDKPRR or SLTRKD). (C) The C-terminal splicing leads to the possibility of two sister families of VEGF-A isoforms; VEGF_xxx and VEGF_xxxb, differing only in the C-terminal.

Figure 2

VEGF₁₂₁b is expressed in human tissue and the expression is reduced in human colon tumours. (A–C) PCR on cDNA from different human tissues using primers to detect VEGF₁₂₁b, VEGF₁₆₅b and VEGF₁₆₅. VEGF₁₆₅ and VEGF₁₆₅b are detected in colon cDNA both in tumour and adjacent control colon (A). VEGF₁₂₁b is detected in colon tissue, aorta, spleen and placenta using VEGF₁₂₁b-specific primers (B). VEGF₁₂₁, ₁₆₅, ₁₂₁b and ₁₆₅b can be found in the retina and to a lesser extent in the human vitreous (C). (D) Densitometry analysis of VEGF₁₂₁b and VEGF₁₆₅b protein expression in matched human control colon samples and colon tumours analysed by western blot using a VEGF_xxxb-specific antibody (n=14 pairs, P=0.0067 paired t-test).

Figure 3

VEGF₁₂₁b inhibits migration of endothelial cells and inhibits serum starved induced cell death in endothelial cells through classical VEGFR-induced pathways. (A) VEGF₁₂₁b is found in the media on transfected cancer cells. (B–C) VEGF₁₂₁b has similar saturation binding kinetics to VEGFR-1 as VEGF₁₆₅b. (D–E) VEGF₁₂₁b inhibits VEGF₁₆₅ (D) or VEGF₁₂₁ (E) induced migration of HUVEC. Cells were allowed to migrate through 8 μm pore inserts with increasing concentrations of VEGF₁₂₁b (0–6 nM) or VEGF₁₆₅b (0–2.5 nM) in the presence of 1 nM VEGF₁₆₅ or 2.9 nM VEGF₁₂₁. Data plotted as percentage migration compared to total number of seeded cells (F) VEGF₁₂₁b is cytoprotective to a similar degree as VEGF₁₆₅ in HUVEC. Release of lactate dehydrogenase as a measurement of cell viability was monitored after 48 h of serum starvation in the presence of increasing amounts of VEGF₁₂₁b (0–3.6 nM), 1 nM VEGF₁₆₅ or full growth media (FGM) with serum and growth factors. (G) Similar cytoprotection of VEGF₁₂₁b was observed in serum starved HUVEC as cell number was not reduced by the same amount after 48 h of cell starvation. (H) VEGF₁₂₁b cytoprotection is mediated via VEGFR-1 and -2, P13-K, MEK1/2. Inhibition of VEGFR1/2=200 nM PTK787, VEGFR-2=10 nM ZM323881, P13- K=15 μM LY294002, p38=10 μM SB203580 and MEK1/2=15 μM PD98059. (One-way ANOVA, Dunnett's post hoc test, 0 nM or control vs addition, ^*P<0.05, ^**P<0.01, ^***P<0.001).

Figure 4

VEGF₁₂₁b reduces tumour growth in nude mice bearing colon carcinoma tumours by reducing tumour vessel ingrowth. (A) LS174t human colon carcinoma cells were transfected with pcDNA₃-VEGF₁₂₁b or empty pcDNA3 plasmid and injected subcutaneously into nude mice and tumour growth was monitored over time. Over-expression of VEGF₁₂₁b resulted in a reduced tumour growth and contained less blood compared with control cells (inserted images). Scale bar=10 mm. (B) Immunohistochemistry staining of tumour sections for VEGFR-2 visualise microvessels (inserted images). Quantification of microvascular density showed significantly fewer blood vessels per unit area than control tumours. Each point represents the mean of 10 random analysed fields and six tumours per treatment were examined (^*P<0.05 unpaired t-test). (C) Cleaved caspase 3 staining of tumour sections (inserted images) showed no significant difference in apoptosis. (D, E) VEGF₁₂₁b has no effect on LS174t colon carcinoma growth in vitro. Over-expression of VEGF₁₂₁b in LS174t colon carcinoma cells had no effect on cell proliferation analysed by FACS (D). Addition of 1 nM VEGF₁₂₁b had no effect of cell number analysed by direct counting of cells over 48 h (E).

Figure 5

In vivo distribution of ¹²⁵I-VEGF₁₂₁b in tumour-bearing mice. Tumour-bearing mice received an intravenous injection of ¹²⁵I-VEGF₁₂₁b and 3D imaged using NanoSPECT/CT. (A–D) Time course for biodistribution of ¹²⁵I-VEGF₁₂₁b after tail vein injection using transverse sections. (E) Coronal. (F) Para-sagittal through the centre of the tumour. The tumour is circled and arrows indicate different organs. (G) Quantification uptake into different organs and tissues over time. Data expressed as % in tissue relative to the total injected dose, per gram of tissue.

Figure 6

VEGF₁₂₁b reduces hypoxia-induced retinal neovascularisation after single intraocular injection. The oxygen-induced retinopathy mouse model leads to neovascularisation induced by hypoxia. (A) Flat mounted mouse retinas with vessels visualised by isolectin staining. Ischaemic and neovascularised areas are circled (i, ischaemic, n, neovascular and r, normal retinal vessels). Normal retinal vessels (vii) are slender compared to neovascular vessels (viii). (B) Quantification of neovascularisation, ischaemic and normal vessel growth compared to matched uninjected control eye (paired t-test, ^*P<0.05, ^**P<0.01). (C–E). Distribution of ischaemic, neovascular and normal vessel growth in injected retinas relative to uninjected eye showed that VEGF₁₂₁b reduced neovascularisation (C) and the ischaemic areas (D) leading to an increase in the revascularisation (E). (One-way ANOVA, Bonferroni post hoc test, ΔP<0.05, ΔΔP<0.01 compared to vehicle injected=0 ng).