VEGF₁₂₁b and VEGF₁₆₅b are weakly angiogenic isoforms of VEGF-A

Abstract

Background: Different isoforms of VEGF-A (mainly VEGF₁₂₁, VEGF₁₆₅ and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGF(xxx)b, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF₁₂₁/₁₆₅b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential.

Results: Recombinant VEGF₁₂₁/₁₆₅b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF₁₆₅. Furthermore, treatment of endothelial cells with VEGF₁₂₁/₁₆₅b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF₁₆₅. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF₁₂₁/₁₆₅b isoforms. A549 and PC-3 cells overexpressing VEGF₁₂₁b or VEGF₁₆₅b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGF(xxx)b isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGF(xxx)b and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGF(xxx)b and total VEGF-A was found.

Conclusions: Our results demonstrate that VEGF₁₂₁/₁₆₅b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF₁₂₁/₁₆₅b-based therapies in patients.

Figure 1

VEGF-A transcripts generated by mRNA alternative splicing. Exon organization of VEGF-A gene (A). Exons 1 to 5 are included in all isoforms (A-C). The 3' alternative exons 8 and 8b, of the same length in base pairs, are differentially included in the "classical" family of VEGF-A isoforms (B) or the novel family of "b isoforms" (VEGF_xxxb) (C).

Figure 2

Purification of recombinant VEGF_xxxb produced in Pichia pastoris. HPLC elution profile showing the absorbance overtime, after production of VEGF₁₂₁b (A). Two peaks are observed for both isoforms: Peak 1 corresponds to proteins in the P. pastoris culture supernatants unbound to the column, whereas peak 2 corresponds to the eluted VEGF₁₂₁b protein. Fractions indicated in the graphs were electrophoresed and stained with Coomassie blue. Bands of the expected size were observed in the fractions corresponding to peak 2 (B). Culture supernatants subjected to electrophoresis for both VEGF₁₆₅b and VEGF₁₂₁b. Under non-reducing conditions the 3-band pattern (light arrows) corresponds to dimers (most likely glycosylated-glycosylated, glycosylated-non-glycosylated and non-glycosylated-non-glycosylated proteins, as previously described for the VEGF-A classic isoforms). Under reducing conditions, bands (2-band pattern, as described for VEGF-A under these conditions) correspond to monomers (C). VEGF_121/165b proteins purified after production in the yeast P. pastoris are strongly immunoreactive with an antibody raised against VEGF_xxxb. The lanes in the blots show bands of 3 clones with different amounts of secreted VEGF₁₂₁b, or 2 clones in the case of VEGF₁₆₅b (D).

Figure 3

VEGF_121/165b isoforms induce proliferation of endothelial cells in culture through VEGFRs activation, although to a lesser extent than VEGF₁₆₅. A: MTT assays. Commercial recombinant VEGF₁₆₅ (R&D systems), VEGF₁₂₁b and VEGF₁₆₅b isoforms produced in P. pastoris (pp), and VEGF₁₆₅b produced in mammalian cells (hs) were added to HUVECs alone or in combination. VEGF₁₆₅ induces proliferation (p < 0.01) of HUVECs by 63% compared to control untreated cells. Co-administration of VEGF₁₂₁b (pp), VEGF₁₆₅b (pp) or VEGF₁₆₅b (hs) with VEGF₁₆₅ does not abrogate this effect. Addition of VEGF_121/165b isoforms alone induces proliferation of HUVECs by ~40% over untreated cells. Co-treatment with VEGF₁₆₅b (hs) and the VEGFR inhibitor GW654652 (1 μM) blocks the effect on proliferation. B: Analysis of DNA synthesis by incorporation of the modified nucleotide EdU. Administration of bFGF and VEGF₁₆₅ increases DNA incorporation into HUVECs by 3-fold compared to untreated controls. Exposure to VEGF₁₆₅b(pp) and VEGF₁₂₁b(pp) also increases significantly DNA incorporation (by almost 2-fold) as compared to controls. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 4

VEGF_121/165b isoforms activate the ERK signalling pathway through VEGF receptors. Effect of VEGF_121/165b isoforms on downstream signalling pathways, using antibodies against Y1175 in VEGFR2 (KDR), S473 in AKT and T202/Y204 in ERK1/2, after treatment of HUVECs with 100 ng/mL of each cytokine. VEGF₁₆₅ stimulates phosphorylation of VEGFR2 and ERK1/2. This effect is not blocked by co-administration of either VEGF₁₂₁b from P. pastoris or VEGF₁₆₅b of different origins (mammalian cells or P. pastoris). Furthermore, addition of VEGF_121/165b isoforms alone produces signalling activation as well. KDR/ERK1/2 phosphorylation was shown to be mediated specifically through VEGF receptors, since treatment with 1 μM of the specific inhibitor GW654652 blocks phosphorylation of the receptor and the intracellular transducer.

Figure 5

VEGF_121/165b isoforms induce vascularization in vivo. A: Angiogenesis analysis in Matrigel plugs with Alexa-647-labelled isolectin. No signal is found in controls, whereas Matrigel plugs carrying any of the VEGF_xxxb isoforms show a strong signal, thus demonstrating angiogenesis in vivo. B: Analysis of Matrigels using FITC-labelled dextran. Control Matrigels do not show fluorescent signal; a large amount of FITC-labelled dextran is observed in VEGF₁₂₁b- and bFGF-containing plugs, whereas VEGF₁₆₅b- and VEGF₁₆₅-containing plugs display a weaker signal.

Figure 6

VEGF_121/165b overexpression accelerates tumorigenesis in A549 xenografts. A: Western blot shows total endogenous VEGF expression in PC-3 and A549 cells, and VEGF_121/165b-overexpression in the transfected cell pools. Two different exposure times were used to develop the blots: Long time (for endogenous VEGF protein levels in PC-3 and A549 cells) and short time (for transfected cells) exposure. Bands corresponding to glycosylated and non-glycosylated proteins can be observed. B: PC3 cells engineered to overexpress VEGF₁₂₁b, VEGF₁₆₅b, or both, injected subcutaneously into athymic mice. No statistical differences between groups are found, but tumors originated from injection of VEGF₁₂₁b-overexpressing cells tend to be larger than those observed for the other groups. C: Tumors resulting from injection of A549 cells overexpressing VEGF_121/165b isoforms are significantly larger (p < 0.01) than controls.

Figure 7

Angiogenesis analysis in sections from xenografted tumors. Immunostaining for CD-31 in A549 tumors revealed a poor vascularization and no differences between controls and VEGF_121/165b-overexpressing tumors. In PC-3 tumors, a significant increase in angiogenesis was found for VEGF₁₂₁b when compared with controls or VEGF₁₆₅b-overexpressing tumors. Par: Parental cells.

Figure 8

VEGF_xxxb protein expression is increased in human breast cancer samples in comparison to healthy mammary glands. VEGF protein levels in breast cancer samples and healthy mammary glands were analyzed in a Tissue Microarray (TMA) by immunohistochemistry using an antibody raised specifically against exon 8b (A) and an anti-total VEGF-A antibody (B). The levels of both VEGF_xxxb (C) and total VEGF-A (D) are higher in the breast cancer samples compared to normal breast tissues (NBT). Differences reach statistical significance when comparing normal breast glands with the Intraductal Carcinoma (IDC) subtype (p < 0.05) for both total VEGF and VEGF_xxxb. Examples of staining include papillary carcinoma (Pap), phyllodes (Phy), infiltrating lobular carcinomas (ILC), and ductal carcinoma in situ (DCIS).