1 Introduction

The vascular endothelial growth factor-A (VEGF or VEGF-A) and its receptors play a fundamental role in angiogenesis. VEGF is a 40 kDa glycoprotein that is secreted to the extracellular compartment. The structure of VEGF is homodimeric, formed by two disulfide-linked monomers arranged in an antiparallel direction, displaying two binding sites at each extreme of the molecule [1]. In mammals, VEGF belongs to a family of five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PlGF (Placental growth factor). The biological functions of VEGF proteins are mediated by a family of tyrosine kinase receptors, known as the VEGF receptors (VEGFR) [1,2,3]. The human VEGF-A gene is arranged in 8 exons and is expressed as multiple isoforms due to differential splicing. The four principal isoforms describe are hVEGF121, hVEGF165, hVEGF189 and hVEGF206 [4,5,6,7]. VEGF121 lacks exons 6 and 7 responsible for the heparin-binding domain and freely diffuse through the extracellular matrix [5].

VEGF is a key regulator of vasculogenesis and angiogenesis during embryogenesis [8, 9]. However, in adults, physiological angiogenesis occurs only during wounds healing and the menstrual cycle [10,11,12]. When VEGF regulatory control is lost, angiogenesis contributes to numerous pathological conditions, such as rheumatoid arthritis [13] psoriasis [14], atherosclerosis [15], macular degeneration [16] and tumorigenesis [17].

VEGF induces cell proliferation and promotes survival and migration of endothelial cells. These biological effects are essential for tumorigenesis as angiogenesis is required for tumor growth and progression [17,18,19,20]. Cancer cells secrete VEGF promoting new blood vessels formation for nutrient and oxygen supply [21,22,23] Particularly, VEGF121 isoforms overexpression has been detected in colorectal cancer and in a cancer mouse model VEGF121 overexpression but not VEGF165 overexpression induced tumor growth [24].

The interest of expressing biologically active VEGF121 by recombinant DNA technology lies in the function of this molecule and its different uses: (i) To study and development of new therapies based on inhibiting VEGF/VEGFR interaction [25,26,27,28,29] and (ii) advanced treatments of patients with diabetic foot and ischemic diseases [30,31,32,33,34,35,36].

Recombinant VEGF has been expressed on different systems, including Escherichia coli [37,38,39], yeast [40, 41], insect cells [42] Chinese hamster ovary cells (CHO) [43]. Nevertheless, VEGF purification in these expression systems requires multiple-steps purification protocols increasing the final product cost [37, 44]. VEGF yields obtained on these systems are in the range of mg/l. However, VEGF expression has focused on VEGF165 isoform.

In this work, we expressed VEGF121 in SiHa cells transduced with adenoviral vectors. The use of replication-defective adenoviral vectors for transient gene expression of human proteins in mammal cell cultures is based on the fact that it is an efficient and safe method for gene transfer and protein expression in vitro and in vivo [45,46,47,48,49,50,51]. We analyzed the biological activity of purified VEGF121 based on proliferation and induction of vascular permeability. VEGF121 proliferative properties were analyzed on human umbilical cord endothelial cells (HUVEC) and ECV-304 cell line. The neoangiogenic activity was studied by in vivo murine model of vascular permeability.

2 Materials and Methods

2.1 Generation of Ad-VEGF Adenoviral Vector

The AdEasy vector system was used to generate the replication-defective vector pAdhVEGF121 [52]. Briefly, a synthetic 500 bp sequence encoding human VEGF plus a 6xHis tag was cloned into XhoI/EcoRV sites of the shuttle vector pAdtrack-CMV. The resulting plasmid was transformed into the E. coli BJ5183 strain along with the pAdEasy vector to generate a defective adenoviral vector by in vivo recombination. The resulted plasmid (pAdhVEGF121) has two cytomegaloviruses (CMV) promoters, one for the green fluorescent protein (GFP) and the other for VEGF. The initial viral pool was prepared by transiently transfecting the pAdhVEGF121 plasmid into HEK-293 cells. Adenovirus stocks were further amplified in HEK-293 cells and harvested by a freeze-thaw lysis protocol. Virus concentration was calculated as gene transfer units (GTU) by GFP expression in HEK-293 cells.

2.2 VEGF121 Production in SiHa Cells

SiHa cells were grown in a 24-well plate in DMEM medium supplemented with 10% fetal bovine serum (FBS). After reaching 80% of confluence, cells were infected with the replication-defective adenovirus at the following multiplicity of infection (MOI): 0.5, 10, 20, 40, 80, 160 GTU/cell. Six hours later, media was removed, cells were washed twice with PBS, and 2 ml of DMEM serum- free were added per well. After 72 h, supernatants were harvested, and VEGF expression was analyzed by SDS-PAGE, Western blot with an anti-Histidine antibody (Sigma, USA). VEGF121 were quantified by the NovexR Human VEGF solid-phase sandwich ELISA (ThermoFisher Scientific, EEUU).

2.3 VEGF121 Purification from the Culture Medium

The expressed protein was purified by immobilized metal affinity chromatography (IMAC) using an ÄktaPurifier liquid chromatography system in the following manner. Cell culture supernatant was centrifuged and filtrated through a 0.2 μm filter to remove debris and floating cells. Clarified medium was applied to a 5 ml Ni-Sepharose fast flow column (GE, USA), previously equilibrated with 5 volumes of equilibrium buffer (0.5 mM PMSF, 100 mM NaH2PO4, imidazole 5 mM pH 7.4). After washing with equilibrium buffer containing 100 mM imidazole, the VEGF121 protein was eluted by raising the imidazole concentration to 250 mM. Protein purification was monitored by UV absorption at A280 nm. Each collected fraction was analyzed by 12% SDS-PAGE/Coomassie staining. VEGF121 was detected by Western blot with an anti-His antibody (Sigma, USA). The eluted fraction was dialyzed in 10 mM NaH2PO4, pH 7.4.

2.4 N-Deglycosylation of VEGF121

The purified VEGF121 was deglycosylated by PNGase F (New England Biolabs). VEGF121 samples were mixed with denaturing glycoprotein buffer and incubated 5 min at 100 °C. The denatured VEGF121 were incubated at 37 °C overnight with PNGase F in the buffer provided by the manufacturer and 1% NP40. VEGF was analyzed by SDS-PAGE/Coomassie blue staining and Western blot.

2.5 Anti-VEGF121 Antibody Binding Affinity Measurement

The Monolith NT.115 device (NanoTemper Technologies GmbH, Germany) was used for analyzing binding affinity by microscale thermophoresis (MST). Bevacizumab antibody was labeled with NT-647 probe and used at a concentration of 6 nM. VEGF121 was titrated from 3 pM to 100 nM. The experiments were performed in binding buffer (Tris–HCl 50 mM, pH 7.4, NaCl 150 mM, MgCl2 10 mM and 0.05% Tween-20). Samples were incubated for 1 h at room temperature before loaded into MST hydrophobic capillaries. The data were analyzed with NT Affinity Analysis software.

2.6 In Vitro Proliferation Assay

VEGF121 biological activity was analyzed by MTT proliferation assays in Human umbilical vein endothelial cells (HUVEC) and ECV-304 cells. HUVEC cells were cultured in M-199 medium supplemented with 10% FBS and ECV-304 cells in DMEM medium supplemented with 10% FBS. HUVEC and ECV-304 cells were seeded in 96-well plates at 1 × 104 cells/well and 2 × 104 cells/well, respectively. After 24 h, cells were washed once with PBS and incubated for 6 h in serum-free medium before adding (0, 10, 20, 40, 80, 160, 320, 640 and 1280 ng/ml) of VEGF121 for 48 h. Then, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 5 mg/ml was added to each well and placed at 37 ° C for 4 h. Media were removed and formazan crystals were solubilized by adding 100 μl of DMSO. The plate was stirred for 15 min at 37 °C and measured the absorbance at 570 nm. EC50 value was calculated by the GraphPad Prism 6 software.

2.7 In Vivo Neovascularization Assay

VEGF121 was mixed with Matrigel (BD Bioscience, USA) at 30 ng/ml and injected subcutaneously into the laterodorsal abdominal region of female CF1-BALB/c mice of 4 weeks of age. After 6 days, PEG IRDye 800CW contrast agent (LI-COR Biosciences, USA) was injected through the tail vein of Matrigel-VEGF121 carrier mice. 24 h post-injection, the leakage of dye from capillaries was quantified by near-infrared fluorescent imaging (NIRF) using the Pearl® Impulse Small Animal Imaging System (LI-COR Biosciences, USA).

3 Results

3.1 VEGF Expression in Adenoviral-Transduced SiHa Cells

The replication-defective adenoviral vector pAdhVEGF121 was constructed by cloning the synthetic VEGF121 gene in pAdtrack followed by recombination with the pAdEasy vector in E. coli BJ5183 strain (Fig. 1a). pAdTrack-hVEGF and pAdEasy-hVEGF plasmids were digested with PacI to check cloning and recombination events. A single band of 4.5 kbp in pAdEasy-hVEGF digested with PacI indicated correct recombination between pAdTrack-hVEGF and pAdEasy (Fig. 1b). The recombinant viral genomes were transfected into the complementing cell line HEK-293. Replication-defective viruses manifest by comet-like fluorescent plaques around 7 days after transfection (Fig. 1c).

Fig. 1
figure 1

Generation of replication-defective adenoviral vectors. a Construction of the recombinant adenoviral expression vector pAdTrack-hVEGF-A121. A synthetic 500 bp band encoding human VEGF and a 6xHis tag was cloned in pAdtrack-CMV. b Restriction enzyme analysis of pAdTrack-hVEGF-A121 (Lane 1), pAdEasy (Lane 2), and homologous recombination product (Lane 3) with PacI. c pAdhVEGF121 was transfected in HEK-293 cells and GFP expression was visualized by fluorescence microscopy. Comet-like fluorescent plaques became apparent at 7 days (red circle). The microphotographs were acquired with an Olympus IX81 DSU microscope (× 40 magnification)

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To determine VEGF121 expression conditions, SiHa cells were infected at MOI of 5, 10, 20, 40, 80 and 160 GTU/ml. Transduction efficiency and cytopathic effect were evaluated by fluorescence microscopy (GFP expression) and phase contrast microscopy, respectively. Seventy-two hours post infection, the number, and fluorescent intensity of cells increased in a GTU dose-dependent manner; however, cytotoxicity and cell death also increased (Fig. 2a). For each MOI tested, we analyzed VEGF121 expression by SDS-PAGE and Western blot, detecting two protein bands, at 17 kDa and at 19 kDa. A linear relationship was also observed between the amount of VEGF121 secreted in the supernatants and the adenovirus load used to infect SiHa cells (Fig. 2b, c). Among 10–80 GTU/cell, the expression levels of VEGF121 underwent an eightfold increase. Therefore, we select 80 UFC/cell for next experiments as the optimal MOI that allows a balance between the fewer cytopathic effect and increased VEGF121 expression.

Fig. 2
figure 2

Viral load effects on cytotoxicity and VEGF121 expression. a SiHa cells were infected at MOI of 0, 10, 20, 40, 80 and 160 GTU/ml. Adenovirus infection resulted in a dose-dependent increase of cytopathic effect and GFP expression. b VEGF121 expression in SiHa cells infected at different MOI. Proteins were separated in 12% SDS-PAGE gel under reducing conditions and immunodetected with an anti-Histidine antibody. At 20 MOI, two bands of VEGF121 were detected, at 17 kDa and 19 kDa. However, at higher viral load VEGF121 overexpression also increases giving rise to a saturation effect that affects the visualization of these 2 bands. c VEGF121 expression levels at different viral load were quantified by NovexR Human VEGF solid-phase sandwich ELISA

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3.2 VEGF121 Purification and Biochemical Characterization

The recombinant VEGF121 was purified by IMAC. The chromatogram illustrates the elution profile of VEGF121 on the IMAC column (Fig. 3a). Initial sample, non-binding fraction, washed fraction and eluted fraction were analyzed by 12% SDS-PAGE/Coomassie staining (Fig. 3b). VEGF121 was purified as a homodimer of approximately 35 kDa with an estimated purity of 98% by densitometry with the Image J software. The protein was recognized by anti-poly-histidine antibody (Fig. 3c). 12 mg of purified VEGF121 were obtained from 100 ml of culture medium using this purification procedure.

Fig. 3
figure 3

Purification and characterization of VEGF121. a Chromatogram of immobilized metal affinity chromatography using a Ni-Sepharose Fast Flow column. b SDS-PAGE analysis of samples from the affinity chromatography steps. Lane 1: Culture medium of SiHa cells (starting material); Lane 2: Flow-through; Lane 3: Proteins from the washing step at 100 mM Imidazole; Lane 4: VEGF121 elution at 250 mM Imidazole. Bands were quantified by densitometry to estimate purity. c SDS-PAGE and Western blotting analysis of VEGF121 produced in SiHa cells, respectively. Lane 1: Non-reducing conditions. Lane 2: Reducing conditions. dN-Deglycosylation assay of VEGF121 by treatment with PNGase F enzyme. VEGF121 (90 μg) was untreated (lane 1) or treated with PNGase F overnight (lane 2) at 37°C and analyzed by SDS-PAGE on a 12% gel under reducing conditions followed by Coomassie blue staining and Western blot with an anti-Histidine antibody

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The glycosylation state of VEGF121 was analyzed with PNGase F enzyme. We demonstrated that the upper band of 19 kDa detected on Western blots correspond to N-glycosylated forms of VEGF121 monomers, whereas the lower band of 17 kDa contains non-N-glycosylated VEGF121. Additionally, a third immunoreactive band of around 15 kDa was identified (Fig. 3d). Furthermore, it was demonstrated by MST that VEGF121 can bind Bevacizumab antibody with a KD of 7 ± 1 nM (Fig. 4).

Fig. 4
figure 4

The VEGF121/Bevacizumab binding analyze by MST. a MST data for VEGF121 interaction with Bevacizumab. b Binding curve between VEGF121 and Bevacizumab. VEGF121 at concentrations from 3 pM to 100 nM was titrated against 6 nM of Bevacizumab labeled with the fluorescent probe NT-650. MST experiments were performed at a LED power of 60%. Plotting of the change in thermophoresis and concomitant fitting of the data yielded a Kd of 7+ 1 nM. Error bars = s.d.; n = 2

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3.3 VEGF121 Induces In Vitro Cell Proliferation in HUVEC and ECV-304 Cells

The biological activity of VEGF121 was studied by its growth-stimulating effect on cells by MTT assays. The effect of recombinant VEGF121 on HUVEC and ECV-304 cells proliferation was dose-dependent (Fig. 5a, b). An increase in VEGF121 concentration caused HUVEC and ECV-304 cells increased proliferation. The half maximal effective concentration (EC50) of VEGF121 to induce cell proliferation was estimated at 11.02 ng/ml and 13.03 ng/ml for HUVEC and ECV-304 cells, respectively.

Fig. 5
figure 5

Cell growth stimulation of HUVEC and ECV-304 cells by VEGF121. HUVEC and ECV-304 cells were treated with increasing amounts of purified VEGF121 (0, 10, 20, 40, 80, 160, 320, 640 and 1280 ng/ml), and cell proliferation was quantified by MTT assay. Error bars = s.d.; n = 3

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3.4 VEGF121 Increase In Vivo Neovascularization in Matrigel-Bearing Mice

VEGF121 induced neovascularization was demonstrated by a matrigel plug assay in mice using IRDye® 800CW PEG as a contrast agent to visualize and quantify vascular permeability. NIRF-800 images showed that mice treated with VEGF121 exhibited greater probe accumulation outside the vasculature than the placebo group, as a result of dye extravasation out of the matrigel plug (Fig. 6a, b).

Fig. 6
figure 6

VEGF121-induces vascular permeability by matrigel plug assays.VEGF121 (30 ng/ml) was mixed with matrigel and implanted on the back of CF1-BALB/c mice. The IRDye 800CW PEG probe was i.v injected 6 days later. Vascular permeability was measured by near-infrared fluorescence. a Representative NIRF-800 images of matrigel alone (upper panel) and matrigel-VEGF121-bearing mice (lower panel) 24 h after NIRF dye inoculation. Extensive leakage of the fluorescent probe outside the matrigel plugs was observed on VEG121-bearing mice. b Relative quantification of NIRF fluorescence of matrigel-VEGF121-bearing mice. The statistical analysis was performed according to a t-test. Error bars = s.d; n = 3. Error bars = s.d; n = 3

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4 Discussion

VEGF is a glycosylated protein that forms homodimers by interchain disulfide bonds. VEGF121 homodimeric form is essential for its biological activity. VEGF is an asymmetric antiparallel homodimer with two receptor binding interfaces found at each pole of the homodimer. Each of the two binding interfaces must be able to contact a receptor monomer, triggering receptor dimerization and activation [1]. In the present work, we reported the expression of biologically active VEGF121 by SiHa cells transduced with replication-defective adenovirus. We select this cell line because they express the Coxsackievirus/Adenovirus receptor (CAR) needed for effective adenoviral transduction [53,54,55]. Non-reducing SDS-PAGE/Western blot results demonstrated that VEGF121 was secreted to the culture medium mainly as a homodimer with a near molecular weight of 35–37 kDa (58% of total protein, Fig. 3c, Lane 1). This result agrees with the molecular weight of VEGF121 obtained from other sources, including yeast and bacteria [37, 40]. We did not detect monomer forms but higher-molecular-mass complexes (Fig. 3c, Lane 1). Expressions of VEGF121 in Pichia pastoris and baculovirus/insect cells has also resulted in multiple VEGF bands, which was interpreted as covalently linked high-molecular-mass molecules [56, 57]. VEGF isoforms have been obtained in various conformations even in E. coli, with roughly equal proportions of monomeric and dimeric VEGF121 forms [58].

Additionally, we demonstrated that VEGF121 was secreted as a glycosylated protein. After N-deglycosylation with PNGase F enzyme, VEGF121 migrated in SDS-PAGE as a band of around 17 kDa corresponding to the non-N-glycosylated VEGF121 form. However, we detected a second immunoreactive band of approximately 15 kDa. We hypothesize that the 15 kDa band represent the N- and O-non-glycosylated form of VEGF121 and the 17 kDa band represents the N-deglycosylated VEGF121 that still contains O-linked oligosaccharides in two solvent-exposed serine residues (S74 and S95). Therefore, we believe that VEGF121 is secreted as an N- and O-linked glycoprotein. While this hypothesis was not demonstrated, the presence of two accessible serine residues at position 74 and 95 in the molecular surface of VEGF121 homodimer supports this hypothesis.

In this work, we did not perform a comparative analysis between glycosylated VEGF derived from SiHa cells and non-glycosylated variants. There is no evidence that VEGF glycosylation is required for dimerization neither binding to its receptor [59]. However, VEGF glycosylation is essential for molecule secretion; demonstrated by reduced secretion when glycosylation sites were mutated in mouse VEGF164 and human VEGF165 [59, 60]. Regarding its biological activity, it was demonstrated that VEGF glycosylation is not required for mitogenic activity [61]. Besides, in vessel permeability assays, there were no differences between VEGF with mutated glycosylation sites and wild-type VEGF [62].

VEGF121 expressed in the supernatant of SiHa cells was purified by a single step of IMAC chromatography. We obtained 12 mg of VEGF121 from 100 ml of clarified culture media. The single-step purification method reported in this paper is simple, fast, and does not require difficult renaturation methods. VEGF121 was expressed and purified from inclusion bodies in E. coli with high yield and purity [37, 63]. However, this procedure involves denaturation/renaturation steps. Conditions for refolding the denatured proteins to its native functional form are cumbersome and have to be standardized by trial-and-error methods, being time-consuming and expensive for scaling-up [64].

Bevacizumab is a therapeutic humanized antibody that blocks angiogenesis by inhibiting VEGF. This antibody recognizes a conformational epitope [1, 65], and bind to VEGF with a KD of 4.45 nM [66]. By MST, we determine that Bevacizumab binds to the purified VEGF121 with a KD of 7 nM. The slight difference between the KD determined by MST and reported KD for Bevacizumab, seem to be a consequence of different experimental conditions. However, this result confirms that the VEGF121 expressed in SiHa cells transduced with adenoviral vector keep the conformational epitope recognized by Bevacizumab, and suggest that VEGF121 is secreted properly folded.

VEGF has two major biological activities: (a) it is mitogenic to vascular endothelial cells and (b) can induce capillary leakage (vascular permeability) [67]. VEGF stimulates HUVEC proliferation and promotes its migration. Therefore, HUVEC cells are generally used for assaying VEGF biological activity [68, 69]. VEGF121 produced in SiHa cells was able to induce mitogenic responses in HUVEC in a dose-dependent manner. The EC50 of VEGF121 was 11.02 ng/ml, similar to the EC50 reported to VEGF purified from bacteria and yeast [37, 40]. Although HUVEC is the most extensively used endothelial cell type to study endothelial functions [68] and responds to VEGF, these cells are isolated as primary cell culture. Working with primary cultures is complex, and it is only possible to keep them in vitro for short periods of time, besides there are increased risks of microorganisms’ contamination. Thus, working with a cell line [70] that has endothelial properties could replace the use of HUVEC.

ECV-304 cell line was first described as a spontaneously transformed cell line originated from a Japanese human umbilical vein endothelial cell culture. Later, it was found to be derived from the human urinary bladder carcinoma T24 cell line [71]. However, ECV-304 cells have many features of endothelial cells [72], such as increasing cell proliferation in the presence of VEGF [73], a characteristic only observed in endothelial cells but not found in T24 cells [74]. Hence, ECV-304 cells would serve as an angiogenesis model allowing us to characterize endothelial functions [75]. Based on this information, we evaluated the mitogenic activity of VEGF121 on ECV-304 cells. VEGF121 produced in SiHa cells was able to enhance ECV-304 cell proliferation in a cell dose-dependent manner, with an EC50 of 13.03 ng/ml. This result suggests that ECV-304 cell can be used to evaluate the biological activity of VEGF121, in replace of HUVEC primary cell culture.

VEGF induced neovascularization is characterized by immature and highly fenestrated blood vessels. There is a direct relationship between neovascularization and vascular permeability. Classically, neovascularization has been measured using the Miles Assay [76]. This test uses a spectrophotometer to quantify the leakage of a dye from the vasculature to the surrounding tissue, with the limitation of the analysis of a single time point, which must be selected empirically. In this work, we present a modified method to visualize and quantify in real-time the vascular leakage using a fluorescent dextran-based dye. VEGF121 mixed in the matrigel plug injected into mice induced the formation of new and highly fenestrated blood vessels, which trigger the leakage of the fluorescent probe from the vasculature to the plugs. Thus, VEGF121 caused an increase in fluorescence accumulation in matrigel plugs.

5 Conclusions

In this study, we demonstrated functional expression of VEGF121 in the culture media of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF121). VEGF121 expression levels were strictly depended on the viral load used, reaching the maximum expression level at 80 GTU/cell. Expression in SiHa cells resulted in a soluble N-glycosylated dimer of 35–37 kDa, and partial formation of high molecular weight oligomers covalently linked. The purified VEGF121 was recognized by the anti-VEGF antibody Bevacizumab, demonstrating that VEGF121 produced in SiHa cells keep the conformational epitope recognized by Bevacizumab. VEGF121 induced cell proliferation in both HUVEC and ECV-304 cell line. Therefore, ECV-304 cells can replace the primary culture model HUVEC for assaying VEGF bioactivity. Finally, a vascular permeability test with mice carrying matrigel plugs and a fluorescent probe made it possible to determine that purified VEGF121 promotes capillary leakage. In conclusion, we have successfully developed an easy and efficient procedure for VEGF121 expression and purification. This molecule could be used for the evaluation of therapies against angiogenic tumors and wound healing.