Table 1.
Cytokines and chemokines used for estimation of the cell immune activation.
Fig 1.
Amino acid sequence of expressed Trx–α2-PI1-8-VEGF121 fusion protein.
His tag = hexahistidine tag; Thr = thrombin cleavage site; FXIIIa = Factor XIIIa substrate sequence NQEQVSPL; VEGF121 = vascular endothelial growth factor A121; resulting recombinant protein α2-PI1-8-VEGF121 after cleavage of the fusion part shown in bold.
Fig 2.
Expression in E. coli Origami B (DE3), purification and removal of the thioredoxin fusion part (Trx) of α2-PI1-8-VEGF121.
Molecular weight markers (1), uninduced cells (2), cells induced with IPTG and L-arabinose, soluble fraction (3), cells induced with IPTG and L-arabinose, insoluble fraction (4), unbound proteins (5), purified fusion protein Trx-α2-PI1-8-VEGF121 (6), preparation of the fusion protein Trx-α2-PI1-8-VEGF121 after cleavage with thrombin (7), purified α2-PI1-8-VEGF121 (8).
Fig 3.
VEGF mitogenic activity evaluation using the xCELLigence system.
The mitogenic activity of α2-PI1-8-VEGF121 and commercial VEGF121 standards was evaluated using real-time monitoring of HUVEC cell proliferation. α2-PI1-8-VEGF121, 50 ng/mL (1), VEGF121 I, 50 ng/mL (2), VEGF121 II, 50 ng/mL (3), negative control (culture medium without VEGF) (4). The cells were incubated for 165 hours. The results shown here are mean ± SEM (n = 4).
Fig 4.
The comparison of endothelial cell proliferation in media containing VEGF121 (50 ng/ mL) from different sources.
Relative cell count in a cultivation medium containing the examined VEGF121 preparations (α2-PI1-8-VEGF121 from this work, VEGF121 I expressed in E. coli, and VEGF121 II expressed in HEK cells) assessed with the fluorescent indicator resazurin after 48 hours (day 2), 96 hours (day 4), and 165 hours (day 7) of incubation, compared to the control with no VEGF (= 0%). The VEGF concentration in all preparations was 50 ng/mL. Results shown as mean ± SEM (n = 4). The statistical significance was determined by the ANOVA, Student–Newman–Keuls method; p<0.05 in comparison with the samples indicated by numbers (in blue) above the columns.
Fig 5.
The effect of thrombin (0.01, 0.05, 0.1 and 1.0 NIH U/mL) on the proliferation of HUVEC cells.
Seeding density 3 500 cells per well of a 96-well E-plate in EBM-2 basal medium supplemented with ascorbic acid, hydrocortisone, heparin, gentamicin, amphotericin-B and 2% fetal bovine serum from EGM-2 SingleQuots and in the presence of the commercial VEGF121 I standard (50 ng/mL) or α2-PI1-8-VEGF121 followed by the xCELLigence system.
Fig 6.
Cell immune activation by VEGF121 determined by Luminex Performance Assay.
Concentration of IL-1α (A), IL-1ß (B), GM-CSF(C, G), TNF-α (D), MCP-1 (E) and IL-8 (F) produced by THP-1 cells (A-F) or HUVEC (G) in the cell culture medium on day 3 (D3) or day 6 (D6) of cultivation in media with 0, 20 and 50 ng/mL of α2-PI1-8-VEGF121 (VEGF) or 10, 100 or 1000 ng/mL of bacterial lipopolysaccharide (LPS). The cells were seeded into 24-well culture plates in 1.5 ml of the media (THP-1: 10 000 cells/well, RPMI-1640 medium; HUVEC: 15 000 cells/well, EGM-2 medium without commercial VEGF121 supplement). The amount of cytokines or chemokines in the media was calculated per 105 cells. Mean ± S.E.M. (standard error of mean) from 3 measurements for each experimental group and time interval. The mean value is shown in italics (in red) above the columns. Values higher than the calibration curve are indicated with “>”. The statistical significance was determined by the ANOVA, Student–Newman–Keuls method; p<0.05 in comparison with the samples indicated by numbers (in blue) above the columns.