Fig 2: Effects of mobilized PBMNCs on CD34+ and CD146+ cell differentiation. CD34+ (A–C) and CD146+ (D–F) positive cells were seeded on cover slips and cultured for 3 weeks after which their endothelial specific antigen expression was examined. Green: CD31 (A and D), CD144 (B and E), CD62E (C and F); red: Dil-LDL; blue: DAPI. (A–F) Original magnification, χ600. CD34 (G–I) and CD146 (J–L) positive selected cells were seeded on Matrigel for the tube formation test. After 4 hrs (G, J), 18 hrs (H, K) and 96 hrs (I, L), images were taken using an inverted microscope at 200χ. CD146+ progenitor cells formed longer tubes (length) and more interconnections (branching) than CD34 progenitor cells under the same culture conditions. M: mean ± S.D. of the length and branching number (sum of the numbers under each of three random low power fields) for the two cell types, respectively; n= 3, *P < 0.05 between two groups.

Fig 3: Oxidative stress induces leukocyte adhesion to HMVEC under shear stress and Nrf2 activation decreases adhesion molecule surface expression. (A) Experimental scheme: HMVEC were plated and laminar shear stress was applied 24 h before TNF-α stimulation. After 22.5 h, cells were either treated with the ROS scavenger N-acetyl-L-cysteine (NAC) or left untreated. Oxidative stress was induced 1 h later by addition of hydrogen peroxide either without (H2O2) or with pretreatment with NAC (N+H2O2). (B) ROS measurement was subsequently acquired by flow cytometry (Mean + SEM; n = 6; * p < 0.05, one-way ANOVA vs. 0 Gy). (C) For adhesion experiments, Cell Tracker Green-stained PBL were added to the medium reservoir of the flow chamber and adhesion to HMVEC was allowed for 30 min under laminar flow (Mean + SEM; n = 6; * p < 0.05, one-way ANOVA vs. 0 Gy). (D) Exemplary pictures, depicting adhered PBL (CellTracker Green CMFDA (5-chloromethylfluorescein diacetate)), either alone or merged with nuclei (DAPI, blue) and F-actin staining of HMVEC cells (phalloidin-TRITC, red). Bar, 100 µm. (E) To analyze the impact of Nrf2 activation on adhesion molecule expression, HMVEC were plated, medium was changed after 24 h and cells were treated with TNF-α and DMSO or the Nrf2 activator AI-1. After 24 h, cells were fixed, stained for Nrf2 (AF488, green) and DAPI (blue). (F) Depicted are exemplary pictures, showing Nrf2 staining alone (green channel) or merged with nuclei (DAPI, blue). (G) Samples were evaluated microscopically for Nrf2 translocation, or (H) adhesion molecules ICAM-1, VCAM-1 and E-Selectin were measured by flow cytometry and values calculated as MFI + SEM relative to DMSO-treated cells. n = 4; * p < 0.05 (Mann-Whitney U test vs. DMSO).

Fig 4: Low-dose X-irradiation modulates mRNA expression of anti-oxidative factors, reduces reactive oxygen species (ROS) and peripheral blood lymphocyte (PBL) adhesion to human microvascular endothelial cells (HMVEC) under laminar conditions. (A) Experimental setup: For analysis of the mRNA expression of anti-oxidative factors, ROS and adhesion molecules under shear stress, laminar flow conditions were applied 24 h after plating of HMVEC. (B) After irradiation and stimulation with tumor necrosis factor alpha (TNF-a), RNA was isolated to measure the mRNA expression of anti-oxidative factors (Nrf2, Nuclear factor-erythroid-2-related factor 2; SOD1, Superoxide Dismutase 1; GPx1, Glutathione Peroxidase 1; Catalase) by quantitative PCR (qPCR) (mean + SEM; n = 6) or (C) cells were subjected to analysis of ROS by flow cytometry (mean + SEM; n = 8–12). (D) For adhesion assays, PBL were added 23.5 h after irradiation and adhesion was allowed for 0.5 h under constant laminar flow. (E) Measurement of adhesion molecules vascular cellular adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and E-selectin expression was performed by qPCR (mean + SEM; n = 6). (F) For adhesion assays, HMVEC were plated on glass cover slips, PBL were stained with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) and added 23.5 h after irradiation for 30 min to the medium reservoir under permanent laminar flow. HMVEC cells and adhered PBL were fixed and stained with 4',6-diamidino-2-dhenylindol (DAPI) and phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin-TRITC). PBL adhesion was evaluated by microscopic counting of PBL relative to the number of endothelial cells (mean + SEM; n = 5–9). * p < 0.05, Kruskal-Wallis test vs. 0 Gy.

Fig 5: Specification of angioblasts induced by endothelial growth factors. (A) Proliferative potential of EPCs derived from ESCs. Morphologic features of angioblast cultures under EGM-2 differentiation are shown at day two (a), day nine (b) and day 15 (c). Panel d indicates the average cell number per colony during the culture. Significant differences existed between ADM and EGM-2 or ECGS; results are expressed as mean ± SD, t-test, n = 20. (B) Antigenic expression of angioblasts specified by different culture media. Flow cytometry results of specified EPCs together with HPP-ECFCs are presented. Single-cell suspensions were stained with the indicated antibody combination; all cells were gated as CD45-. Isotype-matched control antibody staining was performed and used to process the data. (C) Comparison of EGM-2 and ECGS in angioblast specification. Antigenic expression pattern by flow cytometry reveals the differences of angioblast specification under EGM-2 and ECGS, using HPP-ECFCs as a reference. (D) LDL uptake by angioblasts specified by three media. ESC-derived cells were grown in ADM (b), EGM-2 (c) and ECGS (d); and incubated with Dil-LDL to measure their uptake ability. Dil-LDL is labelled in red. Nuclei are stained blue with DAPI 400×. (E) Cellular reactivity to 10 ng/ml TNF-a. Specified EPCs under EGM-2 (dark blue), ECGS (orange), ADM (maroon), or control EC (light blue), were cultured in monolayer and treated with TNF-a for 4 hrs; corresponding controls were treated with an equal amount of PBS. Flow cytometry to detect CD54, CD62E and CD106 was conducted. Percentage positivity is shown. Results were calculated from three experiments and expressed as mean ± SD. ? indicates significant difference between TNF-a-treated cells and corresponding controls (P < 0.05). (F) Vascular-like and microvessel formation on Matrigel. (a) endothelial cells isolated from blood vessels; (b and e) specified EPCs from EGM-2; (c and f) specified EPCs from ECGS; (d) specified EPCs from ADM. a–d, 100×; e–f, 400×.