Journal: Frontiers in Toxicology

Article Title: Nicotine-free electronic vape fluid stimulates angiogenic processes in vitro through ARF6-mediated oxidative stress

doi: 10.3389/ftox.2025.1699112

Figure Lengend Snippet: Nicotine-free e-cigarette condensate (eVape) exposure significantly increases angiogenic processes in human endothelial cells. (a) Human umbilical vein endothelial cells (HUVECs) were exposed to eVape fluid containing 0, 5, and 10 mg/mL of nicotine for 24 h at concentrations ranging from 0% to 2%. Cell viability was measured using the MTT assay, quantified at 570 nm, and calculated as percentage viability normalised to vehicle. (b, c) Endothelial cells were exposed to 2% eVape containing 0 mg/mL of nicotine or vascular endothelial growth factor (VEGF; 100 ng/mL) or vehicle control (H 2 O) for 6 h. (b) Cell adhesion was measured using MTT, while (c) cell migration was assessed using scratch assay and calculated with MiToBo. (d–g) Neovascularisation was measured by culturing cells treated with 2% eVape containing 0 mg/mL of nicotine or VEGF (100 ng/mL) or vehicle control (H 2 O) on Matrigel TM -coated wells. Images were captured at ×20 magnification (scale bar: 100 µm; representative images are shown in (d) ). (e) Angiogenic potential, (f) tube formation, and (g) mesh size were quantified as number of joints, number of tubes, and pixel size, respectively, using Angiogenesis Analyser. The data are presented as mean ± standard error of the mean (SEM), n = 5–6. * p < 0.05 versus vehicle for eVape (0%); # p < 0.05 versus vehicle for VEGF (0 ng/mL).

Article Snippet: NAV2729 and Secin H3 were purchased from Tocris Bioscience (Abingdon, UK); Proteome Profiler Human angiogenesis array and enzyme-linked immunosorbent assay (ELISA) kits for human angiopoietin-2 (DANG20), endothelial growth factor (EGF; DEG00), endoglin (DNDG00), placental growth factor (PIGF; DPG00), prolactin (DY682), and vascular endothelial growth factor (VEGF; DVE00) were all obtained from R&D Systems (Abingdon, UK).

Techniques: MTT Assay, Control, Migration, Wound Healing Assay

Journal: Frontiers in Toxicology

Article Title: Nicotine-free electronic vape fluid stimulates angiogenic processes in vitro through ARF6-mediated oxidative stress

doi: 10.3389/ftox.2025.1699112

Figure Lengend Snippet: Nicotine-free eVape exposure causes significant upregulation of angiopoietin-2, endoglin, placental growth factor (PIGF), and VEGF as well as significant downregulation of endothelial growth factor (EGF) and prolactin in endothelial cells. HUVECs were exposed to 2% nicotine-free eVape fluid for 24 h. (a–c) Cell lysates were used for the Proteome Profiler Angiogenesis array membranes. Changes in the expression of all angiogenesis-related proteins in array (a) (ii) were quantified using the membranes (representative image in (a) (i) ). Specific analyses of the (b) upregulation and (c) downregulation of angiogenesis-related proteins on the membranes were quantified. (d) mRNA expression of angiopoietin-2, EGF, endoglin, PIGF, prolactin, and VEGF were measured by RT-PCR using specific primers. (e) Protein expression of (i) angiopoietin-2, (ii) EGF, (iii) endoglin, (iv) PIGF, (v) prolactin, and (vi) VEGF were measured using specific ELISA arrays. The data are presented as mean ± SEM, n = 5–6. * p < 0.05 versus vehicle for eVape (0%).

Article Snippet: NAV2729 and Secin H3 were purchased from Tocris Bioscience (Abingdon, UK); Proteome Profiler Human angiogenesis array and enzyme-linked immunosorbent assay (ELISA) kits for human angiopoietin-2 (DANG20), endothelial growth factor (EGF; DEG00), endoglin (DNDG00), placental growth factor (PIGF; DPG00), prolactin (DY682), and vascular endothelial growth factor (VEGF; DVE00) were all obtained from R&D Systems (Abingdon, UK).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Toxicology

Article Title: Nicotine-free electronic vape fluid stimulates angiogenic processes in vitro through ARF6-mediated oxidative stress

doi: 10.3389/ftox.2025.1699112

Figure Lengend Snippet: Activation of ARF6 by its guanine nucleotide exchange factor (ARNO) regulates angiogenic processes in endothelial cells induced by nicotine-free eVape. (a, b) HUVECs were exposed to 2% nicotine-free eVape fluid for 24 h, and the cell lysates were analysed by Western blotting for (a) ARF6 or (b) ARNO expression. The Western blotting membranes (representative blots are shown in (i) ) were quantified and normalised to the actin expression shown in (ii) . (c–f) HUVECs were treated with ARF6 inhibitor (NAV2729; 5 µM), ARNO inhibitor (Secin H3; 10 µM), or dimethyl sulfoxide (DMSO) as vehicle control at the same time as eVape exposure. (c) ROS accumulation was assessed by DCFDA incorporation (fluorescence at 485/535 nm) following 2 h of treatment. (d) Cell adhesion, (e) migration, and (f) angiogenic potential were measured following (d, e) 6 h and (f) 24 h of treatments. (g) Protein expression of (i) angiopoietin-2, (ii) EGF, (iii) endoglin, (iv) PIGF, (v) prolactin, and (vi) VEGF were measured using specific ELISA arrays following 24 h of treatment with 2% nicotine-free eVape fluid in the presence (closed circles) and absence (open circles) of Secin H3 (10 µM). The data are presented as mean ± SEM, n = 5–6. * p < 0.05 versus vehicle for eVape (0%); # p < 0.05 versus vehicle for NAV2729 and Secin H3 (DMSO).

Article Snippet: NAV2729 and Secin H3 were purchased from Tocris Bioscience (Abingdon, UK); Proteome Profiler Human angiogenesis array and enzyme-linked immunosorbent assay (ELISA) kits for human angiopoietin-2 (DANG20), endothelial growth factor (EGF; DEG00), endoglin (DNDG00), placental growth factor (PIGF; DPG00), prolactin (DY682), and vascular endothelial growth factor (VEGF; DVE00) were all obtained from R&D Systems (Abingdon, UK).

Techniques: Activation Assay, Western Blot, Expressing, Control, Fluorescence, Migration, Enzyme-linked Immunosorbent Assay

Journal: International journal of oncology

Article Title: The oncometabolite d‑2‑hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

doi: 10.3892/ijo.2018.4649

Figure Lengend Snippet: Figure 2. Cytotoxicity of d‑2HG in BAECs. (A and B) BAECs were treated with various concentrations of d‑2HG (25 µM to 50 mM) for 24 h (A) or 48 h (B). Cell viability was measured by CCK‑8 assays. VEGF (40 ng/ml) was used as a positive control. *P<0.05 versus the control. BAECs, bovine aortic endothelial cells; d‑2HG, d‑2‑hydroxyglutarate; VEGF, vascular endothelial growth factor.

Article Snippet: The amount of VEGF protein secreted by the A549 human lung cancer cells into the medium was determined using a VEGF ELISA kit (R&D Systems, Wiesbaden, Germany).

Techniques: Positive Control, Control

Journal: International journal of oncology

Article Title: The oncometabolite d‑2‑hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

doi: 10.3892/ijo.2018.4649

Figure Lengend Snippet: Figure 3. d‑2HG increases VEGF secretion and the proliferation of BAECs. (A) A549 cells were starved for 16 h in 0.5% FBS‑containing medium, treated with

Article Snippet: The amount of VEGF protein secreted by the A549 human lung cancer cells into the medium was determined using a VEGF ELISA kit (R&D Systems, Wiesbaden, Germany).

Techniques:

Journal: International journal of oncology

Article Title: The oncometabolite d‑2‑hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

doi: 10.3892/ijo.2018.4649

Figure Lengend Snippet: Figure 4. d‑2HG increased migration and tube formation ability in BAECs. (A) BAECs (90% confluence) were starved with 0.5% FBS in DMEM/low glucose for 16 h and treated with mitomycin C (1 µg/ml) for 1 h. The cells were scratched with a 1‑ml micropipette tip and incubated in starvation medium with VEGF (40 ng/ml) or d‑2HG (250 µM). Wound closure from the original location was monitored and graphed. *P<0.05 versus the control. (B) Transwell migration assays were performed in 24‑Transwell plates (6.5 mm diameter, 8 µm pore size membrane). BAECs were seeded in the upper chamber, and VEGF (40 ng/ml) and d‑2HG (250 µM) were added to the lower chambers for 24 h. Migrated cells were fixed with 100% methanol, stained with hematoxylin and eosin, and counted. *P<0.05 versus the control. (C) BAECs were starved in 0.5% FBS in DMEM/low glucose for 16 h, seeded (3x104 cells) on the surface of the Matrigel, and incubated for 8‑16 h with or without VEGF (40 ng/ml) or d‑2HG (250 µM) in DMEM/low glucose containing 0.5% FBS. Morphological changes were observed under a microscope and photographed at x100 magnification. The results are presented as the mean number of branching points. BAECs, bovine aortic endothelial cells; d‑2HG, d‑2‑hydroxyglutarate; VEGF, vascular endothelial growth factor.

Article Snippet: The amount of VEGF protein secreted by the A549 human lung cancer cells into the medium was determined using a VEGF ELISA kit (R&D Systems, Wiesbaden, Germany).

Techniques: Migration, Incubation, Control, Pore Size, Membrane, Staining, Microscopy

Journal: International journal of oncology

Article Title: The oncometabolite d‑2‑hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

doi: 10.3892/ijo.2018.4649

Figure Lengend Snippet: Figure 6. d‑2HG induces VEGFR2 signaling pathway activation and MMP2 activity. (A) BAECs were treated with VEGF (40 ng/ml) or d‑2HG (250 µM) for 10 min, and protein lysates was obtained for western blot analysis. (B) Western blot analysis and gelatin zymography analyses for MMP2 and pro‑MMP2, respectively, were performed with medium obtained from the same samples as described in (A). The relative expression pattern of MMP2 was quantified and graphed below the gel images. *P<0.05 versus the control. (C) Schematic summary of the mechanism through which d‑2HG enhances angiogenesis.

Article Snippet: The amount of VEGF protein secreted by the A549 human lung cancer cells into the medium was determined using a VEGF ELISA kit (R&D Systems, Wiesbaden, Germany).

Techniques: Activation Assay, Activity Assay, Western Blot, Zymography, Expressing, Control