Journal:

Article Title: Thymidine phosphorylase induces angiogenesis in vivo and in vitro : an evaluation of possible mechanisms

doi: 10.1038/sj.bjp.0705216

Figure Lengend Snippet: The effect of thymidine phosphorylase and VEGF165 on HUVECs monolayer regeneration, following a mechanical injury. The top panel of phase contrast micrographs show: (a) the total denudation at the time of injury (T0); (b) basal regrowth in the presence of vehicle (5% FCS); (c) regeneration in the presence of VEGF (1 nM); (d) regeneration in the presence of thymidine phosphorylase (TP) (1 nM); (e) preincubation for 1 h and the continuous presence of TP inhibitor, reverts the TP-induced regeneration to basal level; (f) TP inhibitor has no effect on the VEGF165-induced recovery. Images were captured with a Nikon Diaphot inverted microscope at × 4 objective, coupled to a CCD (JVC), and grabbed using a Q500 Leica software. The calibration bar represents 400 μm. The graphs show the concentration–effect curve of TP and VEGF165, on (g) the recovery of a ‘wounded' area, and (h) the proliferation of endothelial cell. A synchronised monolayer of HUVECs was injured using a multichannel wounder, producing 11 linear lesions, and transferred to fresh media supplemented with 5% FCS, with appropriate treatments. They were incubated for a further 24 h, following which, they were either fixed and image analysed, or trypsinised and counted using a haemocytometer. Data expressed are mean±s.e.m. of at least four separate experiments with quadruplicate wells in each. In the wound recovery experiment, data are shown as percentage of T0 values. *P<0.05, **P<0.01, ***P<0.001 vs vehicle-treated controls, +P<0.05 vs corresponding TP-treated group.

Article Snippet: Human recombinant-thymidine phosphorylase or platelet-derived endothelial cell growth factor (rh PD-ECGF) and human recombinant vascular endothelial growth factor (VEGF) were procured from R&D Systems, USA.

Techniques: Inverted Microscopy, Software, Concentration Assay, Incubation

Journal:

Article Title: Thymidine phosphorylase induces angiogenesis in vivo and in vitro : an evaluation of possible mechanisms

doi: 10.1038/sj.bjp.0705216

Figure Lengend Snippet: Effect of 2-deoxy-D-ribose-1-phosphate (2-DDR-1-P) and thymine on tube formation by endothelial cells in a coculture system. Photomicrographs depict: (a) vehicle-treated cells, (b) VEGF-induced tubulogenesis, (c) suramin-induced inhibition of tubulogenesis, (d) tube formation induced by 2-DDR-1-P (10−6 M), (e) inhibition of tubulogenesis by thymine (10−4 M), and (f) reversal of thymine-inhibition by 2-DDR-1-P (10−4 M). A coculture of endothelial cells and fibroblasts (Angiokit) was used to study the tube formation by endothelial cells. The AngioKit was seeded with cells on day 0, and the optimised growth medium was changed on days 3, 5, 7, 10 and 12. It was then fixed, and stained for CD31, on day 14. Suramin (20 μM) and VEGF (2 ng ml−1) were used as negative and positive controls, respectively. Thymine and 2-DDR-1-P were added on day 4. The appropriate treatments were replenished with each medium change. Graph (g) shows the comparison of venule length following different treatments, as measured using a ‘AngioSys' image analysis system. Four images were grabbed per well, from the four quadrants. Experiments were run in quadruplicate, and data were expressed as the mean±s.e.m. *P<0.05, **P<0.01 vs vehicle control; #P<0.05 vs matched thymine concentration.

Article Snippet: Human recombinant-thymidine phosphorylase or platelet-derived endothelial cell growth factor (rh PD-ECGF) and human recombinant vascular endothelial growth factor (VEGF) were procured from R&D Systems, USA.

Techniques: Inhibition, Staining, Comparison, Control, Concentration Assay

Journal:

Article Title: Thymidine phosphorylase induces angiogenesis in vivo and in vitro : an evaluation of possible mechanisms

doi: 10.1038/sj.bjp.0705216

Figure Lengend Snippet: A proposed mechanism for the angiogenic effects of thymidine phosphorylase. Mechanical stress can lead to cell death (normal inside a solid tumour), and result in the release of thymidine in the microenvironment. Thymidine can enter live carcinoma cells, and is broken down by TP to 2-DDR-1-P, which can be dephosphorylated to 2-DDR. Inside a tumour cell (phase 1), reducing sugars can undergo rearrangement reactions, leading to the generation of free radicals. The concomitant upregulation of HO-1 has been implicated in increasing the expression of VEGF, MMPs and IL-8. These can then act on the host endothelial cells to induce an angiogenic effect in vivo. Furthermore, CO released would stabilise the neo-vessels through an antiapoptotic effect. Additionally, as seen in phase 2, the TP released from injured cells, or added exogenously, can act on thymidine and lead to the generation of 2-DDR, which is a chemotactic/chemokinetic factor, and promote angiogenesis. The 2-DDR that enters the cell can also be incorporated into the glycolytic machinery, and generate energy that can further serve towards a migratory phenotype. This model explains why both 2-DDR and 2-DLR could exert angiogenic effects in vivo, but only 2-DDR was active in vitro. Thin arrows indicate possible links elucidated in this study, while hashed arrows are putative links. The thicker arrows indicate pathways established by other studies (Brown et al., 2000).

Article Snippet: Human recombinant-thymidine phosphorylase or platelet-derived endothelial cell growth factor (rh PD-ECGF) and human recombinant vascular endothelial growth factor (VEGF) were procured from R&D Systems, USA.

Techniques: Expressing, In Vivo, In Vitro

Journal: Journal of Neuro-Oncology

Article Title: Identification of two distinct mesenchymal stromal cell populations in human malignant glioma

doi: 10.1007/s11060-016-2302-y

Figure Lengend Snippet: a VEGF and b PGE 2 production in vitro by two different cell populations from each of two GBMs. The CD90 − populations from GBM-47 and GBM-48 produce higher levels of both VEGF and PGE 2 compared to the CD90 + populations from the same tumors. 1 × 10 5 cells were grown in 1 ml MSC Expansion Media for 24 h and the supernatants were analyzed with ELISA. Each experiment was performed three times in duplicate and VEGF data from both tumors was pooled together

Article Snippet: VEGF levels were determined using the Human VEGF DuoSet kit (R&D Systems).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: CRIP1 Reshapes the Gastric Cancer Microenvironment to Facilitate Development of Lymphatic Metastasis

doi: 10.1002/advs.202303246

Figure Lengend Snippet: VEGFC is upregulated by CRIP1 in GC cells and involved in CRIP1‐mediated lymphangiogenesis and LM. A,B) ELISA showing the effect of CRIP1 overexpression on VEGFC A) and VEGFD B) secretion. C,D) ELISA showing the effect of CRIP1 knockdown on VEGFC C) and VEGFD D) secretion. E,F) RT‐qPCR showing the effect of CRIP1 overexpression E) and knockdown F) on VEGFC mRNA expression. G) Western blot showing the effect of CRIP1 overexpression and knockdown on VEGFC protein expression. H) Western blot showing the expression of CRIP1 and VEGFC in human GC samples. NT, nontumorous tissues; GC, gastric cancer tissues. I) Representative images (left panel) and quantification (right panel) of the Matrigel tube formation assay with human lymphatic endothelial cells (HLECs). HLECs were cultured with conditioned medium derived from GC cells treated as indicated. Scale bars = 200 µm. J) Representative images of enucleated popliteal lymph nodes across groups. K) Histogram analysis of lymph node volumes across groups. L) Ratios of metastatic to total dissected popliteal lymph nodes from mice inoculated with the indicated cells. χ ‐square test was performed to assess the statistical significance in (L). Error bars represent the mean±SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell supernatants were collected to quantify the secretion of VEGFC (DVEC00, R&D systems, MN), VEGFD (DVED00, R&D systems, MN), CCL5 (mlbio, Shanghai, China), and TNF‐ α (mlbio, Shanghai, China) via ELISA assay.

Techniques: Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Tube Formation Assay, Cell Culture, Derivative Assay