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: Translational Vision Science & Technology

Article Title: Sema3A Antibody BI-X Prevents Cell Permeability and Cytoskeletal Collapse in HRMECs and Increases Tip Cell Density in Mouse Oxygen-Induced Retinopathy

doi: 10.1167/tvst.11.6.17

Figure Lengend Snippet: Diagrammatic representation of sprouting angiogenesis in the retina, adapted from Sapieha. Initially, tip cell filopodia are stimulated by proangiogenic factors, such as VEGF-A. Via a combination of pericyte detachment from the vascular basement membrane, matrix metalloproteinase-induced degradation of the basement membrane, and loosening of VE–cadherin junctions, tip cell filopodia migrate through the basement membrane and search for vascular guidance cues. Chemoattractive cues include VEGF-A, whereas chemorepulsive cues include Sema3A. , Subsequently, endothelial cells adjacent to the tip cells (i.e., “stalk” cells) proliferate in response to proangiogenic factors (e.g., VEGF-A) to elongate the neovessels. – Sema3A, semaphorin 3A; VE, vascular endothelial; VEGF-A, vascular endothelial growth factor-A.

Article Snippet: The HRMECs were treated overnight with either 100 ng/mL recombinant human (rh) VEGF-A (293-VE010; R&D Systems, Inc., Minneapolis, MN, USA) or 500 ng/mL rh-Sema3A (1250-S3; R&D Systems) with and without 1 µg/mL of BI-X or 1 µg/mL of anti-trinitrophenol (TNP; 1B7.11 [ATCC TIB-191]; American Type Culture Collection, Manassas, VA, USA).

Techniques: Membrane

Journal: Translational Vision Science & Technology

Article Title: Sema3A Antibody BI-X Prevents Cell Permeability and Cytoskeletal Collapse in HRMECs and Increases Tip Cell Density in Mouse Oxygen-Induced Retinopathy

doi: 10.1167/tvst.11.6.17

Figure Lengend Snippet: Effects of BI-X, Sema3A, and VEGF-A on HRMEC permeability in vitro. Data are presented as mean ± SEM (group size, eight wells/group) and represent one of two independent experiments with comparable results. Statistical analysis was performed by one-way analysis of variance with Dunnett's post hoc multiple comparisons test versus Sema3A (*** P < 0.001). HRMEC, human retinal microvascular endothelial cell; SEM, standard error of the mean; Sema3A, semaphorin 3A; TNP, trinitrophenol; VEGF-A, vascular endothelial growth factor-A.

Article Snippet: The HRMECs were treated overnight with either 100 ng/mL recombinant human (rh) VEGF-A (293-VE010; R&D Systems, Inc., Minneapolis, MN, USA) or 500 ng/mL rh-Sema3A (1250-S3; R&D Systems) with and without 1 µg/mL of BI-X or 1 µg/mL of anti-trinitrophenol (TNP; 1B7.11 [ATCC TIB-191]; American Type Culture Collection, Manassas, VA, USA).

Techniques: Permeability, In Vitro