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Introduction to the TCS Cellworks AngioKit™

A human multicellular in vitro model of angiogenesis

The TCS AngioKit™ model accurately reproduces the different phases of the angiogenesis process using a co-culture of human endothelial cells with other human cell types in a specially developed medium. Capillary development in the model can be conveniently followed by fixing the cultures and staining with antibody to von Willebrand factor or PECAM-1. The endothelial cells start by forming small islands in a monolayer of matrix cells (Fig 1a). The endothelial cells begin to proliferate, then enter a migratory phase where they can be seen to move within the background monolayer to form thread-like, tubular structures by day 7 (Fig 1b). By approximately day 12 to14, these structures gradually join up to form an intricate network of anastomosing tubules resembling the capillary bed of the chick chorioallantoic membrane (Fig1c). The "vessels" formed by this process can be seen to originate from the islands of endothelial cells formed during the proliferative phase. By day 14 the tubules are wider and thicker with patent lumina containing coated pits and caveolae and with tight junctions between interdigitating cells of the vessel walls (Bishop ET et al, 1999. An in vitro model of angiogenesis: basic features. Angiogenesis 3(4): 335-344).

Fig 1a
Day 1 of tubule formation. Endothelial cells stained with antibody to von Willebrand factor (x100)

Fig 1b
Day 7 of tubule formation (x40)

Fig 1c
Day 14 of tubule formation (x40)

Tubules can also be visualised by staining for Collagen IV, a constitutive element of the extracellular matrix in neovascular tissue (Fig 2). The tubules produced also stain positive for the endothelial cell adhesion molecule PECAM-1 (Fig 3) and for ICAM-2 (not shown).

Fig 2
Tubules stained for collagen IV expression

Fig 3
Day 1 of tubule formation. Endothelial cells stained with antibody to von Willebrand factor (x100) Tubules stained for PECAM-1 expression

Tubule formation can be assessed using image analysis and quantified using the TCS 'AngioSys' software package. In summary, stained tubules captured by the analysis software are reduced in width to a single pixel. The total number of pixels in a given field of view therefore represents the length of tubules. Subsequently, means of tubule length, standard deviations and coefficients of variation can be calculated. Total vessel number, total tubule area and the number of branch points formed can be similarly analysed.

Test runs with the assay have shown it to be reproducible and robust when operated in 24-well plates. Using the analysis methods described above to score tubule formation, the observed coefficient of variation between assays is of the order of 10%.

The AngioKit™ provides a model in which human endothelial cell-derived tubules develop slowly over a time course (typically 10- 12 days) which permits the evaluation of both potential angiogenesis inhibitors and activators. No other practical model permits studies of this sort. (Donovan et al, 2001. Comparison of three in vitro human "angiogenesis" assays with capillaries formed in vivo. Angiogenesis 4: 113-121). The co-culture model is set up in 24-well culture dishes and so is suitable for screening studies.

Test Substances

A wide range of test substances have been shown to influence angiogenesis in this model. Some of these affect tubule length and number, some tubule thickness, some the number of branch points and some a combination of these. Some of these are summarised in the table below:

Selection of Molecules Shown to be Active in the TCS AngioKit™ Assay

Note that this represents only the molecules whose identity we know. We have tested  many  proprietary compounds from various laboratories that were active in both senses but which were supplied as coded samples whose structure is not known to us

Examples

Fig 4
Stimulatory effect of VEGF (10ng/ml). Stained for von Willebrand Factor expression

Angiogenesis in the kit is greatly stimulated by addition of VEGF at 10ng/ml (Fig. 4) and inhibited by anti-VEGF neutralising antibody (not shown). Tubule development is strongly inhibited by Suramin. It is now known that Suramin’s mode of action is to prevent VEGF binding to the KDR receptor (Kinase insert Domain containing Receptor) thus inhibiting tyrosine phosphorylation of KDR. Fig 5a shows that at doses of 200 mM and 20 mM, Suramin has a significant anti-angiogenic effect but at 2 mM this is no longer seen.

TNF-a has also been shown previously to inhibit angiogenesis. When used in the AngioKit™, TNF concentrations as low as 0.02 ng/ml showed a significant inhibitory effect (Fig 5b).

Product Format

The TCS angiogenesis model is presented as a kit comprising a pre-seeded 24 well plate containing all components necessary for tubule development and the medium required for medium changes during the period of tubule growth. The plate is shipped on day 2 or 3 of the co-culture and is completely filled with medium for transport. On receipt, the medium should be partially removed in accordance with the instructions enclosed and the cells allowed to equilibrate for 24 hours before further medium changes or reagent additions.

Accessory kits containing validated fixing and staining reagents are also available, as are positive and negative angiogenesis control reagents. Individual antibodies for immunohistochemistry work can also be supplied separately - more details...

A dedicated software package (AngioSys) for scoring image analysis of the assay is also available commercially.
Please contact us for a free demo disc.

Product Pricing and Order Information

» For prices and ordering information, please click here.

Quality Control:

TCS Cellworks carries out comprehensive QC testing on AngioKit™ prior to its release for sale. The graph shows QC data acquired over a large number of manufacturing runs. Test plates from each manufactured batch are subjected to carefully optimised QC assays, quantifying the extent of tubule formation in response to VEGF (angiogenesis stimulator) and suramin (angiogenesis inhibitor).

» Download AngioKit QC Results 2007

Applications

AngioKit™ has many diverse research and development applications, including:

• Biomedical research into the role of angiogenesis in the healthy human body (for example in bone formation, embryogenesis, and the female reproductive cycle).
• Biomedical research into disease states in which angiogenesis is either excessive (for example tumour development, rheumatoid arthritis, psoriasis) or inadequate (for example wound healing, coronary artery disease).
• Anti-ageing research (via effects on the dermal microvasculature).
• Drug discovery – high content / high throughput screening.

Further Technical Information

Click on the links below for additional information:

» AngioKit™ technical presentation

» AngioKit™ protocol

Intellectual Property

Please note that the AngioKit™ is protected by US patent 6225118, GB patent 2331763 and European patent 1023599. Other patents are pending

AngioKit™ References

» Selected General Angiogenesis Publications

1. Delves, G.H et al (2007)  Prostasomes, Angiogenesis and Tissue Factor  Seminars in Thrombosis and Hemostasis 33: 75-9
2. Secchiero, P et al (2007)   Antiangiogenic Activity of the MDM2 Antagonist Nutlin-3. Circulation Research 100(1): 61-9
3. Mavria, G et al (2006)  ERK-MAPK signaling opposes Rho-kinase to promote endothelial cell survival and sprouting during angiogenesis. Cancer Cell 9(1): 33-44
4. Tavolari, S et al (2006).  Selected polychlorobiphenyls congeners bind to estrogen receptor alpha in human umbilical vascular endothelial (HUVE) cells modulating angiogenesis.  Toxicology 218(1): 67-74.
5. Simcock, D.E et al (2006).  Pro-Angiogenesis Activity is increased in Bronchoalveolar Lavage Fluid (BALf) from asthmatics.  Journal of Allergy and Clinical Immunology 117(2): 251
6. Scagliarini, A et al (2006).  In vitro activity of VEGF-E produced by orf virus strains isolated from classical and severe persistent contagious ecthyma.  Veterinary Microbiology 114(1-2): 142-7
7. Cudmore, M et al (2006).  VEGF-E activates endothelial nitric oxide synthase to induce angiogenesis via cGMP and PKG-independent pathways.  Biochemical and Biophysical Research Communications 345(4): 1275- 1282.
8. Chang, H.W et al (2006).  A new role for the anti-apoptotic gene A20 in angiogenesis.  Experimental Cell Research 312(15): 2897-2907.
9. Cooney, R et al (2006).  Adenoviral-Mediated Gene Transfer of Nitric Oxide Synthase Isoforms and Vascular Cell Proliferation.  Journal of Vascular Research 43(5): 462-472
10. Guo, K et al (2006).  PRL-3 Initiates Tumor Angiogenesis by recruiting Endothelial Cells in vitro and in vivo.  Cancer Research 66: 9625-9635
11. Hallock, N (2006).  World News.  Journal of the Association for Laboratory Automation 11(5): 12-38.
12. Eccles, S.A et al (2005).  Cell migration/invasion assays and their application in cancer drug discovery.  Biotechnology Annual Review 11: 391-421
13. Bainbridge, J.W.B et al (2003).  A peptide encoded by exon 6 pf VEGF (EG3306) inhibits VEGF-induced angiogenesis in vitro  and ischaemic retinal neovascularisation in vitro.  Biomedical and Biophysical Research Communications 302(4): 793-9
14. Ho, Y.T et al (2003).  The effects of 2-methoxy oestrogens and their sulphamoylated derivatives in conjunction with TNF-α on endothelial and fibroblast cell growth, morphology and apoptosis.  The Journal of Steroid Biochemistry and Molecular Biology 86(2): 189-196.
15. Tsuzuki, T et al (2007).  Conjugated eicosapentaenoic Acid inhibits vascular endothelial growth factor-induced angiogenesis by suppressing the migration of human umbilical vein endothelial cells.  J.Nutr 137(3):641-6.
16. Guimond et al, (2006). Engineered bio-active polysaccharides from heparin. Macromolecular Biosciences 6, 681-686.
17. Kunz-Schugart, L.A. (2006).  Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro.  Am. J. Cell Physiol. 290; C1385-C1398.
18. Clarke, P.A. et al (2006).  Gastrin enhances the angiogenic potential of endothelial cells via modulation of heparin-binding epidermal-like growth factor.  Cancer Res. 66; 3504-3512.
19. Jones, R.A. et al (2006).  Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumour growth.  Cell Death Differ. 13(9):1442-53
20. Campioni, D. et al (2006).  Immunophenotypic heterogeneity of bone marrow-derived mesenchymal stem cells from patients with haematologic disorders: correlation with bone marrow microenvironment.  Haematologica 91; 364-368
21. Karcher, S. et al (2005).  Different angiogenic phenotypes in primary and secondary glioblastomas.  Int. J. Cancer 118; 2182-2189
22. Ulrich, D. et al (2005).  Effect of chronic wound exudates and MMP-2/-9 on angiogenesis in vitro.  Plastic Reconst. Surg. 116; 539-545
23. Niemisto, A. et al (2005).  Analysis of angiogenesis using in vitro experiments and stochastic growth models.  Physical Rev. E 72: 062902
24. D'Andrea , L.D. et al (2005).  Targeting angiogenesis: Structural characterisation and biological properties of a de novo engineered VEGF mimicking peptide.  Proc. Natl. Acad. Sci. USA 102; 14215-14220.
25. Day, R.M. (2005).  Bioactive glass stimulates the release of angiogenic growth factors and angiogenesis in vitro.  Tissue Engineering 11; 768-777.
26. Wedge, S.R. et al (2005).  AZD2171: A highly potent, orally bioavailable, VEGF Receptor-2 tyrosine kinase inhibitor for the treatment of cancer.  Cancer Research 65; 4389-4400
27. Niemisto, A. et al (2005).  Robust quantitation of in vitro angiogenesis through image analysis.  IEEE Trans. Med. Imaging  24; 549-555.
28. Bapna, A. et al, (2004).  Polymer-assisted, multi-step solution phase synthesis and biological screening of histone deacetylase inhibitors.  Org. Biomol. Chem. 2; 611-620.
29. Courtney, S.M. et al (2004).  2,3-Dihydro-1,3-dioxo-1H-isoindole-5-carboxylic acid derivatives: a novel class of small molecule heparanase inhibitors.  Bioorg. Med. Chem. Lett. 14: 3269-3273
30. Brooks, T.D. et al (2004).  XR5967, a novel modulator of plasminogen activator inhibitor-1 activity suppresses tumour cell invasion and angiogenesis in vitro' Anti-Cancer Drugs 15; 37-44 
31. Nakamura, K.  et al (2004).  KRN633: A selective inhibitor of vascular endothelial growth factor receptor-2 kinase that suppresses tumour angiogenesis and growth.  Mol. Cancer Ther. 3; 1639-1649
32. Beilmann, M., Birk, G. and Lenter, MG  (2004).  Human primary co-culture angiogenesis assay reveals additive stimulation and different angiogenic properties of VEGF and HGF.  Cytokine 26; 178-185
33. Punturieri, M. et al (2004).  Potenziale angiogenico midollare in vitro e analisi citifluorimetrica multiparametrica delle cellule endoteliali midollari in patienzi affetti da sindrome mielodisplastiche.  Lettere GIC 13; 25-29
34. Print, C. et al, (2004).  Soluble factors from human endometrium promote angiogenesis and regulate the endothelial cell transcriptome.  Hum. Reprod. 19; 2356-2366
35. Kumar, S. et al, (2004).  Bone marrow angiogenic ability and expression of angiogenic cytokines in melanoma: Evidence favouring loss of marrow angiogenesis inhibitory activity with disease progression.  Blood 104: 1159-1165
36. Jia, H. et al, (2004).  Vascular Endothelial Growth Factor (VEGF)-D and (VEGF)-A differentially regulate KDR-mediated signalling and biological function in vascular endothelial cells.  J. Biol. Chem. 279; 36148-36157
37. Campioni, D. et al, (2004).  in vitro evaluation of bone marrow angiogenesis in myelodysplastic syndromes: a morphological and functional approach.  Leuk. Res. 28; 9-17
38. Patel, H.B. et al, (2004).  Enhanced angiogenesis following allogeneic blood transfusion.  Clin. Lab. Haematol. 26; 129-134
39. Newman, S. et al (2004).  Inhibition of in vitro angiogenesis by 2-methoxy- and 2-ethyl-estrogen sulfamates.  Int. J. Cancer 109; 533-540
40. Zhang, W. et al (2004).  Effects of insulin-like growth factor-binding proteins 1 through 5 on in vitro angiogenesis assay.  Proc. AACR 45; 4341 March 2004
41. Cai, J. et al, (2003).  Activation of VEGF-R1 sustains angiogenesis and Bcl-2 expression via the PI3-kinase pathway in endothelial cells.  Diabetes 52; 2951-2968
42. Duval, H. et al, (2003).  New insights into the function and regulation of endothelial cell apoptosis.  Angiogenesis  6; 171-183
43.  Shalhoub, J. et al, 2003.  Anti-angiogenic activity of heparins and anti-thrombin.  J. Thromb. Haemostasis 1; Suppl 1 Abstract  P0846
44. Numasaki, M. et al, (2003).  Interleukin-17 promotes angiogenesis and tumour growth.  Blood 101; 2620-2627
45. Burns, P.A. & Wilson, D.J., (2003).  Angiogenesis mediated by metabolites is dependent on VEGF.  Angiogenesis 6; 23-77
46. Sengupta, S. et al (2003).  Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms.  Brit. J. Pharmacol. 139; 219-231
47. Giuliani, N. et al (2003).  Proangiogenic properties of human myeloma cells: production of angiopoietin-1 and its potential relationship to myeloma-induced angiogenesis.  Blood 102; 638-645
48. Oshima, Y. et al, (2003).  Expression and localisation of tenomodulin, a transmembrane type chondomodulin-I-related angiogenesis inhibitor, in mouse eyes.  Invest. Ophthalmol. Vis. Sci. 44; 1814-1823
49. Igagarishi, T. et al (2003).  Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularisation in a murine proliferative retinopathy model.  Gene Therapy 10; 219-226
50. Ghosh, A.K. et al, (2002).  Inhibition by acharan sulphate of angiogenesis in experimental inflammation models.  British Journal of Pharmacology 137; 441-448
51. Rossig, L., et al, (2002).  Inhibitors of histone deacetylation downregulate the expression of endothelial nitric oxide synthase and compromise endothelial cell function in vasorelaxation & angiogenesis.  Circulation Research 91, 837-851
52. Gliki, G. et al, (2002).  Vascular endothelial growth factor induces protein kinase c (PKC)-dependent Akt/PKB activation and phosphatidylinositol 3’-kinase-mediated PKCd phosphorylation: Role of PKC in angiogenesis.  Cell Biology International 26; 751-759
53. Brooks, T. et al (2002).  Proc. AACR 43; 137.
54. Drinkwater, S.L., et al, (2002).  Effect of venous ulcer exudates on angiogenesis in vitro.   Brit. J. Surg. 89; 709-713
55. Urbich, C. et al. (2002).  Dephosphorylation of endothelial nitric oxide synthase contributes to theanti-angiogeneic effects of endostatin.  FASEB J. express article 10.1096/fj.01-0637fje.
56. Urbich, C. et al. (2002).  Double-Edged Role of Statins in Angiogenesis Signalling.  Circulation Research. 90: 737-744.
57. Osugi, T. et al (2002).  Cardiac-specific activation of Signal Transducer and Activator of Transcription-3 promotes vascular formation in the heart.  J. Biol. Chem. 227; 6676-6681
58. Kronenwett, R. et al, (2002).  Inhibition of angiogenesis in vitro by aV integrin-directed antisense oligonucleotides.  Cancer Gene Therapy 9; 587-596
59. Dredge, K. et al (2002).  Novel thalidomide analogues display anti-angiogenic activity independently of immunomodulatory effects.  Brit. J. Cancer 87; 1166-1172
60.  Urbich et al (2002).  CD40 ligand inhibits endothelial cell migration by increasing production of endothelial reactive oxygen species.  Circulation 106; 981-986
61. Svensson, L.A. et al (2002).  The cytolethal distending toxin of Haemophilus ducreyi inhibits endothelial cell proliferation.  Infection & Immunity 70; 2665-2669
62.  Iba, O. et al, (2002).  Angiogenesis by implantation of peripheral blood mononuclear cells into ischaemic limbs.  Circulation 106: 2019
63. Harmey, J.H. et al, (2002).  Lipoylsaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumour invasion.  Int. J. Cancer 101; 415-422
64. Terai, Y. et al, (2001).  Vascular smooth muscle cell growth promoting factor / F-spondin inhibits angiogenesis via the blockade of integrin aVb3 on vascular endothelial cells.  J. Cell. Physiol. 188; 394-402
65. Ohno-Matsui, K. et al, (2001).  Novel mechanism for age-related macular degeneration: an equilibrium shift between the angiogenesis factors VEGF and PEDF.  J. Cell. Physiol. 189; 323-333
66. Jia, H. et al, (2001).  Peptides encoded by exon 6 of VEGF inhibit endothelial cell biological responses and angiogenesis induced by VEGF.  Biochem. Biophys. Res. Commun. 283; 164-173
67. Murphy, J.E. & Fitzgerald, D.J., (2001).  Vascular endothelial growth factor induces cyclooxygenase-dependent proliferation of endothelial cells via the VEGF-2 receptor.  FASEB J. 15; 1667-1
68. Houston, P. et al, (2001).  The transcriptional co-repressor NAB2 blocks Egr-1-mediated growth factor activation and angiogenesis.  Biochem. Biophys. Res. Commun. 283; 480-486
69. Bussolati, B. et al, (2001).  Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide.  Am. J. Path. 159; 993-1008
70. Frandsen, T. et al (2001).  Clin. Cancer Res. 7; Suppl.
71. Donovan et al, (2001).  Comparison of three in vitro human ‘angiogenesis’ assays with capillaries formed in vivo.  Angiogenesis 4; 113-121
72. Bryant,  et al , (2000).  Tissue repair with a therapeutic transcription factor.  Human Gene Therapy 11; 2143-2158
73. Brooks, T.D. et al, (1999).  Antibodies to PAI-1 suppress angiogenesis in vitro.  Clin. Exp. Metastasis 17 (9); 112
74. Grohmann, M et al, (1999).  Nitric oxide released via VEGFR-1 suppresses VEGFR-2 mediated endothelial cell growth and regulates angiogenesis.  J. Reprod. Fertil. (Abs Series 24) No 48

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