Submitted to the Journal of Immunology (2010)

1. Submitted to the Journal of Immunology (2010)

2. ATP as an Extracellular Signal • roles as signal molecule: • DAMP • inflammatory response1, pain sensation2 • synaptic signaling (neurotransmitter)2,3 • neuron-glia signaling4 • muscle contraction5 adenosine-5'-triphosphate (ATP)

3. P2RX7 • ligand-gated ion channel ATP Essential Cell Biology, 2nd Edition (Alberts et al., 2004)

4. in monocytes: inflammatory response ATP + P2RX7

5. Overall Hypothesis If P2RX7 is involved in a monocyte’s role of initiating angiogenesis, then P2RX7 activation in monocytes will result in the generation of VEGF.

6. Results: Figure 1 • Figure 1A and 1B Hypothesis: If P2RX7 receptors in monocytes are involved in the release of VEGF, then VEGF concentrations will increase upon stimulation of monocytes with P2RX7 agonists.

7. Results: Figure 1 Primary human monocytes

8. Results: Figure 1 3′-0-(4-benzoyl) benzoyl ATP (BzATP) adenosine-5'-triphosphate (ATP) Primary human monocytes BzATP & ATP are P2RX7 agonists

9. Results: Figure 1 Modified from www.wikimedia.org Primary human monocytes BzATP & ATP are P2RX7 agonists LPS: known stimulus for VEGF release (positive control)

10. Results: Figure 1 (A) HEPES (control) 100 μM BzATP 300 μM ATP 1 μg/mL LPS (positive control) Primary human monocytes treated with either: VEGF release quantified (Sandwich ELISA) after 24 hours

11. Results: Figure 1 (A) VEGF Anti-VEGF antibody 1 2 3 Enzyme-linked anti-VEGF antibody 4 5 6 quantifying VEGF → sandwich ELISA

12. Results: Figure 1 (A) * = p<0.05 # = p<0.005 VEGF release induced by both BzATP “(> 6-fold) and ATP (> 3-fold)

13. Results: Figure 1 (B) 100 μM BzATP 1 μg/mL LPS • Purpose: • Determine time needed to release significant VEGF levels following P2RX7 agonist-induced activation • Primary human monocytes treated with either: • VEGF release quantified at 1, 2, 4, 8, 16, and 24 hours following treatment

14. Results: Figure 1 (B) * = p<0.05 ** = p<0.01 # = p<0.005 ψ = p<0.001 • Significant VEGF release occurs after: • BzATP – 1 hour • LPS – 16 hours

15. Results: Figure 1 (B) • Problems with Figure 1B: • Why not ATP?

16. Results: Figure 1 (C) • Purpose: • Determine dosage BzATP needed for significant VEGF release after 4 hours • Primary human monocytes treated with varying amounts of BzATP for 4 hours • VEGF release quantified

17. Results: Figure 1 (C) * = p<0.05 ** = p<0.01 # = p<0.005 Significant VEGF release occurs after 4 hours using 20 μM BzATP

18. Results: Figure 1 (C) • Problems: • Std. dev. only visible for 120 μM BzATP • Why not ATP?

19. Results: Figure 1 (C) “Does this give any additional information 1A and B do not give?”

20. Results: Figure 1 (D) 100 μM BzATP 300 μM ATP 1 μg/mL PMA • Purpose: • Determine if short-term exposure to P2RX7 agonists can induce VEGF release • Primary human monocytes treated with either: • Media removed after 5 min. (short- term) or not removed (long-term) • Cells incubated for 4 hours. • VEGF release quantified

21. Results: Figure 1 (D) * = p<0.05 ** = p<0.01 # = p<0.005 Both short-term and long-term exposure to BzATP or ATP results in significant VEGF release

22. Lingering Question: What if P2RX7 agonists (ATP, BzATP) are stimulating other P2 receptors to stimulate VEGF release, not just P2RX7? • problem: BzATP can stimulate other P2 receptors at high concentrations

23. Lingering Question: What if P2RX7 agonists (ATP, BzATP) are stimulating other P2 receptors to stimulate VEGF release, not just P2RX7? solution = use a P2RX7-exclusive antagonist and measure its effect on ATP/BzATP-stimulated VEGF release

24. Results: Figure 2 • Figure 2 Hypothesis: If the BzATP/ATP stimulation of VEGF production is mediated through P2RX7, then a P2RX7 antagonist will attenuate or decrease VEGF production

25. Results: Figure 2 • competitive P2RX7-specific antagonist 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyri dine hydrochloride tocris.com

26. Results: Figure 2 • competitive P2RX7-specific antagonist A438079 tocris.com

27. Results: Figure 2 • competitive P2RX7-specific antagonist The Antagonist tocris.com

28. Results: Figure 2 (A) • Methods: • monocytes pretreated w/ vehicle (HEPES) or antagonist for 30 min at 37°C • monocytes then treated w/ BzATP, ATP or PMA for 4h • VEGF measured • done in triplicate

29. Results: Figure 2 (A) * = p<0.05 ** = p<0.01

30. Results: Figure 2 (A) • problems with Figure 2A: • why graph percent VEGF release on y-axis when Figure 1 just used VEGF concentration? • what VEGF level did they use as the 100% standard for comparison? • could VEGF attenuation be in response to cytotoxicity of the antagonist?

31. Results: Figure 2 (A) • problems with Figure 2A: • why graph percent VEGF release on y-axis when Figure 1 just used VEGF concentration? • what VEGF level did they use as the 100% standard for comparison? • could VEGF attenuation be in response to cytotoxicity of the antagonist? perform viability assay/control (Fig. 2B)

32. Results: Figure 2 (B) • Methods: • pretreated and stimulated similar to experiment in Fig. 2A • after 4 hours, viability assessed using the Non-Radioactive Cell Proliferation Assay (NRCPA) from Promega Corp. • done in triplicate

33. Results: Figure 2 (B) • Methods: • NRCPA Overview • colorimetric method cell density = 106 cells/mL

34. Results: Figure 2 (B) • Methods: • NRCPA Overview • colorimetric method wikipedia.org substrate of one color product of another color

35. Results: Figure 2 (B) • Methods: • NRCPA Overview

36. Results: Figure 2 (B) • Methods: • NRCPA Overview absorbance read @ 490 nm

37. Results: Figure 2 (B)

38. Results: Figure 2 (B) • Figure/Experiment 2 Hypothesis: If the BzATP/ATP stimulation of VEGF production is mediated through P2RX7, then a P2RX7 antagonist will attenuate or decrease VEGF production Results and controls → support hypothesis

39. ATP Essential Cell Biology, 2nd Edition (Alberts et al., 2004) • P2RX7 = ion channel • significant permeability to Ca2+

40. Results: Figure 3 • Figure 3 Hypothesis: If the P2RX7 stimulation of VEGF production is dependent on the increase in intracellular Ca2+ mediated through P2RX7, then a cell permeable Ca2+ chelator will attenuate or decrease VEGF production

41. Results: Figure 3 • BAPTA-AM • cell-permeable Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) tocris.com

42. Results: Figure 3 • BAPTA-AM • cell-permeable Ca2+ chelator Ca2+ wikipedia.org

43. Results: Figure 3 • BAPTA-AM • cell-permeable Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) tocris.com

44. Results: Figure 3 (A) • Methods: • monocytes pretreated w/ vehicle (DMSO) or BAPTA-AM for 20 min at 37°C • monocytes then treated w/ vehicle (HEPES), BzATP, or ATP for 4h • VEGF measured • done in triplicate

45. Results: Figure 3 (A) * = p<0.05 # = p<0.005

46. Results: Figure 3 (B)

47. Results: Figure 3 • problems with Figure 3A and 3B: • better clarity of results if arranged like Fig. 2A and 2B

48. Results: Figure 3 • unpublished observation: • “ionomycin treatment alone unable to stimulate VEGF release” P2RX7 monocyte Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ VEGF

49. Results: Figure 3 • ionomycin = Ca2+ ionophore tocris.com

50. Results: Figure 3 • ionomycin = Ca2+ ionophore wikipedia.org