Submitted to the Journal of Immunology (2010)

ATP as an Extracellular Signal roles as signal molecule: –DAMP inflammatory response 1, pain sensation 2 –synaptic signaling (neurotransmitter) 2,3 –neuron-glia signaling 4 –muscle contraction 5 adenosine-5'-triphosphate (ATP)

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

in monocytes: +P2RX7 inflammatory response

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.

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.

Results: Figure 1 Primary human monocytes

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

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

Results: Figure 1 (A) Primary human monocytes treated with either: VEGF release quantified (Sandwich ELISA) after 24 hours 1.HEPES (control) μM BzATP μM ATP 4.1 μg/mL LPS (positive control)

Results: Figure 1 (A) quantifying VEGF → sandwich ELISA Anti-VEGF antibody VEGF Enzyme-linked anti-VEGF antibody

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

Results: Figure 1 (B) 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 μM BzATP 2.1 μg/mL LPS

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

Results: Figure 1 (B) Problems with Figure 1B: –Why not ATP?

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

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

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

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

Results: Figure 1 (D) 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 μM BzATP μM ATP 3.1 μg/mL PMA

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

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

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

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

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

Results: Figure 2 competitive P2RX7-specific antagonist A tocris.com

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

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

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

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?

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)

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

Results: Figure 2 (B) Methods: –NRCPA Overview colorimetric method cell density = 10 6 cells/mL

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

Results: Figure 2 (B) Methods: –NRCPA Overview

Results: Figure 2 (B) Methods: –NRCPA Overview absorbance 490 nm

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

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

Results: Figure 3 Figure 3 Hypothesis: If the P2RX7 stimulation of VEGF production is dependent on the increase in intracellular Ca 2+ mediated through P2RX7, then a cell permeable Ca 2+ chelator will attenuate or decrease VEGF production

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

Results: Figure 3 BAPTA-AM –cell-permeable Ca 2+ chelator Ca 2+ wikipedia.org

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

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

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

Results: Figure 3 (B)

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

Results: Figure 3 unpublished observation: –“ionomycin treatment alone unable to stimulate VEGF release” VEGF P2RX7 monocyte Ca 2+

Results: Figure 3 ionomycin = Ca 2+ ionophore tocris.com

Results: Figure 3 ionomycin = Ca 2+ ionophore wikipedia.org

Results: Figure 3 Figure 3 Conclusions: –VEGF release attenuated by BAPTA-AM though not dose-dependently –artificial increase in intracellular Ca 2+ ≠ VEGF stimulation

Results: Figure 3 Therefore, P2RX7 agonist-induced VEGF release is Ca 2+ -dependent, but it is not the exclusive signal for VEGF stimulation

P2RX7 role in production of ROS IL-1β nitric oxide synthase ROS production of pro-inflammatory molecules (Lenertz et al., 2009)

P2RX7 role in production of ROS IL-1β nitric oxide synthase ROS production of pro-inflammatory molecules (Lenertz et al., 2009)

Results: Figure 4 Figure 4 Hypothesis: If P2RX7 agonist-induced VEGF release in human monocytes is linked to ROS production, then P2RX7 agonist-induced VEGF release will be curtailed when exposed to an antioxidant

Results: Figure 4 N-AcetylCysteine (NAC) –Antioxidant –Neutralizes reactive oxygen species (ROS) CysteineN-AcetylCysteine

Results: Figure 4 Methods: –Primary human monocytes pretreated with either HEPES or 10mM NAC for 20 min. –Cells then treated with either 100 μM BzATP or 300 μM ATP for 4 hours –VEGF release quantified

Results: Figure 4 NAC pretreatment attenuates P2RX7 agonist-induced VEGF release

Figure 4 Problems: potentially do a viability for NAC treatment

Results: Figure 4 Conclusion: –ROS needed for VEGF production

Results: Figure 4 unpublished observations: –“H 2 O 2 treatment alone, or in combination with ionomycin treatment, is unable to stimulate VEGF release” VEGF P2RX7 ROS monocyte VEGF P2RX7 monocyte Ca 2+ ROS

Results: Figure 4 Figure 4 Conclusions: –VEGF release attenuated by NAC –artificial increase in ROS, intracellular Ca 2+, or both ≠ VEGF stimulation

Results: Figure 4 Therefore, P2RX7 agonist-induced VEGF release is Ca 2+ -dependent and ROS- dependent, but they are not the exclusive signals for P2RX7-mediated VEGF stimulation

Overall Conclusion

VEGF P2RX7 Ca 2+ ATP Ca 2+ ROS ? monocyte

Implications novel action of P2RX7 signaling – aids in monocyte’s role of resolving inflammation via the production of angiogenic factors targeting of P2RX7-dependent VEGF release → tumor treatment (?)

Extra Material

Isolation of Human Blood Monocytes Percoll Density Gradient –Separation of cells and cellular particles –Non-toxic to cells Red blood cells Granulocytes Mononuclear cells (Monocytes)

(1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)- 9a-(Acetyloxy)-1a,1b,4, 4a,5,7a,7b,8,9,9a-decahydro-4a,7b- dihydroxy-3-(hydroxym ethyl)- 1,1,6,8-tetramethyl-5-oxo-1H- cyclopropa[3,4]benz [1,2-e]azulen-9- yl tetradecanoate Phorbol 12-myristate 13-acetate (PMA)