Submitted to the Journal of Immunology (2010)

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)

P2RX7

• ligand-gated ion channel

Essential Cell Biology, 2nd Edition (Alberts et al., 2004)

ATP

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)

O

N

NN

N

NH2

OPOPOPO

O O O

O O OOH

O

OO

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 www.wikimedia.org

Results: Figure 1 (A)

• Primary human monocytes treated with either:

• VEGF release quantified (Sandwich ELISA) after 24 hours

1. HEPES (control)2. 100 μM BzATP3. 300 μM ATP4. 1 μg/mL LPS (positive control)

Results: Figure 1 (A)

• quantifying VEGF → sandwich ELISA

1 2 3

4 65

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

1. 100 μM BzATP2. 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

1. 100 μM BzATP2. 300 μM ATP3. 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

A438079

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 = 106 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 read @ 490 nm

Results: Figure 2 (B)

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 Ca2+

Essential Cell Biology, 2nd 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 Ca2+ mediated through P2RX7, then a cell permeable Ca2+

chelator will attenuate or decrease VEGF production

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

Results: Figure 3

• BAPTA-AM– cell-permeable Ca2+ chelator

Ca2+

wikipedia.org

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

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

monocyteCa2+ Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Results: Figure 3

• ionomycin = Ca2+ ionophore

tocris.com

Results: Figure 3

• ionomycin = Ca2+ ionophore

wikipedia.org

Results: Figure 3

• Figure 3 Conclusions:– VEGF release attenuated by BAPTA-AM

• though not dose-dependently

– artificial increase in intracellular Ca2+ ≠ VEGF stimulation

Results: Figure 3

Therefore, P2RX7 agonist-induced VEGF release is Ca2+-dependent, but it is not the

exclusive signal for VEGF stimulation

• P2RX7 role in production of ROS

IL-1βnitric oxide synthase

ROS

production of pro-inflammatorymolecules

(Lenertz et al., 2009)

• P2RX7 role in production of ROS

IL-1βnitric oxide synthase

ROS

production of pro-inflammatorymolecules

(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)

OHNH3

+

O

SHO

N+

OH

O

SH

H

Cysteine N-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:– “H2O2 treatment alone, or in combination with

ionomycin treatment, is unable to stimulate VEGF release”

VEGF

P2RX7

ROSROSROS

ROSROS

monocyte

VEGF

P2RX7

monocyteCa2+ Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

ROSROSROS

ROSROS

Results: Figure 4

• Figure 4 Conclusions:– VEGF release attenuated by NAC– artificial increase in ROS, intracellular Ca2+, or

both ≠ VEGF stimulation

Results: Figure 4

Therefore, P2RX7 agonist-induced VEGF release is Ca2+-dependent and ROS-

dependent, but they are not the exclusive signals for P2RX7-mediated VEGF stimulation

Overall Conclusion

VEGF

P2RX7

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

ATP

Ca2+

Ca2+

Ca2+ Ca2+

Ca2+

ROSROSROS

ROSROS

?

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

www.gelifesciences.com

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)