implications novel action of p2rx7 signaling – aids in monocyte’s role of resolving inflammation...

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 (?)

Novel action of P2RX7 signaling

Tumor treatment

• Oncogenes induce VEGF release

• ATP-activated monocytes within tumor environment contribute to tumor angiogenesis

• Target P2RX7 dependent VEGF release to inhibit tumor growth

Reviewer Complaints 1) VEGF response may also be activated by other P2 receptors 2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression? 3) The kinetics and dose-responses of BzATP-induced VEGF response in the

absence and presence of the P2RX7 antagonist should be included 4) It has been demonstrated before that P2RX7 receptor stimulation leads to

VEGF expression by Wei et al. in 2008 5) A concentration response curve (dose-response) for ATP should be included

for VEGF release 6) BAPTA-AM use does not differentiate between Ca2+ influx from the

extracellular space or release from intracellular Ca2+ stores 7) Cell membrane integrity and not metabolic activity should be used for the cell

viability assay 8) Unpublished observations should be given in the text or figure 9) Cell viability/membrane integrity following ionomycin and H2O2 stimulation

should be provided 10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in

pg/mL

1) The VEGF response may also be activated by other P2 receptors– eg. P2Y11, P2Y13

1) The VEGF response may also be activated by other P2 receptors– eg. P2Y11, P2Y13

ADDRESSED

1) VEGF response activated by other P2 receptors?

• Figure 3C (Revised) Hypothesis:

If the BzATP/ATP stimulation of VEGF production is mediated through P2RY11, then a P2RY11 antagonist will attenuate or

decrease VEGF production

1) VEGF response activated by other P2 receptors?

• Figure 3D (Revised) Hypothesis:

If the BzATP/ATP stimulation of VEGF production is mediated through P2RY13, then a P2RY13 antagonist will attenuate or

decrease VEGF production

1) VEGF response activated by other P2 receptors?

NF-157 (P2RY11 Antagonist)

tocris.com

2-methylthioadenosine 5′-monophosphate

MeSAMP

(P2RY12/13 Antagonist)

sigmaaldrich.com

1) VEGF response activated by other P2 receptors?

1) VEGF response activated by other P2 receptors?

• unpublished observation:– “co-addition of NF-157 or MeSAMP with

P2RX7 antagonist A438079 resulted in similar attentuation of BzATP induced VEGF as P2RX7 antagonist alone”

2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression?

2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression?

ADDRESSED

Quantitative RT-PCR

mRNA

mRNA

cDNA

5’

5’

5’

5’

5’3’

Reverse Transcription

PCR

2) P2RX7 inducing VEGF expression?

• Figure 4 (Revised) Hypothesis:

If P2RX7 agonists induce VEGF expression, then levels of mRNA encoding VEGF will

increase upon treatment with known P2RX7 agonists

• Primary human monocytes treated for 0.5 or 1 hour with either:

• Lysates analyzed via RT-PCR

2) P2RX7 inducing VEGF expression?

1. 100 μM BzATP2. 300 μM ATP3. 1 μg/mL PMA (positive control)

2) P2RX7 inducing VEGF expression?

• P2RX7 agonists modulate VEGF expression

Figure 4A

2) P2RX7 inducing VEGF expression?

2) P2RX7 inducing VEGF expression?

• Problems: – Statistical significance tests?

2) P2RX7 inducing VEGF expression?

Lingering problem: what if P2RX7 agonists are stimulating other receptors to express VEGF?

2) P2RX7 inducing VEGF expression?

• Methods (Figure 4B):– Pre-incubated monocytes with either:

– Cells then treated for 1 or 1.5 h with either:

– VEGF mRNA quantified via RT-PCR

1. HEPES (control)2. 3 μM antagonist3. 10 μM antagonist

1. HEPES (control)2. 30 μM BzATP3. 100 μM BzATP

2) P2RX7 inducing VEGF expression?

Figure 4B

2) P2RX7 inducing VEGF expression?

Figure Caption:

2) P2RX7 inducing VEGF expression?

Results:

2) P2RX7 inducing VEGF expression?

• Figure 4B:– Does not match the results section– Does not match the figure caption

2) P2RX7 inducing VEGF expression?

• Results (Figure 4B):– Agonist induced VEGF expression is

dependent upon P2RX7 activation

3) The kinetics and dose-responses of BzATP-induced VEGF response in the absence and presence of the P2RX7 antagonist should be included

3) The kinetics and dose-responses of BzATP-induced VEGF response in the absence and presence of the P2RX7 antagonist should be included

NOT ADDRESSED

3) replicate Figure 1B and 1C with P2RX7 antagonist?

3) replicate Figure 1B and 1C with P2RX7 antagonist?

• necessary (?)

4) It has been demonstrated before that P2RX7 receptor stimulation leads to VEGF expression by Wei et al. in 2008

4) It has been demonstrated before that P2RX7 receptor stimulation leads to VEGF expression by Wei et al. in 2008

NOT ADDRESSED

4) past research already demonstrated P2RX7 relationship

w/ VEGF?

Revised Hill Paper

Original Hill Paper

4) past research already demonstrated P2RX7 relationship

w/ VEGF?

Revised Hill Paper

Original Hill Paper

5) A concentration response curve (dose-response) for ATP should be included for VEGF release

5) A concentration response curve (dose-response) for ATP should be included for VEGF release

ADDRESSED

5) replicate Figure 1C with ATP?

5) replicate Figure 1C with ATP?

5) replicate Figure 1C with ATP?VEGF release from each patient after 100 μM BzATP

5) replicate Figure 1C with ATP?

• problems with revised Figure 1D:– no statistical tests performed

6) BAPTA-AM use does not differentiate between Ca2+ influx from the extracellular space or release from intracellular Ca2+ stores

6) BAPTA-AM use does not differentiate between Ca2+ influx from the extracellular space or release from intracellular Ca2+ stores

ADDRESSED

6) extracellular Ca2+ influx or intracellular Ca2+ release?

• Figure 6C (Revised) Hypothesis:

If the P2RX7 stimulation of VEGF release is dependent on the extracellular influx of

Ca2+ mediated through P2RX7, then a cell impermeable Ca2+ chelator will sequester

extracellular Ca2+ and attenuate or decrease VEGF production

6) extracellular Ca2+ influx or intracellular Ca2+ release?

ethylene glycol tetraacetic acid (EGTA)

6) extracellular Ca2+ influx or intracellular Ca2+ release?

7) Cell membrane integrity and not metabolic activity should be used for the cell viability assay

7) Cell membrane integrity and not metabolic activity should be used for the cell viability assay

NOT ADDRESSED

8) Unpublished observations should be given in the text or figure

8) Unpublished observations should be given in the text or figure

ADDRESSED

8) unpublished observations

• ionomycin treatment results

8) unpublished observations

• H2O2 and combined treatment results:

9) Cell viability/membrane integrity following ionomycin and H2O2 stimulation should be provided

9) Cell viability/membrane integrity following ionomycin and H2O2 stimulation should be provided

NOT ADDRESSED

10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL

10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL

NOT ADDRESSED

10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in pg/mL

NOT ADDRESSED

ADDRESSED+

10) addition of data to Fig. 1D and Fig. 2A

Figure 2A / Figure 3A (Revised)

10) modification of y-axis in Fig. 1D and Fig. 2A

Figure 1D / Figure 2A and B (Revised)

Reviewer Complaints 1) VEGF response may also be activated by other P2 receptors 2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression? 3) The kinetics and dose-responses of BzATP-induced VEGF response in the

absence and presence of the P2RX7 antagonist should be included 4) It has been demonstrated before that P2RX7 receptor stimulation leads to

VEGF expression by Wei et al. in 2008 5) A concentration response curve (dose-response) for ATP should be included

for VEGF release 6) BAPTA-AM use does not differentiate between Ca2+ influx from the

extracellular space or release from intracellular Ca2+ stores 7) Cell membrane integrity and not metabolic activity should be used for the cell

viability assay 8) Unpublished observations should be given in the text or figure 9) Cell viability/membrane integrity following ionomycin and H2O2 stimulation

should be provided 10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in

pg/mL

Reviewer Complaints 1) VEGF response may also be activated by other P2 receptors 2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression? 3) The kinetics and dose-responses of BzATP-induced VEGF response in the

absence and presence of the P2RX7 antagonist should be included 4) It has been demonstrated before that P2RX7 receptor stimulation leads to

VEGF expression by Wei et al. in 2008 5) A concentration response curve (dose-response) for ATP should be included

for VEGF release 6) BAPTA-AM use does not differentiate between Ca2+ influx from the

extracellular space or release from intracellular Ca2+ stores 7) Cell membrane integrity and not metabolic activity should be used for the cell

viability assay 8) Unpublished observations should be given in the text or figure 9) Cell viability/membrane integrity following ionomycin and H2O2 stimulation

should be provided 10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in

pg/mL

Reviewer Complaints 1) VEGF response may also be activated by other P2 receptors 2) Is P2RX7 only enhancing secretion of VEGF or is inducing VEGF expression? 3) The kinetics and dose-responses of BzATP-induced VEGF response in the

absence and presence of the P2RX7 antagonist should be included 4) It has been demonstrated before that P2RX7 receptor stimulation leads to

VEGF expression by Wei et al. in 2008 5) A concentration response curve (dose-response) for ATP should be included

for VEGF release 6) BAPTA-AM use does not differentiate between Ca2+ influx from the

extracellular space or release from intracellular Ca2+ stores 7) Cell membrane integrity and not metabolic activity should be used for the cell

viability assay 8) Unpublished observations should be given in the text or figure 9) Cell viability/membrane integrity following ionomycin and H2O2 stimulation

should be provided 10) Data in Fig. 1D and Fig. 2 should also be provided as VEGF release in

pg/mL

Extra Material

Propidium iodide

• Fluorescent molecule

• Intercalating agent

• “Leaky” membrane increases fluorescence

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