Extracellular ATP may contribute to tissue repair by rapidly stimulating purinergic receptor X7-dependent vascular endothelial growth factor release from primary human monocytes

Abstract

Extracellular ATP has been proposed to act as a danger signal to alert the immune system of cell damage. Release of high local concentrations of ATP activates the nucleotide receptor, purinergic receptor X7 (P2RX7), on monocytic cells, which promotes the processing/release of proinflammatory mediators. Although the proinflammatory actions of P2RX7 are well recognized, little is known regarding the potential function of P2RX7 in repair responses. Because the resolution of inflammation is characterized by monocytic cell-dependent production of proangiogenic factors, we evaluated the contribution of P2RX7 to this process. We observed that both short-term and long-term P2RX7 activation promotes the robust release of vascular endothelial growth factor from primary human monocytes. This vascular endothelial growth factor release is calcium dependent and associated with reactive oxygen species production. This previously unrecognized action of P2RX7 suggests that it may not only participate in inflammation and cell death, but that it is also likely to be important in the control of angiogenesis and wound repair.

Fig. 1. P2RX7 agonists stimulate the time- and concentration-dependent release of VEGF from primary human monocytes

(A) Primary human monocytes purified from healthy volunteer donors were treated with control (HEPES), 100 μM BzATP, 300 μM ATP, or 1 μg/mL LPS for 24 hr. (B) Cells were treated with either control, 100 μM BzATP, or 1 μg/mL LPS for 1–24 hr. (C) Cells were treated with control or varying amounts of the P2RX7 agonist BzATP for 4 h. The results depicted in panels A-C each represent at least 3 independent experiments with error bars representing mean +/- SEM. (D) Cells were treated with control or varying amounts of the P2RX7 agonist ATP for 4 h. Single gray data points represent VEGF release (pg/mL) from each donor after 100 μM BzATP treatment. The results depicted in panel D are from 3 independent donors with error bars representing mean +/- range, and numbers in parenthesis indicating fold increase in VEGF release after treatment with 500 μM ATP as compared to control for each donor. *p<0.05, **p<0.01, #p<0.005, ψp<0.001

Figure 2. Short-term stimulation by P2RX7 agonists induces VEGF release from primary human monocytes

Cells were treated with control, 100 μM BzATP, 300 μM ATP, or 1 μg/mL PMA. The media was either removed after 5 min and replaced with fresh, agonist-free media (short-term stimulation), or not removed (long-term stimulation), and the cells were incubated for 4 h. Supernatants were assayed for VEGF as described under Materials and Methods. (A) The results are depicted as fold increase in VEGF compared to vehicle control and are represent of 3 independent experiments (mean +/- SEM). (B) Results are depicted for each individual donor as VEGF release in pg/mL. *p<0.05, **p<0.01, #p<0.005, ψp<0.001

Fig. 3. P2RX7 antagonist A438079 attenuates BzATP and ATP-induced VEGF production

(A) Primary human monocytes were pre-incubated with vehicle or A438079 (3 μM or 10 μM) for 30 min at 37°C. The cells were then treated with control (HEPES), 100 μM BzATP, or 300 μM ATP, or 1 μg/ml PMA for 4 h and supernatants were assayed for VEGF. (B) Cells were pretreated and stimulated in parallel to “A”. After 4 h, cell viability was assessed as detailed in Materials and Methods. Primary human monocytes were pre-incubated with vehicle or either (C) P2Y11 antagonist NF-157 (0.3 μM or 1 μM) or (D) P2Y12/13 antagonist 2-Methylthioadenosine 5′-monophosphate (MeSAMP) (1 μM or 3 μM) for 30 min at 37°C. The cells were then treated with control (HEPES), 100 μM BzATP, or 300 μM ATP, or 1 μg/ml PMA for 4 h and supernatants were assayed for VEGF. The results shown in panels A and B include at least 3 independent experiments (mean +/- SEM), whereas the results presented in panels C and D include at least 2 independent experiments (mean +/- Range). *p<0.05, **p<0.01, #p<0.005, ψp<0.001

Fig. 4. P2RX7 agonists stimulate P2RX7-dependent induction of VEGF mRNA from primary human monocytes

(A) Primary human monocytes were treated with control (HEPES), 100 μM BzATP, 300 μM ATP, or 1 μg/mL LPS for 0.5 and 1 hr. Cells were then lysed in Trizol and the VEGF mRNA levels were measured using quantitative RT-PCR as detailed in Materials and Methods. Data from a representative experiment are shown as averages with error bars representing mean +/- SD. Results are representative of at least 2 analogous experiments. (B) Primary human monocytes were pre-incubated with vehicle or A438079 (3 μM or 10 μM) for 30 min at 37°C. The cells were then treated with control (HEPES), 30 μM BzATP, or 100 μM BzATP for 1 or 1.5 h, lysed in Trizol, and VEGF mRNA levels were measured using quantitative RT-PCR as detailed in Materials and Methods. Data from a representative experiment are shown as averages with error bars representing mean +/- SD. The results are representative of at least 2 analogous experiments.

Fig. 5. LPS-priming or co-stimulation does not synergistically enhance P2RX7 agonist-induced VEGF production

(A) Primary human monocytes were primed with vehicle control (HEPES) or 100 ng/mL LPS for 4 h and stimulated with either vehicle control (HEPES) or 100 μM BzATP for 2 h. (B-C) Primary human monocytes were stimulated with vehicle control (HEPES), 100 μM BzATP, 0.01 μg/mL LPS, 0.1 μg/mL LPS or co-stimulated with 100 μM BzATP/0.01 μg/mL LPS or 100 μM BzATP/0.1 μg/mL LPS for 4 h (B) or 24 h (C). Supernatants were collected and analyzed for VEGF release. The results depicted in panels A-C each represent three independent experiments (mean +/- SEM).

Fig. 6. P2RX7 agonist-induced VEGF production is calcium-dependent

(A) Primary human monocytes were loaded with either vehicle control (DMSO) or BAPTA-AM (3 μM or 5 μM) for 20 min at 37°C. The cells were then treated with control (HEPES), 100 μM BzATP, or 300 μM ATP for 4 h and supernatants were assayed for VEGF. (B) Cells were treated in parallel to panel “A”. Cell viability was measured as noted under Materials and Methods. (C) Primary human monocytes were loaded with either vehicle control (water) or EGTA (1 mM or 3 mM) for 20 min at 37°C. The cells were then treated with control (HEPES), 100 μM BzATP, or 300 μM ATP for 4 h and supernatants were assayed for VEGF. (D) Cells were treated in parallel to panel “C”. Cell viability was measured as noted under Materials and Methods. The results shown in panels A-D include 3 independent experiments (mean +/- SEM). *p<0.05, **p<0.01, #p<0.005, ψp<0.001

Fig. 7. P2RX7 agonist-induced VEGF production is sensitive to NAC

Primary human monocytes were pretreated with either vehicle control (HEPES) or 10 mM NAC for 20 min at 37°C. Following pre-incubation, cells were treated with control, 100 μM BzATP, or 300 μM ATP for 4 h. Supernatants were collected and assayed for VEGF. The results represent 3 independent experiments (mean +/- SEM). *π<0.05, **π<0.01, #π<0.005, ψp<0.001