The TP508 Solution for Nuclear Radiation Exposure

Nuclear Countermeasure Activity of TP508 Linked to Restoration of Endothelial Function and Acceleration of DNA Repair


Barbara Olszewska-Pazdrak 1, Scott D McVicar 1, Kempaiah Rayavara 2, Stephanie M Moya 1, Carla Kantara 1 2, Chris Gammarano 1, Paulina Olszewska 1, Gerald M Fuller 2, Laurie E Sower 2, Darrell H Carney 1 2

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Abstract

There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation.

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Figures

FIG. 1
FIG. 1 Effect of 8 Gy irradiation on NO production in response to TP508 and VEGF. Panel A: Two-day post-confluent cultures of HCAEC were sham-irradiated (solid bars) or 8 Gy gamma irradiated (hatched bars). After 24 h, media was replaced with EBM containing 200 μM L-arginine and cells were treated for 1 h with saline (vehicle; white bars), TP508 (50 μg/ml; dark gray bars) or VEGF (50 ng/ml; light gray bars). Supernatants were collected and analyzed for NO, as described in Materials and Methods. To determine if TP508 could mitigate effects of radiation, HCAEC were treated 1 h postirradiation with TP508 (50 μg/ml; cross-hatched bars) and then assayed for NO production 24 h later, as described above. *P < 0.01 vs. saline alone; #P < 0.01 vs. VEGF-treated; and ^P < 0.05 vs. 8 Gy irradiated cells treated with VEGF without TP508. Panel B: Western blot analysis of eNOS expression from HCAEC 24 h after sham irradiation or 8 Gy irradiation and treated at 1 h postirradiation with saline (vehicle) or TP508 (50 μg/ml; TP). Blot was reprobed with GAPDH as protein loading control.
FIG. 2
FIG. 2 Effect of radiation and TP508 on endothelial cell tube formation. Confluent cultures of HDMEC and human pericytes were 3 Gy irradiated with a 137Cs gamma irradiator as described in Materials and Methods. Cells were harvested 1 h later and combined at a ratio of 1:100 pericytes:HDMEC, then resuspended in EBM containing either 10 μg/ml TP508 or vehicle (0.9% saline). Co-cultures were then incubated on Matrigel for 24 h. Representative images show extent of tube-like networks at 18 h formed in nonirradiated saline-alone treated cells (panel A), nonirradiated TP508-treated cells (panel B), 3 Gy irradiated saline-treated cells (panel C) and 3 Gy irradiated TP508-treated cells (panel D). Mean total tube formation length per well (panel E) was determined using ImagePro 7.0. Total n = 3–8 wells; error bars = 95% CI; ***P < 0.001.
FIG. 3
FIG. 3 Effect of TP508 on DNA repair in HDMEC after 3 Gy irradiation. HDMEC were treated with saline alone (panels A and C) or 200 μg/ml TP508 (B and D) 1 h postirradiation with 3 Gy (see Materials and Methods). Cells were fixed in formaldehyde at 1, 5 and 9 h postirradiation and radiation-induced DNA DSB were visualized using immunofluorescent staining of γ-H2AX foci (red) (panels A and B, 1 h postirradiation; panels C and D, 5 h postirradiation). CellProfiler analysis of eight random fields for each condition and time point was used to determine the percentage of DAPI-stained nuclei in eight random fields with complete DNA repair (no foci) (panel E) and the number of foci per nucleus (panel F). Saline-treated controls (black); TP508 (200 μg/ml) (hatched bars). Total n = 180–240 nuclei counted per condition; error bars = 95% CI; ****P < 0.0001.
FIG. 4
FIG. 4 Effect of TP508 on DNA repair in HDMEC after 6 Gy irradiation. HDMEC were treated with saline alone (panels A and C) or 200 μg/ml TP508 (panels B and D) and 6 Gy gamma irradiated (Materials and Methods). Cells were fixed in formaldehyde at 1, 2.5, 5 and 9 h postirradiation and radiation-induced DNA DSB were visualized using immunofluorescent staining of γ-H2AX foci (red) (panels A and B, 1 h postirradiation; panels C and D, 5 h postirradiation). CellProfiler analysis of eight random fields for each condition and time point were used to determine the percentage of DAPI-stained nuclei in eight random fields with complete DNA repair (no foci) (panel E) and the number of foci per nucleus (panel F). Saline-alone treated (black); TP508 (200 μg/ml) (hatched bars hash). Total n = 180 to 240 nuclei counted per condition; Error bars = 95% CI; ***P < 0.001, ****P < 0.0001.
FIG. 5
FIG. 5 Effect of radiation and TP508 on endothelial cell sprouting from aortic explants. Thoracic aortas were isolated 24 h after sham irradiation and 3–10 Gy irradiation of CD-1 mice that were i.v. injected (tail vein) 1 h postirradiation with saline-alone (open bars) or TP508 (500 μg/ml; black bars). Aortic explants were cultured on Matrigel for 5 days. Panel A: Digital images are representative of the best sprouting from 8 to 10 explants per group. Panel B: Bar graph shows quantification of area of endothelial sprouting (8–10 explants per group). Data are presented as mean ± SD. *P < 0.05 compared to saline at each radiation dose; #P < 0.05 compared to saline with sham irradiation.
FIG. 6
FIG. 6 Dose-dependent effect of TP508 on endothelial cell sprouting from aortic explants. Panel A: Thoracic aortas were isolated 24 h postirradiation from sham irradiated (open bars) or 3 Gy irradiated (black bar) CD-1 mice that were i.v. injected (tail vein) 1 h postirradiation with saline vehicle (black bar) or TP508 (hatched bars) at doses of 20, 100 or 500 μg per mouse. Aortic explants were cultured on Matrigel for 4 days and digital image areas of sprouting outgrowth were quantified. Data are expressed as a mean ± SD, n = 6. *P < 0.05 compared to sham irradiation; #P < 0.05 compared with 3 Gy irradiation with saline Panel B: Thoracic aortas were isolated 24 h postirradiation from sham- (open bars) or 8 Gy irradiated (black bar) CD-1 mice that were i.v. injected (tail vein) 1 h postirradiation with saline alone (black bar) or TP508 (hatched bars) at doses of 20, 100 or 500 μg per mouse. Data are expressed as a mean ± SD, n = 6. *P < 0.05 compared to sham irradiation; #P < 0.05 compared to 8 Gy irradiation with saline alone.
FIG. 7
FIG. 7 Effect of TP508 on survival of CD-1 mice after whole-body gamma irradiation. Male 12-week-old CD-1 mice received 8.5 Gy irradiated, and at 24 h postirradiation injected (sc) with a single 100 μl dose of sterile saline-alone (solid line) or sterile saline containing TP508 (350 μg, 10 mg/kg; dashed line). The data are expressed based on Kaplan-Meyer GraphPad analysis. **P < 0.01 compared to saline; n = 20 mice per group.

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References

  1. Messerschmidt O. Combined effects of radiation and trauma. Adv Space Res. 1989;9:197–201. – PubMed

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Nuclear Countermeasure Activity of TP508 Linked to Restoration of Endothelial Function and Acceleration of DNA Repair
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