Figure 1.
Pathophysiology of Radiation Retinopathy.
Radiation to the eye triggers leukocyte adhesion, blockage, and nutrient/oxygen deprivation of retinal vasculature. Resultant hypoxia leads to dysfunctional retinal neovascularization and vision loss.
Figure 2.
Radiation-induced REC-U937 adhesion.
Panel A) Static-adhesion: Irradiated human RECs were incubated for 24 hours in culture medium containing vehicle (PBS) or KZ-41 (10 µM). Calcein-AM loaded U937 cells were co-cultured with RECs (n = 8/group) for 30 minutes and non-adherent cells were washed from wells; attached U937 cells were quantified with a fluorescence microplate reader (excitation/emission wavelengths of 485/535 ηm). Data demonstrate that KZ-41 inhibits IR-induced leukocyte attachment to RECs (*, **P<0.005) (All data normalized to background fluorescence; data represent % Control fluorescence signal ± SD). Panel B) Flow-Chamber adhesion: RECs were irradiated and cultured for 24 hours. U937 cells were perfused over RECs placed in flow-chamber and digital images were collected after two hours. Data represent mean #-adherent cells/field ± SD. KZ-41 (10 µM) treatment significantly decreased IR-induced adhesion of U937 cells (*, #P<0.05). Panel C) Representative still images A) Control, B) IR-REC and C) IR+KZ-41 show extent of U937 accumulation on surface of RECs.
Figure 3.
ICAM-1 up-regulation in irradiated RECs.
A) Immunoblotting of ICAM-1 from IR-RECs after 24 hours show a significant up-regulation compared to unirradiated cells; treatment with KZ-41 (10 µM) reduces ICAM-1 levels by nearly 24% (Mean ± SD; *P<0.05, **P<0.05; n = 3).B) Confocal microscopy of RECs from flow-chamber slides. Top panels (A-C; Control, IR and IR+KZ-41, respectively) represent overlay images of DAPI and ICAM-1. Lower panels, D-E represents ICAM-1 immunoreactivity.
Figure 4.
Induction of p38MAPK phosphorylation in irradiated RECs.
A) RECs receiving 30 Gy irradiation in a single fraction show increases in phosphorylation of p38MAPK (T180/Y182) that reach a plateau between 4–8 hours compared to control cells at same time-points (*P<0.05). B) Irradiated RECs with or without treatment of KZ-41 (10 µM) were harvested and analyzed for phospho-p38MAPK at 4 hours. KZ-41 treated RECs showed significant reductions in total levels of phosphorylated p38MAPK (T180/Y182), as compared to IR-RECs (**P<0.05).
Figure 5.
Radiation-induced p53 phosphorylation and accumulation.
A–C) Phosphorylation at serine 15, 33, 37 was significantly induced by irradiation (*P<0.05) after 4 hours. The ratio of p53 phosphorylation at Ser 33 and 37 (relative to GAPDH) in KZ-41-treated (10 µM) RECs showed significant reduction (**P<0.05). D) Total p53 protein accumulation revealed significantly reduced levels in KZ-41 treated RECs (**P<0.05).
Figure 6.
Radiation-induced paxillin-dependent proliferative capacity/phenotype.
A) Irradiation-induced REC proliferation was measured after 24 hours using the WST-1 proliferation assay. REC proliferation was enhanced by irradiation and was significantly reduced with treatment of KZ-41 (10 µM) (*P<0.05). B) Paxillin phosphorylation (Y118) was measured 24 hours after irradiation using immunoblotting and showed enhanced levels. Both KZ-41 (10 µM) and p38MAPK inhibitor SB202190 (10 µM) significantly reduced levels of paxillin phosphorylation (*P<0.01, #P<0.05).
Figure 7.
KZ-41 reduces ischemic retinopathy/RNV: Avascular area.
A–D) representative flat-mounted retinas stained for endothelial cells using isolectin-B4 (red) from eyes harvested at P17: Normoxia, OIR, OIR+V, OIR+KZ-41, respectively. Mice received daily ocular administration of either KZ-41 (100 mg/kg) or vehicle (ocular nanoemulsion) from P12 to P17. Avascular area was determined using software-assisted analysis; shown in white. OIR mice show significant avascular area as compared to normoxia controls (*P<0.001). KZ-41 lowered area percent avascular area by nearly 50% (#P<0.001). Images were acquired at 10x magnification and digitally stitched together to show the entire retinal vasculature. Data represent mean (± SD). N = 5/group.
Figure 8.
KZ-41 reduces ischemic retinopathy/RNV: Neovascular area.
A–D) representative flat-mounted images of P17 retinas stained for endothelial cells using isolectin-B4 (red) were analyzed for neovascular tuft formations: Normoxia, OIR, OIR+V, OIR+KZ-41, respectively. Mice received daily ocular administration of either KZ-41 (100 mg/kg) or vehicle (ocular nanoemulsion) from P12 to P17. Analysis was performed after setting threshold limits to disregard non-neovascular networks and larger vessels in and around the optic disc (high intensity neovascular tufts shown in white). Both OIR groups (untreated and vehicle-treated) showed extensive tufting compared to normoxia controls (*P<0.005). KZ-41 lowered area percent neovascular tufts by ∼30% from OIR+Vehicle (#P<0.01). Images were acquired at 10x magnification and digitally stitched together to show the entire retinal vasculature. Data represent mean (± SD). N = 5/group.
Figure 9.
Model of KZ-41 radioprotective mechanism-of-action.
Gamma-(γ) radiation-induced DNA double strand breaks (DSBs) trigger phosphorylation of p38MAPK which in turn results in p53 accumulation enhances ICAM-1 surface levels and incites a proliferative/migratory phenotype through paxillin phosphorylation. KZ-41 reduces phospho-p38MAPK and effectively uncouples p38 MAPK signaling to reduce REC inflammation and halt aberrant cellular motility. Therefore, KZ-41 is able to protect RECs against acute radiation injury and the resultant dysfunction of the retinal vasculature.