Effects of captopril against radiation injuries in the Göttingen minipig model of hematopoietic-acute radiation syndrome

Abstract

Our laboratory has demonstrated that captopril, an angiotensin converting enzyme inhibitor, mitigates hematopoietic injury following total body irradiation in mice. Improved survival in mice is correlated with improved recovery of mature blood cells and bone marrow, reduction of radiation-induced inflammation, and suppression of radiation coagulopathy. Here we investigated the effects of captopril treatment against radiation injuries in the Göttingen mini pig model of Hematopoietic-Acute Radiation Syndrome (H-ARS). Minipigs were given captopril orally (0.96 mg/kg) twice daily for 12 days following total body irradiation (60Co 1.79 Gy, 0.42-0.48 Gy/min). Blood was drawn over a time course following irradiation, and tissue samples were collected at euthanasia (32-35 days post-irradiation). We observed improved survival with captopril treatment, with survival rates of 62.5% in vehicle treated and 87.5% in captopril treated group. Additionally, captopril significantly improved recovery of peripheral blood mononuclear cells, and a trend toward improvement in recovery of red blood cells and platelets. Captopril significantly reduced radiation-induced expression of cytokines erythropoietin and granulocyte-macrophage colony-stimulating factor and suppressed radiation-induced acute-phase inflammatory response cytokine serum amyloid protein A. Using quantitative-RT-PCR to monitor bone marrow recovery, we observed significant suppression of radiation-induced expression of redox stress genes and improved hematopoietic cytokine expression. Our findings suggest that captopril activities in the Göttingen minipig model of hematopoietic-acute radiation syndrome reflect findings in the murine model.

Fig 1. Effects of captopril treatment on survival, weight gain, and temperature in Göttingen minipigs.

Göttingen minipigs, 4–6 months of age, were exposed to 1.79 Gy total body ⁶⁰Co irradiation or sham irradiated. Minipigs either received vehicle (V, water) or captopril (Cap), provided orally twice daily starting 4 h post-irradiation through 12 days post-irradiation. A. Kaplan-Meier Curve of the effect of captopril on survival from total body irradiation. * indicates p<0.05 compared with all other groups; # indicates p<0.05 compared with vehicle-treated survival. B. Baseline weights for all animals were obtained at 5 days prior to irradiation (day -5). Weights were obtained on the indicated dates. Graphs show mean weight gain, ± SEM. C. Baseline temperatures were obtained at 5 days prior to irradiation (day -5). Temperatures were obtained from animals on the indicated dates. Graphs show mean weight gain, ± SEM. Weights and temperatures include all animals extant at the time point.

Fig 2. Effect of delayed captopril treatment on mature blood cell loss and recovery after total body irradiation.

Göttingen minipigs, 4–6 months of age, were exposed to 1.79 Gy total body ⁶⁰Co irradiation or sham irradiated. Minipigs either received vehicle (V, water) or captopril (Cap), provided orally twice daily starting 4 h post-irradiation through 12 days post-irradiation. Blood was obtained on day -3 (pre-irradiation), and on the indicated time points post-irradiation for analysis and quantification of A. red blood cells (RBC), B. platelets, C. red blood cells (RBC), D. hematocrit (HCT), E. absolute neutrophil count (ANC), and F. absolute lymphocyte count (ALC). Data show means ± standard error of the mean, n = 3–4 pigs per group. Average blood cell levels for sham irradiated animals are shown at B (basal) for clarity. * p < 0.05 for captopril treatment vs radiation + vehicle.

Fig 3. Effect of delayed captopril treatment on circulating hematopoietic progenitors after total body irradiation.

Göttingen minipigs, 4–6 months of age, were exposed to 1.79 Gy total body ⁶⁰Co irradiation or sham irradiated. Minipigs either received vehicle (V, water) or captopril (Cap), provided orally twice daily starting 4 h post-irradiation through 12 days post-irradiation. Blood was obtained on day -3 (pre-irradiation, shown at B), and on days 6, 9, 13, 16, and 23 post-irradiation for analysis and quantification of A. total colony forming units (CFU) and B. granulocyte-macrophage colony forming units (CFU-CM). Average blood cell levels for sham irradiated animals are shown at B (basal) for clarity. Data show means ± standard error of the mean, n = 3–4 pigs per group.

Fig 4. Effect of delayed captopril treatment on blood chemistry after total body irradiation.

Göttingen minipigs, 4–6 months of age, were exposed to 1.79 Gy total body ⁶⁰Co irradiation or sham-irradiation. Minipigs either received vehicle (V, water) or captopril (Cap), provided orally twice daily starting 4 h post-irradiation through 12 days post-irradiation. Blood was obtained on day -3 (pre-irradiation, shown at B), and on days 3, 6, 9, 13, 16, 20, 23, 30, and 35 post-irradiation for analysis and quantification of A. alanine aminotransferase (ALT), B. alkaline phosphatase (ALK/PHOS), C. creatinine, D. blood urea nitrogen (BUN), and E. glucose. Average blood cell levels for sham irradiated animals are shown at B (basal) for clarity. Data show means ± standard error of the mean, n = 3–4 pigs per group. * p < 0.05 difference from sham control.

Fig 5. Effect of delayed captopril treatment on serum cytokine levels after total body irradiation.

Göttingen minipigs, 4–6 months of age, were exposed to 1.79 Gy total body ⁶⁰Co irradiation or sham-irradiation. Minipigs either received vehicle (V, water) or captopril (Cap), provided orally mixed with yogurt twice daily starting 4 h post-irradiation through 12 days post-irradiation. Serum was obtained on days pre- or post-irradiation for ELISA for A. erythropoietin (EPO), B. granulocyte-macrophage colony stimulating factor (GM-CSF), or C. serum amyloid A (SAA). Average blood cell levels for sham irradiated animals are shown at B (basal) for clarity. Data show means ± standard error of the mean, n = 4 pigs per group for all time points except the last two time points (27 and 35 days post-irradiation) which included only 3 animals for the 1.79 Gy, vehicle-treated group. * p < 0.05 difference from sham control. # p < 0.05 difference from radiation + vehicle.

Fig 6. Effect of delayed captopril treatment on cytokine gene expression in the bone marrow after total body irradiation.

Göttingen minipigs, 4–6 months of age, were exposed to 1.79 Gy total body ⁶⁰Co irradiation or sham irradiation. Minipigs either received vehicle (V, water) or captopril (Cap), provided orally twice daily starting 4 h post-irradiation through 12 days post-irradiation. Bone marrow tissue was obtained at the time of euthanasia. All sham irradiated animals were euthanized at 32–35 days after sham irradiation. Radiation + V animals were euthanized at 17, 21, 34 and 35 days post-irradiation. Radiation + captopril animals were euthanized at 21 and 33, 34 and 35 days post-irradiation. RNA was prepared from bone marrow tissue and used for qRT-PCR for A. JAM2 (junctional adhesion molecule B), B. CCL5 (C-C motif chemokine ligand 5), C. IL5 (interleukin 5), D. IL2 (interleukin 2), and E. CCL2 (C-C motif chemokine ligand 2). Graph shows means of gene expression normalized to GAPDH ± standard error of the mean, n = 4 pigs per group. * p < 0.05 difference from sham control.