Selective Insulin-like Growth Factor Resistance Associated with Heart Hemorrhages and Poor Prognosis in a Novel Preclinical Model of the Hematopoietic Acute Radiation Syndrome

Abstract

Although bone marrow aplasia has been considered for the past decades as the major contributor of radiation-induced blood disorders, cytopenias alone are insufficient to explain differences in the prevalence of bleeding. In this study, the minipig was used as a novel preclinical model of hematopoietic acute radiation syndrome to assess if factors other than platelet counts correlated with bleeding and survival. We sought to determine whether radiation affected the insulin-like growth factor-1 (IGF-1) pathway, a growth hormone with cardiovascular and radioprotective features. Gottingen and Sinclair minipigs were exposed to ionizing radiation at hematopoietic doses. The smaller Gottingen minipig strain was more sensitive to radiation; differences in IGF-1 levels were minimal, suggesting that increased sensitivity could depend on weak response to the hormone. Radiation caused IGF-1 selective resistance by inhibiting the anti-inflammatory anti-oxidative stress IRS/PI3K/Akt but not the pro-inflammatory MAPK kinase pathway, shifting IGF-1 signaling towards a pro-oxidant, pro-inflammatory environment. Selective IGF-1 resistance associated with hemorrhages in the heart, poor prognosis, increase in C-reactive protein and NADPH oxidase 2, uncoupling of endothelial nitric oxide synthase, inhibition of nitric oxide (NO) synthesis and imbalance between the vasodilator NO and the vasoconstrictor endothelin-1 molecules. Selective IGF-1 resistance is a novel mechanism of radiation injury, associated with a vicious cycle amplifying reactive oxygen species-induced damage, inflammation and endothelial dysfunction. In the presence of thrombocytopenia, selective inhibition of IGF-1 cardioprotective function may contribute to the development of hemostatic disorders. This finding may be particularly relevant for individuals with low IGF-1 activity, such as the elderly or those with cardiometabolic dysfunctions.

FIG. 1.

IGF-1 levels. Plasma IGF-1 concentrations in adult male GMP (n = 22) and SMP (n = 24) were quantified using ELISA. Basal IGF-1 levels before irradiation were marginally higher in SMP compared to GMP (P > 0.043; Mann-Whitney U test).

FIG. 2.

Plasma IGF-1 levels increased in decedent GMP. IGF-1 levels were quantified longitudinally in survivors (n = 10) and decedents (n = 12) using ELISA (panels A and B). Values in scatter plots are IGF-1 levels from individual animals; each symbol represents an individual animal. Average IGF-1 levels are compared between preirradiation and euthanasia in survivors (n = 15) and decedents (n = 17) (panel C). IGF-1 was significantly increased in decedent animals at euthanasia compared to their preirradiation levels (**P < 0.01, Student’s paired t test, two tails). Values plotted in panel C are mean ± SEM.

FIG. 3.

Dynamic changes in the expression of IGF-1 and CRP. Plasma IGF-1 and CRP levels were compared longitudinally in survivors and decedents (panels A, B, D and E). Representative IGF-1 and CRP levels from one survivor and one decedent (panels A and B, respectively) show that the expression of IGF-1 paralleled that of CRP, however, the equilibrium was lost when the animals became sick due to radiation injury (panel B). Fold changes in the IGF-1-to-CRP ratio were higher at euthanasia than preirradiation in decedents (panel C). IGF-1 and CRP expression levels from individual animals were normalized to their preirradiation levels and are shown as the relative fold changes for survivors (panel D) and decedents (panel E). Panel F: The ratio of CRP to IGF-1 in animals with and without hemorrhages (hem) in heart and also decedents compared to survivors. Values plotted in panels C and F are mean average ratios after normalization. Panels G–I: Plasma angiotensin II concentration from individual survivors (panel G), decedents (panel H) and average angiotensin II levels at preirradiation and at euthanasia (panel I). There were no statistically significant differences in the concentration of angiotensin II in decedents compared to survivors. Values plotted in panels C, F and I are mean ± SEM. Data analyzed using Student’s paired t test; n = 10–12/group. *P < 0.05; **P < 0.01. Each symbol in panels D, E, G and H represent an individual animal.

FIG. 4.

Deregulated IGF-1R signaling in decedents. Immunoblots were performed using antibodies against p-IGF-1R, IGF-1R, Akt, p-Akt, p-ERK1/2 and ERK1/2 (panels A, C and E; n = 7–12/group). Shown here are representative images. Protein bands were quantified using densitometry measurements (panels B, D and F). Expression of p-IGF-1R was significantly reduced in decedents, whereas total IGF-1R was increased in the heart (panels A and B). p-Akt levels were also decreased, however, the expression of total Akt remains unchanged (panels C and D). Differences were observed in the expression of total ERK1/2 and p-ERK-1/2 but they were not statistically significant (panels E and F). Na⁺/K⁺-ATPase (membrane) or GAPDH (cytosolic) was used as the loading control. Values plotted are mean relative fold change ± SEM. Student’s t test was used for comparison of survivors vs. decedents. **P < 0.01; ***P < 0.001.

FIG. 5.

Increased oxidative stress in decedents. Immunoblots were performed using α-p67-phox, α-SOD1 and α-catalase antibody and were normalized to Na⁺/K⁺-ATPase, GAPDH and β-tubulin, respectively. NADPH oxidase subunit, p67-phox expression was increased in decedents. The expression of superoxide dismutase (SOD) was decreased, whereas the catalase levels were not altered in decedents compared to survivors (panel A). Protein bands were quantified using densitometry (panels B and C). Values plotted are mean relative fold changes ± SEM; Student’s t test was used for comparison of survivors vs. decedents. n = 8–11/group.*P < 0.05; **P < 0.01.

FIG. 6.

Altered expression and the activity of eNOS in decedents. Panel A: Representative Western blots show the expression of eNOS in the heart membrane fractions of survivors and decedents. Immunoblots were performed using antibodies against eNOS and p-eNOS (S1177) and were normalized to Na⁺/K⁺-ATPase. Panel B: Densitometric analysis of p-eNOS, total eNOS and the ratio of p-eNOS to eNOS. The expression of total p-eNOS and eNOS was increased in decedents, however, the increase in p-eNOS was not statistically significant. All data are expressed as mean relative fold changes ± SEM; n = 10–13/group. Student’s t test was used for comparison of survivors vs. decedents. *P < 0.05.

FIG. 7.

BH4, NO and ET-1 levels were altered in decedents. Plasma BH4, NO and ET-1 concentrations were measured longitudinally in survivors and decedents from preirradiation through necropsy. Significant reduction in the production of BH4 (panels A and B) as well as NO (panels D and E) was evident in decedents compared to survivors. The concentration of BH4 and NO at preirradiation vs. euthanasia are shown in panels C and F, respectively. Plasma ET-1 levels from individual survivors (panel G) and decedents (panel H) and average ET-1 levels at preirradiation and at euthanasia (panel I). There was no statistically significant difference in the concentration of ET-1 in decedents compared to survivors (panel I). Values shown at day 45 in survivors (panels A, D and G) and decedents (panels B, E and H) are BH4, total nitrite and ET-1 levels at euthanasia (Euth). Student’s paired t test was used for comparison of preirradiation to euthanasia. n = 8/group. *P < 0.01; **P < 0.001. BH4 = tetrahydrobiopterin; NO = nitric oxide; ET-1 = endothelin-1.

FIG. 8.

Increased ratios of ET-1 to BH4 and ET-1 to nitric oxide. Fold changes in the ratios of individual animals in the expression of ET-1 to BH4 (panels A and B) and ET-1 to NO (panels D and E). Each symbol represents an individual animal. Average ratios of ET-1 to BH4 (panel C) and ET-1 to NO (panel F) were increased at euthanasia compared to their preirradiation levels. n = 6-8; Wilcoxon signed rank test, *P < 0.05. BH4 = tetrahydrobiopterin; NO = nitric oxide; ET-1 = endothelin-1.