Mitigation of Multi-Organ Radiation Injury with ACE2 Agonist Diminazene Aceturate

Abstract

The renin-angiotensin system (RAS) is known to regulate the pathogenesis of radiation-induced injury as inhibitors of the RAS enzyme angiotensin converting enzyme (ACE) have established function as mitigators of multi-organ radiation injury. To further elucidate the role of RAS signaling during both the acute and delayed syndromes of radiation exposure, we have evaluated whether pharmacologic modulation of alternate RAS enzyme angiotensin converting enzyme 2 (ACE2) reduces the pathogenesis of multi-organ radiation-induced injuries. Here, we demonstrate pharmacologic ACE2 activation with the small molecule ACE2 agonist diminazene aceturate (DIZE) improves survival in rat models of both hematologic acute radiation syndrome (H-ARS) and multi-organ delayed effects of acute radiation exposure (DEARE). In the H-ARS model, DIZE treatment increased 30-day survival by 30% compared to vehicle control rats after a LD50/30 total-body irradiation (TBI) dose of 7.75 Gy. In the mitigation of DEARE, ACE2 agonism with DIZE increased median survival by 30 days, reduced breathing rate, and reduced blood urea nitrogen (BUN) levels compared to control rats after partial-body irradiation (PBI) of 13.5 Gy. DIZE treatment was observed to have systemic effects which may explain the multi-organ benefits observed including mobilization of hematopoietic progenitors to the circulation and a reduction in plasma TGF-beta levels. These data suggest the ACE2 enzyme plays a critical role in the RAS-mediated pathogenesis of radiation injury and may be a potential therapeutic target for the development of medical countermeasures for acute radiation exposure.

FIG. 1.

ACE2 agonism with DIZE mitigates H-ARS. Panel A. Survival after 7.75 Gy total-body irradiation (TBI) in adult female WAG/RijCmcr rats treated with daily subcutaneous injection of DIZE (N = 27) or water vehicle (N = 29). P value for log-rank analysis: P = 0.046. Panels B–F: Day 10 assessment of a cohort of female rats after 7.25 Gy TBI and treatment with DIZE (N = 9) or water vehicle (N = 9). Five age-matched non-irradiated animals were included as controls. Panel B: Scatter plot of complete blood cell counts. Panel C: Scatter plot of total cells per femur. Panel D: Left, representative FACS plots depicting the percentage of bone marrow cells within the hematopoietic stem cell-enriched population of CD71⁻OX82⁺CD90⁺ cells. Right, scatter plots of percent CD71⁻OX82⁺CD90⁺. Panel E: Calculated total CD71⁻OX82⁺CD90⁺ cells per femur. Panel F: Colony forming units per 10,000 whole bone marrow cells. Solid bar indicates median, significance values determined by ANOVA with multiple comparison tests.

FIG. 2.

ACE2 agonism with DIZE mitigates multi-organ DEARE. Panel A: Survival after 13.5 Gy partial-body irradiation (PBI) in adult female WAG/RijCmcr rats treated with subcutaneous injection of DIZE (N = 15) or water vehicle (N = 16) 3×/week (MWF). Gray region from days 50–120 indicates period of risk for morbidity due to lung-DEARE, while beige region from days 120–200 indicates period of risk for morbidity due to kidney-DEARE. Median survival for each group is indicated with a dashed line. P value for log-rank analysis: P = 0.0005. Panel B: Corresponding mean percentage body weight change for each treatment group. Panel C: Breathing rates at day 70 after 13.5 Gy TBI and treatment with DIZE (N = 4) or water vehicle (N = 4). Five age-matched non-irradiated animals were included as controls. Panel D: Blood urea nitrogen (BUN) levels at days 90–180 after 13.5 Gy TBI and treatment with DIZE (N = 9) or water vehicle (N = 7). Solid bar indicates median, significance values determined by ANOVA with multiple comparison tests.

FIG. 3.

DIZE increases ACE2 Signaling. Panel A: RT-PCR analysis performed on RNA isolated from whole lung lysates at day 42 after 13.5 Gy PBI in female rats treated with DIZE (N = 5) or water vehicle (N = 4). Data represent means ± SEM. P values determined by two-way ANOVA analysis with multiple comparison test. Panel B: Enzymatic ACE activity (left), ACE2 activity (middle), and the calculated ratio of ACE:ACE2 activity (right) in whole lung lysates at day 70 after 13.5 Gy PBI in female rats treated with DIZE (N = 5) or water vehicle (N = 4). Solid bar indicates median, significance values determined by ANOVA with multiple comparison tests.

FIG. 4.

DIZE regulates systemic TGF-beta and IL-10 levels. ELISA analysis was used to determine protein concentrations at days 42 and 70 after 13.5 Gy PBI in female rats treated with subcutaneous injection of DIZE (N = 5) or water vehicle (N = 4). A. TGF-beta levels in the bronchoalveolar fluid (BALF, left) and plasma (right). B. IL-10 levels in the BALF (left) and plasma (right). Four age-matched non-irradiated animals were included as controls. Significance values determined by two-way ANOVA with multiple comparison tests.

FIG. 5.

DIZE treatment induces mobilization of bone marrow progenitor cells. At 6 h after a single injection of either vehicle (n = 5), Neulasta (N = 5; 0.55 mg/kg), DIZE (n = 5, 15 mg/kg) in non-irradiated adult rats, peripheral blood was assessed for the following: Panel A: Complete blood cell counts including WBC differential. Panel B: Circulating progenitor cell populations as identified by FACS analysis (representative plots left) of CD71⁻OX82⁺ myeloid progenitors and CD71⁻OX82⁺CD90⁺ HSCs. C. Colony forming units per 1 ml whole blood. Solid bar indicates median, significance values determined by ANOVA with multiple comparison tests.

FIG. 6.

ACE2 signaling pathway. ACE2 metabolizes the conversion of angiotensin peptide Ang II to Ang (1–7). This both reduces the amount of Ang II available to bind the AT1R and increases signaling of Ang (1–7) through AT2R and MasR. Diminazene aceturate (DIZE) treatment increased ACE2 activity and increases AT2R and MasR transcription. We hypothesize this mechanism promoted the observed increase in survival by suppressing pro-inflammatory and pro-fibrotic signaling cascades.