Animals

All studies described were performed in accordance with an approved Institutional Animal Care and Use Committee protocol. WAG/RijCmcr rats were bred and maintained in a barrier facility at our institution. Two weeks prior to irradiation, rats were switched to a moderate antioxidant diet (Teklad Global 2018 diet) which is more representative of antioxidant levels in a human diet (18). All rats were provided reverse osmosis hyper-chlorinated water ad libitum.

Diminazene Aceturate (DIZE) Administration

Diminazene aceturate (DIZE) was purchased from Sigma-Aldrich (St. Louis, MO, D7770) and MedChemExpress (HY-12404, Monmouth Junction, NJ) and reconstituted in sterile water at a stock concentration of 10 mg/ml. Rats were weighed immediately prior to drug administration and administered 15 mg/kg of the 10 mg/ml DIZE solution via subcutaneous injection (19). Vehicle control rats received subcutaneous injections of an equivalent volume of sterile water.

Total-Body Irradiation Model

Adult female WAG/RijCmcr rats (11–12 weeks old) were exposed to 7.25–7.75 Gy TBI. All rats were irradiated without the use of anesthetics by being placed in a plastic jig and the entire body was exposed using a XRAD 320 kV orthovoltage x-ray system (Precision X-Ray, Madison, CT). The X-ray system was operated at 320 kVp and 13 mAs with a half-value layer of 1.4 mm copper and a dose rate of 169 cGy/min. For survival studies to day 30 postirradiation, rats were exposed to a total dose of 7.75 Gy TBI which is the LD50/30 in this model. Rats were dosed daily subcutaneously starting at 24 h postirradiation with either 15 mg/kg DIZE or sterile water until the end of the study. To examine the recovery of peripheral blood and bone marrow compartment, a subset of rats was exposed to a myelosuppressive dose of 7.25 Gy TBI which is approximately the LD10/30 in this model. At 24 h postirradiation, 15 mg/kg DIZE or sterile water were dosed daily subcutaneously until harvest on day 10 postirradiation.

Partial-Body Irradiation Model

Adult female WAG/RijCmcr rats were exposed to 13.5 Gy PBI with bone marrow shielding to one hind-limb (X-RAD 320 Precision, 320 kVp; 169 cGy/min). This dose is based on the established LD50/120 radiation dose for female all-cause mortality through lung-DEARE in this strain (20). All rats received supportive care after irradiation consisting of antibiotics (enrofloxacin ∼10 mg/kg/day) in the drinking water from days 2–14, subcutaneous saline (40 ml/kg) days 2–10, and powdered diet days 35–70. Rats were dosed subcutaneously starting at 24 h after PBI with either 15 mg/kg DIZE or sterile water (3×/week [Monday-Wednesday-Friday (MWF)] for the duration of the study. Daily health checks were performed by trained staff. Additionally, body weight, breathing rate and renal function were monitored to assess the health of the rats. Rats were followed for survival through gastrointestinal, hematopoietic, lung and renal injury up to 190 days.

Mobilization Assay

To assess for the mobilization of bone marrow progenitor cells to the peripheral blood, normal non-irradiated adult male rats were treated with a single subcutaneous dose of either 15 mg/kg DIZE, 0.55 mg/kg Neulasta^® (Amgen, Thousand Oaks, California), or vehicle control. The dose for Neulasta^® was selected based on prior studies in our lab using PEGylated G-CSF (21).

At 6 h after injection, the peripheral blood was collected, a complete blood cell count (CBC) was performed, and the mononuclear fraction was isolated by Ficoll-Paque separation. Mononuclear cells were then analyzed by flow cytometry and CFU assay as described below.

Blood Collection

Blood was collected from anesthetized rats via the jugular vein. Briefly, rats were restrained with one hand by positioning the forelimbs in the caudodorsal direction with the thumb and middle finger. The head of the rat was secured with the index finger, creating a straight line along the ventral side of the rat. A 23-gauge needle attached with a syringe was carefully inserted into either the right or left external jugular vein and blood was collected. Syringes were coated with EDTA to prevent clotting for all CBC blood draws.

Peripheral Blood Analysis

Complete blood counts were determined using a Heska Element 5 Veterinary Hematology Analyzer.

Blood Urea Nitrogen (BUN) Determination

BUN levels were monitored starting at day 90 postirradiation and every 30 days thereafter as using a urease-nitroprusside colorimetric assay as described previously (22, 23). Irradiated rats with BUN>120 mg/dl were euthanized and given a value of 120 mg/dl to account for attrition, since such rats were previously confirmed to have severe and irreversible renal damage (9, 24).

Breathing Rate Measurement

Breathing rates were measured using a MouseOx Plus Pulse Oximeter (Starr Life Sciences Corp) in accordance with manufacturer's instructions. Rats were acclimated to the recording processing on the day prior to recording. After calibration for movement, the breathing rate was measured over a five-minute period and averaged to obtain the reported rate for each rat.

Bronchoalveolar Lavage Fluid (BALF) Collection

BALF was collected immediately after euthanasia as previously described (25). Lavage fluid was centrifuged at 300g for 5 min at 4°C to remove cellular components and the resulting BALF supernatant was 10× concentrated (Millipore Amicon Ultra-4 Centrifugal Filter Unit) prior to ELISA analysis.

ACE and ACE2 Activity Assays

ACE and ACE2 activity were determined in whole lung digests with commercially purchased kits: ACE2 (MAK377, Sigma Aldrich) and ACE (CS0002, Sigma-Aldrich).

Bone Marrow Isolation

Bone marrow was harvested from the femurs of euthanized rats. Each femur was carefully excised from the animal and flushed with PBS containing 10% FBS and 1% penicillin-streptomycin using a 1 ml syringe with a 25G needle, making sure to flush from both ends to ensure all cells were removed. Cells were pelleted by centrifugation and the red blood cell fraction was removed with ACK lysis buffer. Total viable cells were determined using a Countess Automated Cell Counter Hemocytometer C10227 (Invitrogen) with trypan blue exclusion.

RT-PCR Assay

Lung total RNA was isolated using RNeasy Micro Kit (74004, Qiagen) and quantified on Nanodrop 2000 Spectrophotometer (Model: 840-274200, Thermo Fisher Scientific). C- DNA was prepared using the high-capacity RNA-to-cDNA^™ Kit (4387406, Thermo Fisher Scientific) and used to examine respective transcript expression using TaqMan Gene Expression Assay primer probes (4331182, Thermo Fisher Scientific). The rat primer probes used were ACE (Rn00561094_m1), ACE2 (Rn01416293_m1), AT2R (Rn00560677_s1), MASR (Rn00562673_s1) and the reference GAPDH (Rn01775763_m1). Expression of target genes was normalized to GAPDH. The relative expression of the gene was calculated with respect to control (0 Gy) and presented as fold change using the 2-ΔΔ^CT method.

Flow Cytometry Analysis

Bone marrow or peripheral blood mononuclear cells were stained with the following fluorescence-conjugated antibodies used to differentiate rat hematopoietic stem and progenitor cell populations (26): APC-CD45 (17-0461-82, Thermo Fisher Scientific), PE/Cy7-CD71 (204424, Biolegend), BV510-CD90 (202535, Biolegend), FITC-Ox82 (205103, Biolegend).

Samples were acquired live on MACSQuant 10 Analyzer Flow Cytometer (Miltenyi) with 7-AAD used to exclude dead cells. Data was analyzed using FlowJo software version 10.0 (BD Life Sciences).

Colony Forming Unit (CFU) Assay

CFU assays were conducted using MethoCult (R3774, Stem Cell Technologies, Vancouver, BC, Canada) media in six-well Smartdish^™ (Stem Cell Technologies) plates at a seeding density of 10,000 bone marrow mononuclear cells per well or the mononuclear cell fraction isolated by Ficoll-Paque (Stem Cell Technologies) gradient separation from 0.5 ml of whole blood.

Cytokine Estimation by ELISA

Rat TGF-beta (BMS623-3, Invitrogen) and IL-10 (ab100764, Abcam) cytokine concentrations were determined per the manufacturer's instruction.

Statistical Analyses

Statistical analysis was performed using the GraphPad Prism version 9 (GraphPad Software, Inc.). P values for comparison of survival curves were determined using Log-rank test.

Statistical analysis of multiple groups and time points was conducted using 2-way ANOVA with Tukey's multiple comparison test. A one-way ANOVA with Tukey's multiple comparison was used to analyze multiple groups at a single time point. P values of <0.05 were considered statistically significant.

H-ARS

We first evaluated survival during H-ARS in adult female rats exposed to 7.75 Gy TBI and treated with daily subcutaneous injection of water vehicle or 15 mg/kg DIZE starting at 24 h after TBI. DIZE treated rats had significantly improved survival to day 30 after TBI vs. water treated controls (Fig. 1A, P = 0.046). We then assessed recovery of peripheral blood complete blood counts (CBCs) and the bone marrow compartment in irradiated and DIZE treated rats at day 10 after a myelosuppressive dose of 7.25 Gy. DIZE treatment increased total white blood cell counts (WBCs) compared to vehicle treated controls (Fig. 1B). In the bone marrow, DIZE improved recovery of total cellularity (Fig. 1C). In rats, the CD71-CD45RA-OX82⁺CD90⁺ population is enriched for hematopoietic stem and progenitor cells (26). At day 10 after 7.25 Gy, this population is significantly decreased compared to non-irradiated controls at day 10 (Fig. 1D). There was a non-significant trend towards an increase in the percentage of CD71-CD45RA-OX82⁺CD90⁺ cells in DIZE treated bone marrow vs. vehicle control (Fig. 1D); however, the absolute numbers of CD71-CD45RA-OX82⁺CD90⁺ cells were significantly increased in DIZE-treated rats (Fig. 1E). Consistent with improved bone marrow cellularity, myeloid colony forming potential (CFU-GM) was also significantly increased in DIZE versus vehicle-treated rats (Fig. 1F).

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.

Multi-Organ Late Effects

Next, to see if ACE2 agonism with DIZE mitigates radiation pneumonitis, we employed a rat partial body irradiation model with shielding to one hind limb. This model reproduces the major sequelae of radiation injury to the bone marrow, gastrointestinal tract, lungs, and kidney (8, 27, 28). Pneumonitis manifests at days 50–120 postirradiation as evidenced by morbidity due to increased respiration, pleural effusion, and weight loss. Rats which survive past 120 days eventually succumb to renal failure evidenced by elevated blood urea nitrogen (BUN) levels. In this model, we have previously demonstrated lisinopril mediated ACE inhibition supports multi-organ recovery after radiation injury (8, 28). Here, we evaluated whether long-term administration of DIZE could similarly mitigate radiation injury. DIZE or water vehicle were administered subcutaneously starting at 24 h after PBI and was administered three times weekly (MWF) until endpoint. DIZE treatment after 13.5 Gy PBI in adult female rats significantly improved survival through 190 days (Fig. 2A). DIZE treatment increased median survival from 147 days in control-irradiated rats to 177 days. Long-term DIZE treatment had no effect on body weight compared to irradiated controls (Fig. 2B). Consistent with the observed improvement in survival during pneumonitis, DIZE treatment significantly reduced the radiation-induced elevation in breathing rate at day 70 (Fig. 2C). As part of our IACUC protocol, BUN levels are assessed monthly in all irradiated rats starting at day 90 and rats in renal failure (BUN > 120 mg/dL) are humanely euthanized. DIZE treatment significantly reduced BUN levels at days 90, 120 and 150 relative to 13.5 Gy PBI alone rats (Fig. 2D).

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.

Modulation of ACE/ACE2 Activity

We assessed the effects of DIZE treatment on modulation of ACE/ACE2 transcription and protein activity in the whole lung after 13.5 Gy PBI. At day 42 after 13.5 Gy PBI, ACE2 transcript levels in the lung are decreased relative to non-irradiated rats (Fig. 3A). Although DIZE treatment did not significantly alter ACE2 expression compared to vehicle treatment, DIZE treatment did decrease transcription of ACE relative to vehicle treatment. Additionally, DIZE increased mRNA expression of two receptors known to act as downstream targets of ACE2 enzymatic products: AT2R and MasR (Fig. 3A). At day 70 after 13.5 Gy PBI, we then assessed enzymatic activity levels of ACE and ACE2 in whole lungs (Fig. 3B). Although DIZE treatment did not alter ACE activity, DIZE increased ACE2 activity by more than twofold and decreased the ratio of ACE/ACE2 activity (Fig. 3B).

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.

Modulation of TGF-Beta and IL-10 Levels

At days 42 and 70 after 13.5 Gy PBI, we measured cytokines levels of two proteins known to affect radiation-induced lung injury: TGF-beta and IL-10. Both factors were measured in the plasma and bronchoalveolar lavage fluid (BALF). Levels of the pro-fibrotic factor TGF-beta were elevated in the plasma and BALF at both time points after 13.5 Gy PBI compared to non-irradiated control levels (Fig. 4A). However, treatment with DIZE reduced both plasma and BALF TGF-b levels vs. 13.5 Gy PBI alone (Fig. 4A). Conversely, anti-inflammatory cytokine IL-10 was reduced after 13.5 Gy PBI in the BALF at day 42 compared to non-irradiated levels (Fig. 4B). Treatment with DIZE increased levels of IL-10 in both the BALF and plasma at the day 70 timepoint (Fig. 4B).

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.

Bone Marrow Mobilization

Prior studies have suggested agonism of ACE2/Ang (1-7) signaling promotes mobilization of bone marrow progenitors with vasoreparative function (19, 29). Here, we evaluated whether a single subcutaneous injection of 15 mg/kg DIZE in non-irradiated rats induces bone marrow mobilization at 6 h. Data were compared to mobilization with a single dose of 0.55 mg/kg pegylated-G-CSF (Neulasta). Treatment with either DIZE or Neulasta increased total white blood cell and neutrophil counts at 6 h (Fig. 5A) and the percentage of hematopoietic progenitors present in the circulation (Fig. 5B). Both DIZE treatment and Neulasta also increased the numbers of circulating cells capable of forming hematopoietic colonies in a colony forming unit assay (Fig. 5C).

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.