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.
In the event of a radiological or nuclear disaster, victims of radiation exposure will experience dose-dependent toxicity to multiple organ systems. In the early days to weeks after acute radiation exposure, medical treatment must focus on the critical need to mitigate life-threatening injury to the hematopoietic and gastrointestinal systems. In accordance with this need, the FDA has approved the use of filgrastim (Neupogen®), pegfilgrastim (Neulasta®), sargramostim (Leukine®), and romiplostim (Nplate®) for treatment of the hematopoietic acute radiation syndrome (H-ARS). However, survivors of ARS are likely to experience the delayed effects of acute radiation exposure (DEARE) which encompass late morbidities and progressive functional decline of multiple organ systems. While there are no FDA-approved treatments to date for mitigation of DEARE, pharmacologic modulation of the renin-angiotensin system (RAS) has therapeutic potential.
RAS is known to regulate the response of several organ systems to radiation injury as pharmacologic inhibition of the key RAS enzyme angiotensin converting enzyme (ACE) is known to reduce hematopoietic, cardiac, respiratory, and renal damage after radiation exposure (1–9). In the regulation of vascular homeostasis, RAS signaling enzymes ACE and ACE2 are thought to act in opposition with ACE promoting vasoconstriction and ACE2 mediating vasorelaxation. ACE catalyzes the conversion of hormone peptide angiotensin I (Ang I) to the vasoconstrictor product angiotensin II (Ang II). Conversely, ACE2 counterbalances ACE-mediated vasoconstriction via reduction of Ang II to Ang (1–7) (10). The local RAS system and specifically the balance of ACE/ACE2 activity is thought to play a key role in the pathogenesis of many disease processes and has most recently been implicated in the pathogenesis of respiratory distress syndrome in COVID-19 patients (11, 12).
Here we have evaluated whether pharmacologic agonism of ACE2 mitigates early and late radiation injury in a similar manner to ACE inhibition. To pharmacologically promote ACE2 signaling in vivo, we have administered the small molecule ACE2 agonist diminazene aceturate (DIZE). DIZE is a well-tolerated anti-parasitic medication commonly used for the treatment of trypanosomiasis in livestock (13–16). Previously, DIZE treatment prior to radiation exposure has been shown to reduce acute radiation-induced renal injury in rats by increasing ACE2/Mas signaling (17). In this study, we have evaluated the efficacy of DIZE as a mitigator of radiation injury using two adult rat models: total-body irradiation (TBI) for H-ARS and partial-body irradiation (PBI) for lung and renal DEARE.
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.
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 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.
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-ΔΔCT 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 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.
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).
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).
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).
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).
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).
Diminazene aceturate (DIZE) is an aromatic diamidine compound developed in 1955 as anti-Trypanosoma medication for domestic livestock under the trade name Berenil (13). In addition to its anti-protozoal activity, DIZE has been observed to reduce immune activation and systemic inflammatory cytokine production (30). More recently, molecular docking and enzymatic activity assays have shown DIZE strongly enhances ACE2 catalytic activity (31). Based on this finding, DIZE has been employed as a pharmacologic ACE2 agonist in animal models of hypertension (19, 32–34) and cardiac dysfunction (35–37). Here, we have observed DIZE administration after radiation injury promotes multi-organ recovery without any overt tissue toxicity. To our knowledge, this is the first study to demonstrate DIZE mitigates lethal radiation injury in either an H-ARS or DEARE model. Moreover, the observation that DIZE can improve multi-organ survival in acute and delayed settings is a key translational finding as this may be a potential therapeutic mechanism to promote multi-organ recovery after radiation injury.
The ACE2/Ang (1-7) signaling pathway is known to play an important role in bone marrow regeneration after radiation or chemotherapy injury as administration of Ang (1-7) accelerates bone marrow recovery (38–41). Recently, ACE2 has been shown to be increased in highly purified human hematopoietic stem cell populations (42). Direct agonism of Ang (1-7) signaling promotes HSC expansion in vitro (39). Consistent with these findings, we observed DIZE-mediated ACE2 agonism significantly increased recovery of total bone marrow cellularity and functional colony forming capacity at both early and late time points after radiation injury. Additionally, we observed a survival benefit in DIZE-mediated ACE2 agonism in rats after 7.75 Gy TBI.
In addition to mediating an improved survival after hematopoietic injury, we observed that DIZE treatment also mitigated late morbidities due to pulmonary and renal failure. After 13.5 Gy PBI, rats in this model manifest morbidity from radiation pneumonitis characterized by rapid respiration (25). Here, we observed DIZE treatment reduces pneumonitis related morbidities and mitigates the increase in respiration rate at day 70 after PBI seen in irradiated control rats. DIZE treatment also significantly delays morbidity from renal failure as evidenced by significantly reduced BUN levels and increased overall median survival. These data are consistent with a prior report which demonstrated prophylactic DIZE treatment reduced acute renal damage after radiation injury (17).
Our observations with ACE2 agonism after radiation injury bear many similarities to ACE inhibitor treatment in radiation injury models. For example, ACE inhibitors have been shown to increase survival during H-ARS in rodent and pig models (1, 43, 44). Additionally, extensive studies by our group in the rat PBI model have demonstrated ACE inhibitors mitigate lung and kidney DEARE (8, 25, 28). It is possible ACE2 agonism may be acting to negate downstream ACE signaling by reducing the available amount of Angiotensin II (Ang II) to bind and activate Angiotensin II type 1 receptor (AT1R). Indeed, like ACE inhibitor treatment, DIZE treatment is known to lower blood pressure in animal models (45).
However, several findings from this study suggest ACE2 agonism with DIZE elicits a unique downstream signaling mechanism summarized in Fig. 6. As anticipated, DIZE induces high levels of ACE2 activity in the lung, but we also observed increased transcription of RAS receptors AT2R and MasR. Both AT2R and MasR are thought to directly counterbalance AT1R receptor function by promoting vasodilation, reducing inflammation, and reducing fibrosis (46). The necessity for AT2R and MasR in mediating the observed effects will be the focus of future studies. In keeping with a systemic suppression of pro-fibrotic signaling, we observed a dramatic reduction in both circulating and BALF TGF-beta levels in DIZE-treated rats. This is a key finding as TGF-beta is a master regulator in the initiation of radiation fibrosis. It is also consistent with prior experimental lung and cardiac fibrosis models where DIZE treatment was observed to decrease fibrotic progression and lower TGF-beta expression (47, 48). Increased levels of IL-10 in DIZE treated rats suggests DIZE treatment reduces inflammation as IL-10 is an anti-inflammatory mediator and is considered a candidate therapy for treatment of idiopathic pulmonary fibrosis (49).
One of the most interesting DIZE-mediated effects observed was the mobilization of hematopoietic progenitors into the circulation. At 6 h after DIZE administration, we saw a marked increase in circulating hematopoietic progenitors which was similar to Neulasta-mediated mobilization. Our observation is consistent with DIZE treatment in a murine model of myocardial infarction, in which DIZE was observed to increase circulating CD90+ cells thought to act as reparative endothelial progenitor cells (19). Additionally, agonism of Ang (1-7) signaling has also been observed to increase circulating hematopoietic progenitor cell populations (39, 50). Moreover, in diabetic patients the loss of vasoreparative function has been linked to decreased ACE2 expression in CD34+ hematopoietic progenitors as lentiviral-mediated ACE2 gene transfer in CD34+ cells derived from diabetic patients rescued vasoreparative function in hind-limb ischemia models (29). Together these studies suggest DIZE may promote multi-organ recovery via activation of hematopoietic progenitors with vasoreparative function.
In conclusion, we have observed DIZE treatment promotes multi-organ and long-term recovery after radiation injury. Since ACE2 is likely to promote recovery via mechanisms distinct from ACE pathway inhibition, it is likely the combination of ACE inhibitors and ACE2 agonists may have additive effects as radiation mitigators. In the mitigation of multi-organ injury after 13.5 Gy PBI, we observe that median survival in DIZE-treated female rats is 177 days. While this survival benefit is less than previously observed in female rats treated with ACE inhibitor lisinopril (213 days, unpublished data), we have not fully optimized the dose or schedule of either DIZE or lisinopril. Mitigation will also need to be validated in male rats as there are established sex differences in radiation sensitivity to DEARE (20). For this reason, future studies are warranted to optimize the potential combination of ACE inhibitors with ACE2 agonists for mitigation of multi-organ injury.
This work was supported by funding from NIH/NIAID U01AI133594, U01AI138331, the MCW Department of Radiation Oncology, and the MCW Cancer Center.