Biological sex differences in renin angiotensin system enzymes ACE and ACE2 regulate normal tissue response to radiation injury

Abstract

Introduction: In experimental animal models, biological sex-differences in the manifestation and severity of normal tissue radiation injury have been well-documented. Previously we demonstrated male and female rats have differential and highly reproducible responses to high-dose partial body irradiation (PBI) with male rats having greater susceptibility to both gastrointestinal acute radiation syndrome (GI-ARS) and radiation pneumonitis than female rats. Methods: In the current study, we have investigated whether differential expression of the renin-angiotensin system (RAS) enzymes angiotensin converting enzyme (ACE) and ACE2 contribute to the observed sex-related differences in radiation response. Results: During the period of symptomatic pneumonitis, the relative ratio of ACE to ACE2 (ACE/ACE2) protein in the whole lung was significantly increased by radiation in male rats alone. Systemic treatment with small molecule ACE2 agonist diminazene aceturate (DIZE) increased lung ACE2 activity and reduced morbidity during radiation pneumonitis in both sexes. Notably DIZE treatment also abrogated morbidity in male rats during GI-ARS. We then evaluated the contribution of the irradiated bone marrow (BM) compartment on lung immune cell infiltration and ACE imbalance during pneumonitis. Transplantation of bone marrow from irradiated donors increased both ACE-expressing myeloid cell infiltration and immune ACE activity in the lung during pneumonitis compared to non-irradiated donors. Discussion: Together, these data demonstrate radiation induces a sex-dependent imbalance in the renin-angiotensin system enzymes ACE and ACE2. Additionally, these data suggest a role for ACE-expressing myeloid cells in the pathogenesis of radiation pneumonitis. Finally, the observed sex-differences underscore the need for consideration of sex as a biological variable in the development of medical countermeasures for radiation exposure.

Keywords: ACE; ACE2; GI-ARS; biological sex; medical countermeasures; myeloid cells; radiation pneumonitis; renin angiotensin system.

FIGURE 1

Characterization of Peripheral Blood ACE Activity Following Partial Body Irradiation (PBI). (A) Total peripheral blood white blood cell (WBC) counts and (B) percentage peripheral blood neutrophils (%NEU) at days 0, 14, 42, and 70 following 12.5 Gy partial body irradiation (PBI) with shielding of 5%–8% BM in one hind limb. (C) ACE activity within CD45⁺ peripheral blood mononuclear cells (PBMCs) at days 0, 42, and 70 following 12.5 Gy PBI. (D) Plasma AngII and Ang(1-7) levels at day 70 following irradiation. (N = 5 rats per group). (E) Blood plasma levels of testosterone and progesterone at day 70 following 12.5 Gy. (N= 5 rats per group). For all graphs, error bars indicate standard error of the mean *p < 0.05; **p < 0.01.

FIGURE 2

Sex Differences in Lung Immune Infiltrate During Radiation Pneumonitis. (A) Breathing rate at days 42 and 70 following 12.5 Gy Partial Body Irradiation (PBI). (B) Left, representative lung sections stained with mast cell marker tryptase (brown) in 0 Gy and 12.5 Gy PBI male and female rats at 70 days post radiation. Right, quantification of tryptase⁺ mast cells. (C) Representative flow cytometric analysis of the lung CD45⁺CD11b⁺ myeloid cell population at day 42 post-irradiation. (D) Quantification of percentage lung CD45⁺ cells and percentage CD45⁺ CD11b⁺ myeloid cells. (E) ACE activity within the lung CD45⁺ cell population at day 42 following 12.5Gy PBI. (N = 5 rats/group). For all graphs, error bars indicate standard error of the mean *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 3

Radiation-induced changes in ACE and ACE2 protein expression in the whole lung. (A) Representative, cropped day 70 western blot analysis of ACE (A) and ACE2 (B) protein in whole lung lysates in 0 Gy and 12.5 Gy partial body irradiated rats with corresponding GAPDH control. Uncropped blots are provided in Supplementary Figure S3. Western blotting bands of ACE (C) and ACE2 (D) were quantified by densiometric analysis and normalized with respect to GAPDH bands. (E) The ratio of ACE and ACE2 protein expression. (N = 5 rats/group). For all graphs, error bars indicate standard error of the mean ***p < 0.001; ****p < 0.0001.

FIGURE 4

Treatment with ACE2 agonist diminazene aceturate (DIZE) reduces morbidity following 13.5 Gy partial body irradiation (PBI). Survival following 13.5 Gy PBI in male and female rats following subcutaneous (SQ) treatment with vehicle or DIZE three times per week (MWF) starting at 72 h post-irradiation and continued through endpoint. Log-rank analysis was used to compare survival between male vehicle (N = 10 rats), female vehicle (N = 16 rats), male + DIZE (N= 11 rats) and female + DIZE (N = 10 rats) through day 120.

FIGURE 5

Radiation-induced changes in ACE and ACE2 protein expression in the bone marrow (BM). (A) Representative H&E-stained sections of male and female femurs prior to irradiation (0 Gy) and at day 70 following 12.5 Gy PBI. (B) Total viable bone marrow cells per femur at day 70 in non-irradiated control and irradiated rats. (C) BM enzymatic activity of ACE (left) and ACE2 (middle) at day 70. Right, calculated ratio of ACE/ACE2 at day 70. (D) Percent of total bone marrow positive for mature bone marrow lineage markers: CD11b, B220 and CD3 markers in control and irradiated rats at day 70. (E) Representative flow cytometry of BM CD45⁺CD11b⁺ myeloid cell population. (F) Representative histograms of ACE cell surface staining within the BM CD45⁺CD11b⁺ population. (G) Left, quantification of ACE⁺ expression within the CD45⁺CD11b⁺ population. Right, quantification of ACE⁺CD45⁺CD11b⁺ population. N = 5 rats/group. For all graphs, error bars indicate standard error of the mean *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 6

Recipients of irradiated bone marrow exhibit elevated myeloid ACE activity in the whole lung. (A) Bone marrow mononuclear cells (BM MNCs) from either control (0 Gy) or 7.75 Gy irradiated donors were transplanted into recipient rats exposed to 13 Gy. At day 70, the lung myeloid compartment was assessed by FACS analysis. (B) Representative flow cytometry gating of the CD45⁺CD11b⁺ myeloid cells in the lungs of BM-recipient rats. (C) Left, quantification of total lung CD45⁺ and CD45⁺ CD11b⁺ cells. (D) Representative histograms of ACE⁺ cells within the CD45⁺CD11b⁺ myeloid cell fraction. (E) Left, quantification of ACE⁺ expression within the CD45⁺CD11b⁺ population. Right, quantification of ACE⁺CD45⁺CD11b⁺ population. (F) Whole lung enzymatic ACE activity. (N = 9 rats/group). For all graphs, *p < 0.05.