Components of the renin-angiotensin system have been investigated for hematopoietic radiation protection because of the role of this system in hematopoietic cell regulation. Since ACE inhibitors are used extensively in the general population, it is critical to understand the effects of these drugs on radiation-induced hematopoietic injury, since radiotherapy is a common therapeutic modality for treating cancer, leukemia, and lymphoma [28
]. Paradoxically both ACE inhibitors and Ang II peptides have been shown to provide protection of the hematopoietic system [16
]. Here, we demonstrate that treatment of mice with the ACE inhibitor captopril resulted in either radiation protection or radiation sensitization, depending upon the time of captopril administration. Captopril treatment in nonirradiated mice had a biphasic affect on the cycling of ST-HSC with transient quiescence after 2 days of treatment followed by increased proliferation by 7 days of treatment. Our experiments demonstrate that captopril administration beginning 1 hour or 24 hours after irradiation and continuing for 7 to 30 days increased survival. Therefore, captopril-induced radiation protection correlated with transient quiescence (increased G0
) of the ST-HSC population and prevention of stem cell pool exhaustion. However, when captopril was initiated 7 days before irradiation and continued either to the time of irradiation or for an additional 30 days postirradiation, a significant increase in mortality was observed compared to untreated irradiated mice. In this case, radiation sensitization was correlated with increased cycling (increased G2
/M) of the ST-HSC population at the time of radiation exposure.
HSC quiescence sustains long-term hematopoiesis by protecting the HSC pool from radiation-induced injury and from premature exhaustion under conditions of hematopoietic stress. On the other hand, premature entry of HSC into the cell cycle following radiation exposure exhausts the stem cell pool and leads to hematological failure [36
]. The G2
/M phases of the cell cycle as well as increased rate of cell cycling are associated with increased sensitivity to radiation [5
]. Examination of the response of HPC in nonirradiated mice indicated that two days of captopril administration resulted in HPC transiently withdrawing from the cell cycle with increased percentages of cells in the G0
phases. However, in mice that received captopril for seven days, the HPC re-entered the cell cycle resulting in higher percentages of cells in the radiation-sensitive G2
/M phases of the cycle. Therefore, exposure of the more rapidly cycling cells to radiation resulted in greater damage to the cells. This was reflected by the significant decreases in CFU of multilineage progenitor cells observed at 5 days postirradiation that ultimately resulted in increased hematopoietic failure. These findings suggest that HSC from captopril pretreated mice exhibit a higher activated state that is associated with a loss of HSC cell cycle quiescence and increased susceptibility to radiation therapy. This may have led to impaired long-term repopulating potential, hematopoietic exhaustion, bone marrow failure, and decreased survival.
Previous studies demonstrated that ACE inhibition is associated with decreased hematocrit, CD34+
cell apoptosis, and decreased cycling of hematopoietic stem cells [14
]. In some patient populations, captopril was shown to cause granulocytopenia [31
], aplastic anemia [33
], and pancytopenia [34
], highlighting the role of ACE activities in hematopoietic cell homeostasis. Inhibition of ACE prevents proteolytic inactivation of the hemoregulatory peptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of hematopoietic stem cells cycling in vivo
]. It was thought that AcSDKP regulation of the hematopoietic stem cell cycle could be the mechanism radiation protection by ACE inhibitors. However, administration of AcSDKP did not provide radiation protection, whereas Ang II receptor antagonists provided radioprotective effects similar to ACE inhibition [20
], suggesting that blockade of Ang II maturation is the primary radioprotective effect of ACE inhibitors.
Cell culture experiments by others indicated that Ang II induces proliferation of ST-HSC and increased the formation of CFU-GM and CFU-GEMM colonies [11
]. Our results demonstrate that radiation protection by administration of captopril after irradiation was correlated enhanced repopulation of bone marrow-derived CFU-GM and CFU-M clonogenic progenitor cells. In contrast, radiation sensitization due to the administration of captopril before irradiation was associated with decreased numbers of stem/progenitor cells (CFU-GEMM, CFU-GM, and CFU-M) in the bone marrow and subsequent dampened mature blood cell recovery. Thus our findings of radiation protection and sensitization in vivo
are similar to in vitro
findings for Ang II effects on specific progenitor populations. The differences between these effects may be due to indirect activities of ACE inhibition in vivo
on hematopoietic progenitor cell proliferation.
It is of considerable importance that populations of erythrocytes, reticulocytes, and platelets, in addition to leukocytes, exhibited accelerated recovery in mice that received captopril postirradiation. Recent studies have demonstrated that the ability to rapidly reconstitute these lineages plays a key role in rescuing lethally irradiated recipients [35
]. Upon reaching a critically low threshold platelet level or severe thrombocytopenia, hemorrhage develops in multiple organs and is a predictor of mortality [4
]. The magnitude and duration of severe thrombocytopenia is hypothesized to be as important as severe neutropenia for survival from the radiation hematopoietic syndrome [25
]. In our mouse irradiation model, it appears that the threshold level of severe thrombocytopenia was approximaetly 30,000 platelets/μl. Severe thrombocytopenia (below the threshold level) occurred on days 10 and 14 postirradiation in untreated irradiated mice (22,000 and 25,000 platelets/μl) and in the mice that received captopril before irradiation (15,000 and 12,000 platelets/μl). In both groups, reduced platelet levels were associated with gross macroscopic brain hemorrhage (petechiae & ecchymoses) as well as microscopic hemorrhages. In contrast, mice in the group that received captopril treatment only after irradiation did not have a period of severe thrombocytopenia and evidence of hemorrhage was not observed in any organ. Decreased platelet counts and increased brain hemorrhages observed in mice pretreated with captopril were associated with increased mortality. Conversely, mice administered captopril only postirradiation exhibited improved platelet counts and brain hemorrhage and death were mitigated.
Captopril has been proposed to function as an antioxidant [37
], a free radical scavenger [38
], and as an inhibitor of other proteases such as matrix metalloproteinases [39
]. Captopril and other ACE inhibitors when administered postirradiation have been shown to modify a variety of late radiation-induced tissue injuries in the kidney, lung, skin, and heart [40
]. The mechanism of these agents in other tissue types is not completely known. Importantly, the enhanced marrow repopulation capacity observed in our in vivo
radiation protective studies further supports our hypothesis that the necessary signals required to efficiently promote HSC repopulation may be potentiated through captopril treatment postirradiation. Irradiation of the bone marrow compartment before transplantation produces cytotoxic effects on the nonhematopoietic cellular constituents in the bone marrow “hematopoietic niche” as well on the hematopoietic cells [42
]. Kopp et al
) reported that regeneration of damaged sinusoidal endothelial cells in the bone marrow niche is the rate-limiting step in hematopoietic regeneration from myelosuppressive therapy [45
]. Although not the focus of this paper, captopril-induced radiation protection could possibly support enhanced nonhematopoietic cell repopulation resulting in accelerated reconstitution of the requisite hematopoietic supportive osteoblastic niche [46
]. Furthermore, our findings suggest that the ability of captopril treatment in vivo
to regulate the state of stem cell quiescence and the potential repopulating/regenerative ability of hematopoietic progenitors might have a useful application in clinical transplantation. On the other hand, patients undergoing ACE inhibitor therapy may be at a higher risk for impaired/delayed HSC engraftment and successful recovery of normal hematopoiesis. Further understanding of the mechanisms of action of captopril and other ACE inhibitors for radiation protection in these tissues will help in the development of future agents for prevention and mitigation of radiation-induced injuries.