After the events of September 11, 2001, a decade of research on the development of medical countermeasures (MCMs) to treat victims of a radiological incident has yielded two FDA-approved agents to mitigate acute radiation syndrome. These licensed agents specifically target the mitigation of radiation-induced neutropenia and infection potential, while the ramifications of the exposure event in a public health emergency incident could include the entire body, causing additional acute and/or delayed organ/tissue injuries. Anecdotal data as well as recent findings from both radiation accident survivors and animal experiments implicate radiation-induced injury or dysfunction of the vascular endothelium leading to tissue and organ injuries. There are significant gaps in our understanding of the disease processes and progression, as well as the optimum approaches to develop medical countermeasures to mitigate radiation vascular injury. To address this issue, the Radiation and Nuclear Countermeasures Program of the National Institute of Allergy and Infectious Diseases (NIAID) organized a one-day workshop to examine the current state of the science in radiation-induced vascular injuries and organ dysfunction, the natural history of the pathophysiology and the product development maturity of potential medical countermeasures to treat these injuries. Meeting presentations were followed by a NIAID-led open discussion among academic investigators, industry researchers and government agency representatives. This article provides a summary of these presentations and subsequent discussion from the workshop.

Intestinal radiation toxicity occurs during and after abdominopelvic radiotherapy. Endothelial cells play a significant role in modulating radiation-induced intestinal damage. We demonstrated that the endothelial cell surface receptor thrombomodulin (TM), a protein with anticoagulant, anti-inflammatory and antioxidant properties, mitigates radiation-induced lethality in mice. The goal of this study was to determine whether recombinant TM (Solulin) can protect the intestine from toxicity in a clinically relevant rat model. A 4 cm loop of rat small bowel was exposed to fractionated 5 Gy X radiation for 9 consecutive days. The animals were randomly assigned to receive daily subcutaneous injections of vehicle or Solulin (3 mg/kg/day or 10 mg/kg/day) for 27 days starting 4 days before irradiation. Early intestinal injury was assessed two weeks after irradiation by quantitative histology, morphometry, immunohistochemistry and luminol bioluminescence imaging. Solulin treatment significantly ameliorated intestinal radiation injury, made evident by a decrease in myeloperoxidase (MPO) activity, transforming growth factor beta (TGF-β) immunoreactivity, collagen-I deposition, radiation injury score (RIS) and intestinal serosal thickening. These findings indicate the need for further development of Solulin as a prophylactic and/or therapeutic agent to mitigate radiation-induced intestinal damage.

Results from our recent studies have led to the novel hypothesis that radiation-induced coagulopathy (RIC) and associated hemorrhage occurring as part of the acute radiation syndrome (ARS) is a major cause of death resulting from radiation exposure in large mammals, including humans. This article contains information related to RIC, as well as potential strategies for the prevention and treatment of RIC. In addition, new findings are reported here on the occurrence of RIC biomarkers in humans exposed to radiation. To determine whether irradiated humans have RIC biomarkers, blood samples were obtained from radiotherapy patients who received treatment for different types of malignancies. Blood samples from allogeneic hematopoietic cell transplantation (allo-HCT) patients obtained before, during and after irradiation indicated that exposure led to prolonged clot formation times, increased levels of thrombin-antithrombin III (TAT) complex and increased circulating nucleosome/histone (cNH) levels, which suggest potential coagulopathies in the allo-HCT patients. Since these allo-HCT patients received chemotherapy prior to radiotherapy, it is possible that the chemical agents could have influenced the observed results. Frozen plasma samples from radiotherapy patients with prostate, lung and breast cancer were also obtained for analyses of cNH levels. The results indicated that some of these patients had very high cNH blood levels. Analysis of cNH levels in plasma samples from irradiated ferrets also indicated increased cNH levels compared to preirradiation baseline levels. The results from irradiated animals and some radiotherapy patients suggest the possibility that anti-histone antibodies, which block the toxic effects of elevated cNH levels in the blood, might be useful as therapeutic agents for adverse biological radiation-induced effects. The detection of increased levels of cNH in some radiotherapy patient blood samples demonstrates its potential as a biomarker for diagnosing and/or predicting the propensity for developing coagulopathies/hemorrhage, offering possible treatment options with personalized medicine therapies for cancer patients.

Ionizing radiation exposure can cause acute radiation sickness (ARS) by damaging the hematopoietic compartment. Radiation damages quiescent hematopoietic stem cells (HSCs) and proliferating hematopoietic cells, resulting in neutropenia, thrombocytopenia and increased risk for long-term hematopoietic dysfunction and myelodysplasia. While some aspects of the hematopoietic response to radiation injury are intrinsic to hematopoietic cells, the recovery of the HSC pool and overall hematopoiesis is also dependent on signals from bone marrow endothelial cells (BM ECs) within the HSC vascular niche. The precise mechanisms through which BM ECs regulate HSC regeneration remain unclear. Characterization of the altered EC gene expression that occurs in response to radiation could provide a roadmap to the discovery of EC-derived mechanisms that regulate hematopoietic regeneration. Here, we show that 5 Gy total-body irradiation substantially alters the expression of numerous genes in BM ECs within 24 h and this molecular response largely resolves by day 14 postirradiation. Several unique and nonannotated genes, which encode secreted proteins were upregulated and downregulated in ECs in response to radiation. These results highlight the complexity of the molecular response of BM ECs to ionizing radiation and identify several candidate mechanisms that should be prioritized for functional analysis in models of hematopoietic injury and regeneration.

Exposure to ionizing radiation induces not only apoptosis but also senescence. While the role of endothelial cell apoptosis in mediating radiation-induced acute tissue injury has been extensively studied, little is known about the role of endothelial cell senescence in the pathogenesis of radiation-induced late effects. Senescent endothelial cells exhibit decreased production of nitric oxide and expression of thrombomodulin, increased expression of adhesion molecules, elevated production of reactive oxygen species and inflammatory cytokines and an inability to proliferate and form capillary-like structures in vitro. These findings suggest that endothelial cell senescence can lead to endothelial dysfunction by dysregulation of vasodilation and hemostasis, induction of oxidative stress and inflammation and inhibition of angiogenesis, which can potentially contribute to radiation-induced late effects such as cardiovascular diseases (CVDs). In this article, we discuss the mechanisms by which radiation induces endothelial cell senescence, the roles of endothelial cell senescence in radiation-induced CVDs and potential strategies to prevent, mitigate and treat radiation-induced CVDs by targeting senescent endothelial cells.

There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation.

Hemodynamic shear stress is defined as the physical force exerted by the continuous flow of blood in the vascular system. Endothelial cells, which line the inner layer of blood vessels, sense this physiological force through mechanotransduction signaling and adapt to maintain structural and functional homeostasis. Hemodynamic flow, shear stress and mechanotransduction signaling are, therefore, an integral part of endothelial pathophysiology. Although this is a well-established concept in the cardiovascular field, it is largely dismissed in studies aimed at understanding radiation injury to the endothelium and subsequent cardiovascular complications. We and others have reported on the differential response of the endothelium when the cells are under hemodynamic flow shear compared with static culture. Further, we have demonstrated significant differences in the gene expression of static versus shear-stressed irradiated cells in four key pathways, reinforcing the importance of shear stress in understanding radiation injury of the endothelium. This article further emphasizes the influence of hemodynamic shear stress and the associated mechanotransduction signaling on physiological functioning of the vascular endothelium and underscores its significance in understanding radiation injury to the vasculature and associated cardiac complications. Studies of radiation effect on endothelial biology and its implication on cardiotoxicity and vascular complications thus far have failed to highlight the significance of these factors. Factoring in these integral parts of the endothelium will enhance our understanding of the contribution of the endothelium to radiation biology. Without such information, the current approaches to studying radiation-induced injury to the endothelium and its consequences in health and disease are limited.

Radiation therapy is commonly used to treat patients with head and neck squamous cell carcinoma (HNSCC). One of the major side effects of radiotherapy is injury to the salivary glands (SG), which is thought to be mediated by microvascular dysfunction leading to permanent xerostomia. The goal of this study was to elucidate the mechanism of radiation-induced microvasculature damage and its impact on SG function. We measured bovine aortic endothelial cell (BAEC) apoptosis and ceramide production in response to 5 Gy irradiation, either alone or with reactive oxygen species (ROS) scavengers. We then investigated the effect of a single 15 Gy radiation dose on murine SG function. BAECs exposed to 5 Gy underwent apoptosis with increased ceramide production, both prevented by ROS scavengers. Among the 15 Gy irradiated mice, there was considerable weight loss, alopecia and SG hypofunction manifested by reduced saliva production and lower lysozyme levels. All of these effects, except for the lysozyme levels, were prevented by pretreatment with ROS scavengers. Microvessel density was significantly lower in the SG of irradiated mice compared to the control group, and this effect was significantly attenuated by pretreatment with Tempol. This study demonstrates that radiation-induced SG hypofunction is to a large extent mediated by microvascular dysfunction involving ceramide and ROS generation. These findings strongly suggest that ROS scavengers may serve as potential radioprotectors of SG function in patients undergoing radiotherapy for HNSCC.

Current therapeutic approaches for treatment of exposure to radiation involve the use of antioxidants, chelating agents, recombinant growth factors and transplantation of stem cells (e.g., hematopoietic stem cell transplantation). However, exposure to high-dose radiation is associated with severe damage to the vasculature of vital organs, often leading to impaired healing, tissue necrosis, thrombosis and defective regeneration caused by aberrant fibrosis. It is very unlikely that infusion of protective chemicals will reverse severe damage to the vascular endothelial cells (ECs). The role of irradiated vasculature in mediating acute and chronic radiation syndromes has not been fully appreciated or well studied. New approaches are necessary to replace and reconstitute ECs in organs that are irreversibly damaged by radiation. We have set forth the novel concept that ECs provide paracrine signals, also known as angiocrine signals, which not only promote healing of irradiated tissue but also direct organ regeneration without provoking fibrosis. We have developed innovative technologies that enable manufacturing and banking of human GMP-grade ECs. These ECs can be transplanted intravenously to home to and engraft to injured tissues where they augment organ repair, while preventing maladaptive fibrosis. In the past, therapeutic transplantation of ECs was not possible due to a shortage of availability of suitable donor cell sources and preclinical models, a lack of understanding of the immune privilege of ECs, and inadequate methodologies for expansion and banking of engraftable ECs. Recent advances made by our group as well as other laboratories have breached the most significant of these obstacles with the development of technologies to manufacture clinical-scale quantities of GMP-grade and human ECs in culture, including genetically diverse reprogrammed human amniotic cells into vascular ECs (rAC-VECs) or human pluripotent stem cells into vascular ECs (iVECs). This approach provides a path to therapeutic EC transplantation that can be infused concomitantly or sequentially with hematopoietic stem cell transplantation more than 24 h after irradiation to support multi-organ regeneration, thereby improving immediate and long-term survival, while limiting long-term morbidity resulting from nonregenerative damage repair pathways.

This article reviews our current knowledge about cell-derived extracellular vesicles (EVs), including microparticles and exosomes, and their emergence as mediators of a new important mechanism of cell-to-cell communication. Particular emphasis has been given to the increasing involvement of EVs in the field of radiation-induced vascular injury. Although EVs have been considered for a long time as cell “dust”, they in fact precisely reflect the physiological state of the cells. The role of microparticles and exosomes in mediating vascular dysfunction suggests that they may represent novel pathways in short- or long-distance paracrine intercellular signaling in vascular environment. In this article, the mechanisms involved in the biogenesis of microparticles and exosomes, their composition and participation in the pathogenesis of vascular dysfunction are discussed. Furthermore, this article highlights the concept of EVs as potent vectors of biological information and protagonists of an intercellular communication network. Special emphasis is made on EV-mediated microRNA transfer and on the principal consequences of such signal exchange on vascular injury and radiation-induced nontargeted effect. The recent progress in elucidating the biology of EVs has provided new insights for the field of radiation, advancing their use as diagnostic biomarkers or in therapeutic interventions.