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Mechanisms of radiation-induced endothelium damage: Emerging models and technologies

Published:February 10, 2021DOI:https://doi.org/10.1016/j.radonc.2021.02.007

      Highlights

      • Exposure to ionizing radiation (IR) often results in vascular/endothelial injury.
      • The microvasculature is sensitive to IR-induced damage and can initiate organ damage.
      • Novel insight is gained from emerging in vitro models of endothelial cell function.
      • Only a few radiotherapeutic agents have been approved by the FDA. 5.Therapeutics that protect against IR-induced endothelial damage are urgently needed.

      Abstract

      Radiation-induced endothelial/vascular injury is a major complicating factor in radiotherapy and a leading cause of morbidity and mortality in nuclear or radiological catastrophes. Exposure of tissue to ionizing radiation (IR) leads to the release of oxygen radicals and proteases that result in loss of endothelial barrier function and leukocyte dysfunction leading to tissue injury and organ damage. Microvascular endothelial cells are particularly sensitive to IR and radiation-induced alterations in endothelial cell function are thought to be a critical factor in organ damage through endothelial cell activation, enhanced leukocyte-endothelial cell interactions, increased barrier permeability and initiation of apoptotic pathways. These radiation-induced inflammatory responses are important in early and late radiation pathologies in various organs. A better understanding of mechanisms of radiation-induced endothelium dysfunction is therefore vital, as radiobiological response of endothelium is of major importance for medical management and therapeutic development for radiation injuries. In this review, we summarize the current knowledge of cellular and molecular mechanisms of radiation-induced endothelium damage and their impact on early and late radiation injury. Furthermore, we review established and emerging in vivo and in vitro models that have been developed to study the mechanisms of radiation-induced endothelium damage and to design, develop and rapidly screen therapeutics for treatment of radiation-induced vascular damage. Currently there are no specific therapeutics available to protect against radiation-induced loss of endothelial barrier function, leukocyte dysfunction and resulting organ damage. Developing therapeutics to prevent endothelium dysfunction and normal tissue damage during radiotherapy can serve as the urgently needed medical countermeasures.

      Graphical abstract

      Abbreviations:

      ARS (Acute radiation syndrome), AJ (Adherens junctions), ATM (Ataxia telangiectasia-mutated), bMFA (biomimetic microfluidic assay), CNS (Central nervous system), CVD (Cardiovascular disease), DAMP (Damage-associated molecular patterns), G-CSF (Granulocyte-colony stimulating factor), GM-CSF (Granulocyte/Macrophage Colony Stimulating Factor), GI (Gastrointestinal), GGA (Geranylgeranylacetone), HUVEC (Human umbilical vein endothelial cells), ICAM-1 (Intercellular adhesion molecule 1), IL-1 (Interleukin-1), IR (Ionizing radiation), EC (Endothelial cell), JAK1 (Janus kinase 1), JAM-C (Junctional adhesion molecule-C), LET (Linear energy transfer), MHC (Major histocompatibility complex), M-CSF (macrophage colony-stimulating factor), MnSOD (Manganese-dependent superoxide dismutase), MPS (Microphysiological systems), NETS (Neutrophil extracellular traps), PAI-1 (Plasminogen activator inhibitor type 1), PAF (Platelet activating factor), PDGF (Platelet-derived growth factor), PKCδ (Protein Kinase C-delta), ROS (Reactive oxygen species), SRS (Stereotactic radiosurgery), TEER (Transendothelial electrical resistance), TGFβ (Transforming Growth Factor-Beta), TM (Thrombomodulin), TJ (Tight junctions), TBI (Total body irradiation), TNF-α (Tumor necrosis factor-α), TYK2 (Tyrosine Kinase 2), WBC (White blood cells), WMD (Weapons of mass destruction)

      Keywords

      Healthy tissue may be exposed to ionizing radiation (IR) during radiotherapy [
      • Singh V.K.
      • Seed T.M.
      A review of radiation countermeasures focusing on injury-specific medicinals and regulatory approval status: part I. Radiation sub-syndromes, animal models and FDA-approved countermeasures.
      ], nuclear accidents, or by weapons of mass destruction (WMD) [

      Cerezo, L., Radiation accidents and incidents. What do we know about the medical management of acute radiation syndrome? Rep Pract Oncol Radiother, 2011. 16: p. 119 DOI: https://doi.org/10.1016/j.rpor.2011.06.002.

      ]. IR produces oxidative stress resulting in acute and chronic cellular damage. The vascular endothelium, which plays an important role in organ homeostasis, is a key target of radiation damage. Microvascular endothelial cells (EC), in particular, are sensitive to radiation and radiation-induced alterations in EC structure and function. Damage to the endothelium is an important regulator of radiation damage in both targeted radiotherapy and whole-body irradiation resulting from exposure to WMD [
      • Satyamitra M.M.
      • DiCarlo A.L.
      • Taliaferro L.
      Understanding the pathophysiology and challenges of development of medical countermeasures for radiation-induced vascular/endothelial cell injuries: report of a NIAID workshop, August 20, 2015.
      ]. However, the signaling pattern in these two cases may be different due to, for example, the differential upregulation of cytokines in local vs. whole body irradiation. IR-induced activation of EC leads to enhanced leukocyte-EC interactions, increased permeability, and initiation of apoptotic pathways [
      • Korpela E.
      • Liu S.K.
      Endothelial perturbations and therapeutic strategies in normal tissue radiation damage.
      ,
      • Rossaint J.
      • Zarbock A.
      Tissue-specific neutrophil recruitment into the lung, liver, and kidney.
      ]. Radiation-induced endothelial damage and its associated vascular changes often lead to chronic lesions when organs at risk (e.g. lung, kidney, heart and brain) are exposed to sufficiently high doses [
      • Satyamitra M.M.
      • DiCarlo A.L.
      • Taliaferro L.
      Understanding the pathophysiology and challenges of development of medical countermeasures for radiation-induced vascular/endothelial cell injuries: report of a NIAID workshop, August 20, 2015.
      ], and patients with IR-induced tissue damage may die of organ failure.
      Moreover, exposure to IR can accelerate atherosclerosis adversely affecting normal tissues resulting in coronary artery disease, peripheral vascular disease, radiation pneumonitis and fibrosis, and cerebrovascular disease. This effect can be increased with higher doses per fraction [
      • Venkatesulu B.P.
      • et al.
      Radiation-induced endothelial vascular injury: a review of possible mechanisms.
      ,
      • Parikh R.B.
      • et al.
      Association of utilization management policy with uptake of hypofractionated radiotherapy among patients with early-stage breast cancer.
      ]. This is clinically relevant as contemporary treatments move increasingly towards hypofractionation [
      • Park H.J.
      • et al.
      Radiation-induced vascular damage in tumors: implications of vascular damage in ablative hypofractionated radiotherapy (SBRT and SRS).
      ,
      • Konski A.
      • et al.
      Radiation oncology practice: adjusting to a new reimbursement model.
      ] in treatments such as stereotactic brain and body radiosurgery (single fraction) and radiotherapy (typically 3–5 fractions) for primary and metastatic brain tumors, early-stage non-small cell carcinoma of the lung and breast conservation irradiation. The radiation target volumes for these treatments must include the planning target volume plus a margin which includes some surrounding normal tissue as defined in the ICRU reports 50 [

      ICRU, L.E.T., International Commission on Radiation Units and Measurements. ICRU report, 1993. 62.

      ], 62 [
      • Landberg T.
      • et al.
      Report 62.
      ] and 83 [
      • Hodapp N.
      Der ICRU-Report 83: Verordnung, Dokumentation und Kommunikation der fluenzmodulierten Photonenstrahlentherapie (IMRT).
      ]. This normal tissue can be dose limiting potentially compromising the target volume or resulting in increased short- and long-term secondary effects from EC damage.
      Monitoring these vascular damages have become greatly helpful for diagnosis purposes. Recently, many novel methodologies have been developed to monitor the vascular damages. Angiography, magnetic resonance angiography (MRA), computed tomography angiography (CTA) and ultrasound are used clinically for imaging diseases that involve endothelial dysfunction resulting from an inflammatory response [
      • Chan J.M.
      • et al.
      MRI detection of endothelial cell inflammation using targeted superparamagnetic particles of iron oxide (SPIO).
      ]. Compared to other techniques, MRA has the advantage of providing high resolution images of the vessel wall. In addition, the recent development of targeted micro-sized particles of iron oxide (MPIO) targeting intercellular adhesion molecules like ICAM-1, VCAM-1, P-selectin and E-selectin have significantly improved the sensitivity and specificity of molecular MRA for imaging endothelial activation compared to other techniques such as plasma biomarkers [

      Gauberti M. et al., Molecular magnetic resonance imaging of endothelial activation in the central nervous system. Theranostics, 2018. 8(5): p. 1195 DOI: http://www.thno.org/v08p1195.htm.

      ].
      Protecting the endothelium protects tissue from radiation damage [
      • Kiang J.G.
      • Olabisi A.O.
      Radiation: a poly-traumatic hit leading to multi-organ injury.
      ] and therapeutics that specifically prevent EC dysfunction ultimately protect tissue from radiation injury [
      • Korpela E.
      • Liu S.K.
      Endothelial perturbations and therapeutic strategies in normal tissue radiation damage.
      ] thereby reducing side effects of radiation therapy. Given the central role of the endothelium in normal vascular function, a better understanding of mechanisms of radiation-induced dysfunction is vital in developing therapeutics for radiation-induced endothelium damage [
      • Satyamitra M.M.
      • DiCarlo A.L.
      • Taliaferro L.
      Understanding the pathophysiology and challenges of development of medical countermeasures for radiation-induced vascular/endothelial cell injuries: report of a NIAID workshop, August 20, 2015.
      ].

      Endothelial dysfunction in irradiated tissue

      Normal endothelial cell function

      The vascular endothelium is composed of a single layer of cells lining all blood vessels, which acts as a semipermeable barrier regulating the delivery of nutrients, oxygen and cellular components to local tissues and the removal of carbon dioxide and waste products (Fig. 1). The EC barrier regulates the transport of molecules between vascular and tissue compartments through cell–cell junctions and vesicular transport [
      • Cahill P.A.
      • Redmond E.M.
      Vascular endothelium–gatekeeper of vessel health.
      ]. The endothelium produces several vasoactive substances such as nitric oxide that are vital in regulation of vascular control and growth [
      • Barton M.
      • Baretella O.
      • Meyer M.R.
      Obesity and risk of vascular disease: importance of endothelium-dependent vasoconstriction.
      ]. With its secretory, synthetic, metabolic, immunologic and surface expression functions, the endothelium plays a critical role in many physiological processes, including control of vascular tone, trafficking of the blood cells between vasculature and underlying tissue, regulation of immune responses, permeability, and angiogenesis [
      • Aird W.C.
      Endothelial cell heterogeneity.
      ]. EC show significant heterogeneity in structure, function, cell morphology, gene expression and antigen composition across organs and species [
      • Aird W.C.
      Endothelial cell heterogeneity.
      ,
      • Aird W.C.
      Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.
      ]. This heterogeneity is in part dictated by intrinsic signals as well as the organ-specific microenvironment leading to distinct differences in barrier properties and interactions with circulating immune cells that result in differential sensitivities and responses to radiation.
      Figure thumbnail gr1
      Fig. 1Overview of vascular endothelium and mechanisms by which IR impacts endothelial cell regulation. Top panel: Normal tissue - Endothelial cells act as a semipermeable barrier that regulates the delivery of nutrients and oxygen to tissue and the removal of carbon dioxide and waste products. Normal endothelial cells have basal levels of some adhesion molecules. Bottom panel: Irradiated tissue – Ionizing radiation increases the production of ROS leading to DNA and mitochondrial damage and increased apoptosis. IR also alters endothelial permeability by acting on tight and adherens junctions allowing excess extravasation of proteins to cross into the extracellular tissue. Radiation exposure also increases the release of proinflammatory cytokines and chemokines and upregulation of adhesion molecules resulting in increased leukocyte-endothelial cell interaction and trafficking to vital organs.

      Endothelial cell dysfunction in early and late radiation damage

      Radiation-induced endothelium injury can manifest acutely/early or late after IR exposure. Acute/early radiation syndrome (ARS) encompasses three types of ‘radiation sicknesses’ in bone marrow, gastrointestinal (GI), and cardiovascular/central nervous system (CNS) [
      • Hall E.
      • Giaccia A.
      Radiobiology for the radiologist.
      ]. GI-ARS is attributed to the radiation sensitivity of the microvascular EC [
      • Paris F.
      • et al.
      Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice.
      ] and endothelial dysfunction and increased inflammatory mediators inhibit the recovery of the villus epithelium leading to the breakdown of the epithelial barrier [
      • Satyamitra M.M.
      • DiCarlo A.L.
      • Taliaferro L.
      Understanding the pathophysiology and challenges of development of medical countermeasures for radiation-induced vascular/endothelial cell injuries: report of a NIAID workshop, August 20, 2015.
      ]. Moreover, fluid and nutrient absorption across EC is reduced, and mucosal ulcerations develop, resulting in diarrhea, nausea, abdominal pain, and mucous discharge. If ulceration continues, it can lead to bacterial translocation and systemic inflammation [
      • Huh J.W.
      • et al.
      Long-term consequences of pelvic irradiation: toxicities, challenges, and therapeutic opportunities with pharmacologic mitigators.
      ].
      Late vascular effects such as capillary collapse, thickening of basement membrane, scarring and fibrosis may occur weeks to months post-irradiation. Depending on the radiation dose, late pathological syndromes may be delayed in expression, chronic in nature, and associated with evolving pathologies within multiple organ systems, including the bone marrow, GI tract, lung, heart, kidney and the CNS. In the brain, up to 3 weeks after single dose of 15–30 Gy, leukocyte–EC interactions were upregulated [
      • Acker J.
      • et al.
      Serial in vivo observations of cerebral vasculature after treatment with a large single fraction of radiation.
      ]. Although hyperpermeability to albumin was observed in the mesentery up to 6 hours after radiation exposure [
      • Panés J.
      • et al.
      Role of leukocyte-endothelial cell adhesion in radiation-induced microvascular dysfunction in rats.
      ] and inhibition of intercellular adhesion molecule (ICAM-1) blocked the hyperpermeability response to radiotherapy, the increase in leukocyte–EC interaction and hyperpermeability can persist for days to weeks after radiotherapy [
      • Ashcraft K.A.
      • et al.
      Application of a novel murine ear vein model to evaluate the effects of a vascular radioprotectant on radiation-induced vascular permeability and leukocyte adhesion.
      ]. The hyperpermeability of injured vessels facilitates extravasation of fibrinogen into the extravascular space and fibrinogen undergoes fibrinopeptide cleavage by thrombin, which then results in crosslinking of fibrin by enzymes such as tissue transglutaminase [
      • Laurens N.
      • Koolwijk P.D.
      • De Maat M.
      Fibrin structure and wound healing.
      ,
      • Haroon Z.A.
      • et al.
      Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis.
      ]. Crosslinking of tissue transglutaminase activates latent TGFβ, which further induces tissue fibrosis by promoting deposition of collagen [
      • Nunes I.
      • et al.
      Latent transforming growth factor-β binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-β.
      ].

      Ionizing radiation upregulates endothelial cell mediated inflammatory response

      IR-induced cellular damage triggers apoptosis and cell death leading to the release of damage-associated molecular patterns (DAMP) and activation of the systemic inflammatory response producing significant changes in the microvascular network structure and function [
      • Kiani M.F.
      • Ansari R.
      • Gaber M.W.
      Oxygen delivery in irradiated normal tissue.
      ,
      • Nguyen V.
      • et al.
      Late effects of ionizing radiation on the microvascular networks in normal tissue.
      ,
      • Roth N.M.
      • Sontag M.R.
      • Kiani M.F.
      Early effects of ionizing radiation on the microvascular networks in normal tissue.
      ,
      • Stoecklein V.M.
      • et al.
      Radiation exposure induces inflammasome pathway activation in immune cells.
      ]. EC acquire a pro-inflammatory phenotype leading to increased cytokine release (Table 1), ROS production, and enhanced EC adhesion molecule expression resulting in increased recruitment of immune cells of myeloid and lymphoid origin [
      • Venkatesulu B.P.
      • et al.
      Radiation-induced endothelial vascular injury: a review of possible mechanisms.
      ,
      • Panés J.
      • et al.
      Role of leukocyte-endothelial cell adhesion in radiation-induced microvascular dysfunction in rats.
      ,
      • Guipaud O.
      • et al.
      The importance of the vascular endothelial barrier in the immune-inflammatory response induced by radiotherapy.
      ,
      • Yuan H.
      • et al.
      Radiation-induced up-regulation of adhesion molecules in brain microvasculature and their modulation by dexamethasone.
      ,

      Prabhakarpandian B. et al., Expression and functional significance of adhesion molecules on cultured endothelial cells in response to ionizing radiation. Microcirculation, 2001. 8(5): p. 355-364 DOI: https://doi-org.libproxy.temple.edu/10.1111/j.1549-8719.2001.tb00182.x.

      ] (Fig. 1). Increased recruitment of neutrophils, monocytes, and macrophages, as well as Th1 and Th17 lymphocytes, contribute to IR-induced inflammation [
      • Venkatesulu B.P.
      • et al.
      Radiation-induced endothelial vascular injury: a review of possible mechanisms.
      ,
      • Guipaud O.
      • et al.
      The importance of the vascular endothelial barrier in the immune-inflammatory response induced by radiotherapy.
      ,
      • Kang J.-H.
      • et al.
      Radiation potentiates monocyte infiltration into tumors by ninjurin1 expression in endothelial cells.
      ,
      • Wirsdörfer F.
      • Jendrossek V.
      The role of lymphocytes in radiotherapy-induced adverse late effects in the lung.
      ]. This inappropriate influx of immune cells across the vascular endothelium, initiation of vascular EC damage and loss of barrier function have been implicated in the pathogenesis of radiation injury and organ dysfunction [
      • Yang X.
      • Chang Y.
      • Wei W.
      Endothelial dysfunction and inflammation: immunity in rheumatoid arthritis.
      ].
      Table 1Cytokines in radiation-induced inflammatory response.
      ClassificationCytokinesResponse after Radiation ExposureReferences
      Interleukin and TNF FamilyIL-1-β, IL-1-α, IL-8, IL-6, TNF-α, IL-4, IL-13
      • Plays a role in generating reactive nitrogen and oxygen species such as nitric oxide or hydroxyl radicals
      • Induces proto-oncogene expression post-radiation
      • Promotes T-cell differentiation towards T-helper types 1, 2 and 17
      • Initiates neutrophil adhesion, migration and extravasation into tissues
      • Promotes pro-inflammatory transcription factors such as NF-κB and AP-1
      • Enhances vascular adhesion molecule (CAMs, cadherins etc.) expression
      • Kany S.
      • Vollrath J.T.
      • Relja B.
      Cytokines in inflammatory disease.
      ,
      • Diehl S.
      • Rincón M.
      The two faces of IL-6 on Th1/Th2 differentiation.
      ,
      • Schaue D.
      • Kachikwu E.L.
      • McBride W.H.
      Cytokines in radiobiological responses: a review.
      ,
      • Gulati K.
      • et al.
      Cytokines and their role in health and disease: a brief overview.
      ,
      • Gao H.
      • et al.
      Effects of various radiation doses on induced T-helper cell differentiation and related cytokine secretion.
      ,
      • Di Maggio F.M.
      • et al.
      Portrait of inflammatory response to ionizing radiation treatment.
      Type I and II Interferon familyIFN-α, IFN-β, IFN-γ
      • Expressed on WBCs, signal TYK2 and JAK1 and activates STATs. Activation of STATs leads to IL-1β phosphorylation and IL-6 production, leading to inflammation. Activation of STATS can also inhibit cell division, activate and drive WBC formation in inflammation.
      • Leads to expression of MHC class II on endothelial and immune cells
      • Gulati K.
      • et al.
      Cytokines and their role in health and disease: a brief overview.
      ,

      Muñoz-Carrillo JL. et al., Cytokine profiling plays a crucial role in activating immune system to clear infectious pathogens, in Immune Response Activation and Immunomodulation. 2018, IntechOpen.

      Colony stimulating factor familyG-CSF, GM-CSF, M-CSF
      • Induces WBC production, leading to increased trafficking of leukocytes in response to inflammation
      • Increases adhesion molecule expression on EC
      • Gulati K.
      • et al.
      Cytokines and their role in health and disease: a brief overview.
      Growth factor familyTGFβ, PDGF
      • Increased expression leading to increased collagen production and tissue remodeling
      • Strong stimulators of fibrosis
      • Straub J.M.
      • et al.
      Radiation-induced fibrosis: mechanisms and implications for therapy.
      ,
      • Kishi M.
      • et al.
      Blockade of platelet-derived growth factor receptor-β, not receptor-α ameliorates bleomycin-induced pulmonary fibrosis in mice.
      A key step in IR-induced organ damage is excessive adhesion to and migration of activated leukocytes across vasculature (Supplementary Data Video 1 and Video 2) and a reduction in neutrophil infiltration is associated with better outcomes following skin irradiation [
      • Korpela E.
      • Liu S.K.
      Endothelial perturbations and therapeutic strategies in normal tissue radiation damage.
      ]. Neutrophil recruitment is a multi-step process, which requires crosstalk between neutrophils and EC and includes five discrete steps: capture/attachment, rolling, firm arrest, spreading and extravasation/migration and each step requires crosstalk between leukocytes and EC [
      • Filippi M.-D.
      Neutrophil transendothelial migration: updates and new perspectives.
      ] (Figure 2). Ultimately, arrested neutrophils extravasate to inflamed tissues across EC regulated by concurrent chemoattractant-dependent signals, adhesive events and hemodynamic shear forces [
      • Filippi M.-D.
      Neutrophil transendothelial migration: updates and new perspectives.
      ].
      Figure thumbnail gr2
      Fig. 2Multi-step process of neutrophil recruitment that includes rolling, adhesion and transmigration. On endothelial cells, selectins (e.g., P- & E-selectin) are responsible for neutrophil capture and rolling, while adhesion molecules ICAM-1, VCAM-1 and PECAM-1 are critical regulators of neutrophil firm attachment and migration.
      A number of molecules involved in the leukocyte adhesion cascade are also involved in radiation-induced tissue damage. For example, we demonstrated by intravital microscopy that adhesion molecules (ICAM-1, E-selectin) were upregulated in irradiated tissue in vivo and the resulting increased leukocyte adhesion was modulated with administration of an anti-ICAM-1 antibody [
      • Yuan H.
      • et al.
      Radiation-induced up-regulation of adhesion molecules in brain microvasculature and their modulation by dexamethasone.
      ,

      Prabhakarpandian B. et al., Expression and functional significance of adhesion molecules on cultured endothelial cells in response to ionizing radiation. Microcirculation, 2001. 8(5): p. 355-364 DOI: https://doi-org.libproxy.temple.edu/10.1111/j.1549-8719.2001.tb00182.x.

      ,
      • Yuan H.
      • et al.
      Radiation-induced permeability and leukocyte adhesion in the rat blood–brain barrier: modulation with anti-ICAM-1 antibodies.
      ]. We and others have shown that irradiation of EC in vitro significantly increased the expression of the adhesion molecules VCAM-1 and ICAM-1 [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ,

      Haubner F. et al., Effects of radiation on the expression of adhesion molecules and cytokines in a static model of human dermal microvascular endothelial cells. Clinical hemorheology and microcirculation, 2013. 54(4): p. 371-379 DOI: https://doi-org.libproxy.temple.edu/10.3233/CH-2012-1626.

      ].
      EC also synthesize the neutrophil agonist, platelet activating factor (PAF) [
      • Kimura H.
      • et al.
      Inhibition of radiation-induced up-regulation of leukocyte adhesion to endothelial cells with the platelet-activating factor inhibitor, BN52021.
      ], which is co-expressed with P-selectin within minutes, while it takes a few hours to co-express E-selectin with IL-8. These co-expression pairs, in turn, upregulate integrin expression on leukocytes. Administration of a PAF antagonist, BN52021, blocked the upregulation of leukocyte adhesion [
      • Kimura H.
      • et al.
      Inhibition of radiation-induced up-regulation of leukocyte adhesion to endothelial cells with the platelet-activating factor inhibitor, BN52021.
      ].
      Neutrophils activated in response to IR-induced systemic inflammation can release neutrophil extracellular traps (NETs) [
      • Kennedy A.R.
      • Maity A.
      • Sanzari J.K.
      A review of radiation-induced coagulopathy and new findings to support potential prevention strategies and treatments.
      ]. NETs are composed of nuclear chromatin filaments studded with histones and granular proteins, such as neutrophil elastase and myeloperoxidase. While important for bactericidal activities, NETs can also damage EC and further upregulate the inflammatory response [
      • Soroush F.
      • et al.
      Protein kinase C-delta (PKCδ) tyrosine phosphorylation is a critical regulator of neutrophil-endothelial cell interaction in inflammation.
      ,
      • Gupta A.K.
      • et al.
      Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death.
      ]. Ability of peripheral blood neutrophils from cervical cancer patients to form NETs increased from 53.6% before radiation therapy to 66.7% after radiation therapy [
      • Fomenko Y.
      • et al.
      Influence of combined therapy on generation of neutrophil extracellular traps in patients with cervical cancer.
      ] but the mechanisms by which IR activates NETs and the interconnection between NETs and radiation-induced EC damage have not been fully delineated.

      IR-induced endothelium activation increases barrier dysfunction

      Damage to and/or the denudation of EC after radiation exposure produces changes in permeability of EC. The vasculature becomes leaky within hours post-IR but the degree to which EC of various vessel types become permeable varies in vitro [
      • Sharma P.
      • Templin T.
      • Grabham P.
      Short term effects of gamma radiation on endothelial barrier function: uncoupling of PECAM-1.
      ]. IR also increases the permeability of the blood–brain-barrier allowing for ionic movement, excess extravasation of inflammatory cells, proteins and biologic response molecules (e.g. growth factors, cytokines) into the brain parenchyma causing brain damage [
      • Fauquette W.
      • et al.
      Radiation-induced blood–brain barrier damages: an in vitro study.
      ,
      • Yuan H.
      • et al.
      Effects of fractionated radiation on the brain vasculature in a murine model: blood–brain barrier permeability, astrocyte proliferation, and ultrastructural changes.
      ]. IR alters endothelial permeability by acting on tight and adherens junctions [

      Kabacik S, Raj K. Ionising radiation increases permeability of endothelium through ADAM10-mediated cleavage of VE-cadherin. Oncotarget, 2017. 8(47): p. 82049 DOI: https://doi.org/10.18632/oncotarget.18282.

      ]. Tight junctions adhesion is mediated by the claudins family of proteins, which are connected to the cytoskeleton by tight junctions proteins while adherens junctions are formed by classical cadherins that are linked to the cytoskeleton by proteins belonging to the catenin family [
      • Steed E.
      • Balda M.S.
      • Matter K.
      Dynamics and functions of tight junctions.
      ,
      • Dejana E.
      • Vestweber D.
      The role of VE-cadherin in vascular morphogenesis and permeability control.
      ]. Among these, vascular endothelial-cadherin (VE-cadherin, a substrate of ADAM10), an important regulator of vascular integrity, which when activated, increases EC permeability [
      • Kouam P.N.
      • et al.
      Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin.
      ]. In vitro, IR activates ADAM10 and cleavage of VE-cadherin, leading to increased human endothelial permeability to macromolecules of various sizes in a radiation dose dependent manner [

      Kabacik S, Raj K. Ionising radiation increases permeability of endothelium through ADAM10-mediated cleavage of VE-cadherin. Oncotarget, 2017. 8(47): p. 82049 DOI: https://doi.org/10.18632/oncotarget.18282.

      ]. Consistent with other studies [
      • Kouam P.N.
      • et al.
      Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin.
      ], we demonstrated that exposure of human EC under shear flow to IR in a novel microphysiological system significantly increases permeability and decreases transendothelial electrical resistance (TEER) across the endothelial barrier [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ].

      Radiation exposure leads to mitochondrial dysfunction

      In many mammalian cells, mitochondria produce cellular energy but mitochondrial content in EC is limited and EC ATP production is primarily via aerobic glycolysis [
      • Fitzgerald G.
      • Soro-Arnaiz I.
      • De Bock K.
      The Warburg effect in endothelial cells and its potential as an anti-angiogenic target in cancer.
      ]. When HUVECs were exposed to 5–20 Gy of IR, the mitochondrial membrane potential decreased and mitochondrial ROS production increased [
      • Hu S.
      • et al.
      New insight into mitochondrial changes in vascular endothelial cells irradiated by gamma ray.
      ]. Furthermore, murine cardiac microvascular EC acquired protein expression profiles related to mitochondrial dysfunction when they were irradiated with 8 and 16 Gy X-rays [
      • Azimzadeh O.
      • et al.
      Integrative proteomics and targeted transcriptomics analyses in cardiac endothelial cells unravel mechanisms of long-term radiation-induced vascular dysfunction.
      ]. It is hypothesized that EC mitochondria functions more as important signaling organelles [
      • Quintero M.
      • et al.
      Mitochondria as signaling organelles in the vascular endothelium.
      ] and the three main mitochondrial functions, Ca2+ regulation, control of cell death, and oxidative stress signaling are disturbed following IR exposure [
      • Baselet B.
      • et al.
      Pathological effects of ionizing radiation: endothelial activation and dysfunction.
      ]. Mitochondrial Ca2+ signaling is altered by IR [
      • Kam W.W.-Y.
      • Banati R.B.
      Effects of ionizing radiation on mitochondria.
      ], and at IR doses high enough to overcome cellular antioxidant responses, oxidative stress can lead to mitochondrial dysfunction [
      • Zorov D.B.
      • Juhaszova M.
      • Sollott S.J.
      Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release.
      ].

      IR increases apoptotic cell death

      Apoptotic cell death due to radiation exposure can be primarily mediated by either p53 or the sphingomyelin/ceramide pathways [
      • Venkatesulu B.P.
      • et al.
      Radiation-induced endothelial vascular injury: a review of possible mechanisms.
      ]. It’s shown that p53 is important in response to DNA damage, which activates mitochondrial-mediated cell apoptosis [
      • Liu Z.
      • et al.
      PDGF-BB and bFGF ameliorate radiation-induced intestinal progenitor/stem cell apoptosis via Akt/p53 signaling in mice. American Journal of Physiology-Gastrointestinal and Liver.
      ]. Endothelial cell apoptosis at doses > 5 Gy can be induced by persistent DNA damage, resulting in leading to p53 accumulation and resulting in activation of the caspase pathway, [
      • Langley R.
      • et al.
      Radiation-induced apoptosis in microvascular endothelial cells.
      ] but mechanisms of endothelial cytotoxicity at lower radiation doses are less known [
      • Pluder F.
      • et al.
      Low-dose irradiation causes rapid alterations to the proteome of the human endothelial cell line EA. hy926.
      ]. It has been proposed that EC apoptosis after high-dose single-fraction IR is primarily modulated by the sphingomyelin ceramide pathway [
      • Seideman J.H.
      • et al.
      Alpha particles induce apoptosis through the sphingomyelin pathway.
      ]. Sphingomyelin is a phospholipid present in the cell membrane which is hydrolyzed by TNF activation after IR exposure. Extracellular acidic sphingomyelinase then leads to radiation-induced, ceramide-mediated EC apoptosis [
      • Sathishkumar S.
      • et al.
      Elevated sphingomyelinase activity and ceramide concentration in serum of patients undergoing high dose spatially fractionated radiation treatment: implications for endothelial apoptosis.
      ]. A classical study has shown the requirement of the sphingomyelinase encoding gene, ASMase, for mediating ceramide generation in induction of microvascular endothelial apoptosis [
      • Paris F.
      • et al.
      Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice.
      ] and high dose IR triggering of ASMase/ceramide signaling in EC has been shown to play a major role in membrane signaling after radiation exposure [
      • Corre I.
      • Guillonneau M.
      • Paris F.
      Membrane signaling induced by high doses of ionizing radiation in the endothelial compartment. Relevance in radiation toxicity.
      ]. However, the precise relationship between IR dose and EC apoptosis has not been clearly established [
      • Brown J.M.
      • Koong A.C.
      High-dose single-fraction radiotherapy: exploiting a new biology?.
      ].
      IR also induces differential effects on microRNA (miRNA) levels in EC. In vitro studies demonstrated that miRNAs have a role in EC clonogenic survival and cell growth, as well as impacting EC radio-sensitivity indicating an important role of miRNAs in the EC response to radiation [
      • Wagner-Ecker M.
      • et al.
      MicroRNA expression after ionizing radiation in human endothelial cells.
      ].

      Emerging models used for studying the effects of IR on endothelium

      In vivo animal models

      Although no single animal model can completely represent the human condition, in vivo models including mouse, rat, rabbits, and pigs have been widely used to study radiation-induced endothelium damage [
      • Baselet B.
      • et al.
      Pathological effects of ionizing radiation: endothelial activation and dysfunction.
      ]. The choice of an appropriate animal model for a specific study is generally dictated by factors such as anatomy, physiology, genetics, and immune response of the animal model being considered. Mice show similarities to human physiological and genome (>95%) [
      • Singh V.K.
      • et al.
      Animal models for acute radiation syndrome drug discovery.
      ] and have been used widely to study radiation induced upregulation of cell adhesion molecules, leukocyte-endothelial interaction and EC damage [
      • Yuan H.
      • et al.
      Radiation-induced up-regulation of adhesion molecules in brain microvasculature and their modulation by dexamethasone.
      ,
      • Pena L.A.
      • Fuks Z.
      • Kolesnick R.N.
      Radiation-induced apoptosis of endothelial cells in the murine central nervous system: protection by fibroblast growth factor and sphingomyelinase deficiency.
      ]. However, the mouse may not be the optimum animal model for studying radiation induced damage for multiple reasons. A major disadvantage is their small body thickness, which does not account for the heterogeneity of radiation dose distribution characteristic to human exposure [
      • Singh V.K.
      • et al.
      Animal models for acute radiation syndrome drug discovery.
      ]. Additionally, despite the phylogenic relatedness, translating studies from mouse immune system to human disease is complicated by many factors including size, metabolic rate, WBCs composition, and differences in pathophysiology and drug LD50 values that are significantly different from humans [
      • Satyamitra M.M.
      • DiCarlo A.L.
      • Taliaferro L.
      Understanding the pathophysiology and challenges of development of medical countermeasures for radiation-induced vascular/endothelial cell injuries: report of a NIAID workshop, August 20, 2015.
      ,
      • Perlman R.L.
      Mouse models of human diseaseAn evolutionary perspective.
      ]. Given the complexities inherent in the use of animal models, several in vitro systems have been developed that use human cell-based assays to provide a better understanding of radiation-induced cell damage in humans.

      In vitro cell culture systems

      EC grown as a homogeneous 2D monolayer are traditionally used to complement animal models. A number of these in vitro EC models have been used to study effects of IR on activation of the NFκB pathway [
      • Dong X.
      • et al.
      NEMO modulates radiation-induced endothelial senescence of human umbilical veins through NF-κB signal pathway.
      ], alterations in mitochondrial membrane potential [
      • Hu S.
      • et al.
      New insight into mitochondrial changes in vascular endothelial cells irradiated by gamma ray.
      ] and the effect on vascular tone [
      • Beckman J.A.
      • et al.
      Radiation therapy impairs endothelium-dependent vasodilation in humans.
      ]. The effect of IR on EC permeability and barrier function can also be monitored in 2D models through measurement of TEER, EC adherens junctional integrity, and EC monolayer permeability [
      • Gabryś D.
      • et al.
      Radiation effects on the cytoskeleton of endothelial cells and endothelial monolayer permeability..
      ]. Although using primary EC can provide important insight into various physiological and pathological processes, cell culture presents significant challenges. For example, almost all primary EC have an average life span of 5–10 serial passages in vitro, after which these cells often stop proliferating when they form multinucleated cells and eventually die [
      • Bouïs D.
      • et al.
      Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research.
      ]. Another drawback is the phenotypic differences between large vessel-derived EC (e.g. HUVEC) vs. EC of microvascular origin (e.g., human dermal microvascular cells) but also between EC derived from different organs. These mostly static cell culture systems fail to reproduce many of the complexities of the in vivo conditions including three-dimensional EC morphology, cell–cell and cell-matrix interactions, and shear flow conditions [
      • Coluccio M.L.
      • et al.
      Microfluidic platforms for cell cultures and investigations.
      ]. Nevertheless, these low cost, easy to use, and high-throughput systems are considered standard for analysis of cellular responses [
      • Coluccio M.L.
      • et al.
      Microfluidic platforms for cell cultures and investigations.
      ]. Flow-based 2D cultures offer advantages as they have more precise spatial/temporal dynamics, sensor integration and continuous monitoring [

      Prabhakarpandian B. et al., Expression and functional significance of adhesion molecules on cultured endothelial cells in response to ionizing radiation. Microcirculation, 2001. 8(5): p. 355-364 DOI: https://doi-org.libproxy.temple.edu/10.1111/j.1549-8719.2001.tb00182.x.

      ,
      • Coluccio M.L.
      • et al.
      Microfluidic platforms for cell cultures and investigations.
      ].

      Microphysiological systems

      The inability of 2D monolayer cultures to recreate the appropriate microenvironment for cell-matrix, cell–cell, cell-tissue and cell-organism interactions has led to the development of an emerging new class of in vitro models that better mimic the in vivo conditions. These new models not only provide novel approaches for studying radiation damage to EC but also offer a new tool for the rapid development and screening of therapeutics. Microphysiological systems, often synonymous with “organ-on-chip”, commonly consist of an interconnected set of 2D and 3D cell cultures in microfluidic devices. These emerging organ-on-a-chip systems can increasingly recapitulate the complex microenvironment of different human tissues by recreating organ-specific geometries and co-culturing of multiple cell types, mimicking the physiological and pathological conditions and responses to therapeutics. Microphysiological systems can serve as in vitro models of brain, GI tract, lung, liver, vasculature, and skin microvasculature [
      • Wikswo J.P.
      The relevance and potential roles of microphysiological systems in biology and medicine.
      ] and are rapidly becoming the new standard tool for better understanding vascular biology, pharmacology and toxicology [
      • Wikswo J.P.
      The relevance and potential roles of microphysiological systems in biology and medicine.
      ]. Several microphysiological systems have been used to study the effects of radiation on different organs and screen various therapeutics [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ,
      • Jalili-Firoozinezhad S.
      • et al.
      Modeling radiation injury-induced cell death and countermeasure drug responses in a human Gut-on-a-Chip.
      ]. Employing both human and mouse cells, together with omic and in silico modeling approaches, will help better predict how therapeutics developed in animal models may work in clinical settings as well as screen potential therapeutics on patient cells for personalized medicine [
      • Kilpatrick L.E.
      • Kiani M.F.
      Experimental approaches to evaluate leukocyte-endothelial cell interactions in sepsis and inflammation.
      ].

      Organoids, spheroids, and scaffolds

      Recent advances in tissue engineering have facilitated the development of novel 3D models for radiobiological research including organoids, spheroids and scaffolds. Organoids consist of a collection of organ-specific cell types that spatially organize to restrict lineage commitment similar to in vivo [
      • Simian M.
      • Bissell M.J.
      Organoids: a historical perspective of thinking in three dimensions.
      ]. Spheroids are aggregated, mutually adherent population of cells with spherical shapes [
      • Fennema E.
      • et al.
      Spheroid culture as a tool for creating 3D complex tissues.
      ]. Although organoids have in vivo like cellular architecture and spheroids have advantages of high reproducibility [
      • Fang Y.
      • Eglen R.M.
      Three-dimensional cell cultures in drug discovery and development.
      ], they are still not widely used in studying radiation-induced EC damage in part due to the major challenges associated with developing organoids and spheroids. A key limitation in using organoid and/or spheroid based approaches to generate functional tissue is that upon reaching a certain size, organoids switch from a proliferative, stem-like state to a non-proliferative, terminally differentiated one and develop a necrotic core [
      • Grebenyuk S.
      • Ranga A.
      Engineering organoid vascularization.
      ]. With the exception of a few avascular tissues, any efforts at producing tissues would be limited to a length scale of approximately 150 μm, the natural diffusion limit of oxygen in tissue. However, scalable 3D microfabrication technologies like layer-by-layer deposition of materials and selective removal of materials to form tubular voids, as well as a range of hybrid approaches utilizing sacrificial materials [
      • Grebenyuk S.
      • Ranga A.
      Engineering organoid vascularization.
      ], allow for generation of free-form vascular structures in organoids [
      • Nashimoto Y.
      • et al.
      Integrating perfusable vascular networks with a three-dimensional tissue in a microfluidic device.
      ,
      • Mansour A.A.
      • et al.
      An in vivo model of functional and vascularized human brain organoids.
      ].
      Scaffold/matrix-based 3D cultures (Fig. 3A) are formed by seeding cells on a 3D synthetic-based matrix or by dispersing cells in a liquid matrix followed by polymerization to mimic the tissue microenvironment [
      • Fang Y.
      • Eglen R.M.
      Three-dimensional cell cultures in drug discovery and development.
      ]. Scaffolds have high reproducibility and allow for coculture of other cell types but frequently have a simplified architecture [
      • Fang Y.
      • Eglen R.M.
      Three-dimensional cell cultures in drug discovery and development.
      ]. While 3D cultures are considered more informative for studying radiation damage, they are not as easy to propagate and standardize as monolayer cultures. These 3D in vitro models must incorporate both the spatial organization and differentiated function of the tissue in vivo to allow reproduction of cell–cell interactions and create the appropriate microenvironment for cellular proliferation and gene expression [
      • Schmeichel K.L.
      • Bissell M.J.
      Modeling tissue-specific signaling and organ function in three dimensions.
      ]. For example, a 3D capillary model using human umbilical vein EC (HUVEC) grown in a collagen gels showed more complex and persistent 53BP1 DNA damage foci when exposed to high LET (linear energy transfer) as compared to low LET [
      • Grabham P.
      • et al.
      Effects of ionizing radiation on three-dimensional human vessel models: differential effects according to radiation quality and cellular development.
      ]. However, due in part to the complexity and lack of reproducibility of some 3D models, more realistic in vitro assays using human cells are needed to better mimic the in vivo microenvironment.
      Figure thumbnail gr3
      Fig. 3Use of microphysiological systems in radiobiological research: (A) micro engineered 3D scaffolds; (B) a novel in vitro biomimetic microfluidic assay (bMFA) developed to study radiation-induced endothelium damage; (C) map of microvascular networks in animals obtained using intravital microscopy; (D) vascular network reproduced on polydimethylsiloxane device; (E) the bMFA includes vascular channels that are connected to the tissue compartment through a 3 μm barrier; (F) EC are aligned in the direction of flow in the bMFA (scale bar 250 μm); (G) confocal microscopy demonstrates that EC form a complete 3D lumen in the vascular channel. F-actin is labeled in green, and nuclei are labeled in red. [C–G: reproduced with permission from reference
      [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ]
      ].

      Microfluidic systems

      Microfluidic systems (Fig. 3B) permit the study of complex vascular and microvascular processes, such as neutrophil-EC interactions and the inflammatory response, using in vitro 3D models that more realistically reproduce the tissue microenvironment. For example, Gut-on-a-chip models have been used to study the effects of gamma (γ)-radiation on villus intestinal epithelium [
      • Jalili-Firoozinezhad S.
      • et al.
      Modeling radiation injury-induced cell death and countermeasure drug responses in a human Gut-on-a-Chip.
      ]. Exposure to γ-radiation increased the generation of ROS, cell cytotoxicity, apoptosis, and led to compromised intestinal barrier integrity. A microvasculature-on-a-chip was used to investigate the effects of IR on HUVEC forming 3D perfusable networks mimicking the human microvasculature [
      • Guo Z.
      • et al.
      Validation of a vasculogenesis microfluidic model for radiobiological studies of the human microvasculature.
      ]. Importantly, a systematic comparison between HUVEC cultured in this system with a traditional 2D monolayer model showed significant differences upon IR exposure, particularly at high doses that are typically used in stereotactic body radiation therapy (SBRT) and stereotactic radiosurgery (SRS). At high IR doses up to 25 Gy, VE‐cadherin cell–cell adherens junctions did not change significantly in the 3D fluidic model but were significantly damaged in the 2D monolayer model. Similarly, increased apoptosis was observed in the 2D monolayer model as compared to the 3D fluidic model. An advantage of the 3D models is that ECs form 3D tube-like structures in these model systems mimicking a more realistic in vivo environment and behave quite differently than those cultured in a 2D environment [
      • Guo Z.
      • et al.
      Validation of a vasculogenesis microfluidic model for radiobiological studies of the human microvasculature.
      ]. The fact that many of the in vitro studies of effects of IR has traditionally resulted from studies conducted with clonogenic assays in a 2D environment should give impetus to validate these findings in microfluidic systems mimicking the 3D in vivo microenvironment.
      Our group has developed a novel biomimetic microfluidic assay (bMFA) that facilitates real-time assessment of neutrophil rolling, firm arrest, spreading and migration to the extravascular tissue in a realistic microvasculature geometry under physiologic shear conditions in a single assay (Fig. 3 C–F). This is one of the few in vitro systems that realistically model in vivo geometrical features, including vascular morphology and flow conditions such as converging or diverging flow at bifurcations of the vasculature [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ,
      • Prabhakarpandian B.
      • et al.
      Microfluidic devices for modeling cell–cell and particle–cell interactions in the microvasculature.
      ,
      • Prabhakarpandian B.
      • et al.
      Synthetic microvascular networks for quantitative analysis of particle adhesion.
      ,
      • Rosano J.M.
      • et al.
      A physiologically realistic in vitro model of microvascular networks.
      ]. This integrated microfluidic assay provides a novel platform for investigating the EC inflammatory response, neutrophil-EC interaction, and EC damage in response to various stimuli including IR (Supplementary Data, Video 3). We have used bMFA to demonstrate that IR exposure upregulated human neutrophil-EC interaction, increased ICAM-1 and VCAM-1 (but not E-selectin) expression, and increased permeability under shear flow conditions [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ].

      Therapeutics

      Radioprotective agents are classified into three broad categories: radioprotectors, radiomitigators and radiotherapeutics. Radioprotectors are administered prior to radiation exposure to protect downstream injury and include molecules with thiol functionalities and antioxidant properties. Radiomitigators are given during or quickly after radiation exposure, primarily to mitigate normal tissue toxicity, stop/alleviate the consequences of tissue exposure to radiation and to hasten repair (Table 2). Radiotherapeutics are given after the clinical appearance of normal tissue toxicity to ameliorate radiation damage and to initiate tissue regeneration or repair [
      • Mishra K.
      • Alsbeih G.
      Appraisal of biochemical classes of radioprotectors: evidence, current status and guidelines for future development. 3.
      ]. Radiomitigators and radiotherapeutics can also serve as radiation countermeasures during mass casualty events resulting from radiation exposure.
      Table 2Therapeutics developed to treat ionizing radiation induced vascular injury.
      TherapeuticHow has it been tested?ResultsClinical trials? FDA approved?
      Neupogen (Radio mitigator)Nonhuman primates, minipig
      • Farese A.M.
      • et al.
      Filgrastim improves survival in lethally irradiated nonhuman primates.
      ,
      • Moroni M.
      • et al.
      The Gottingen minipig is a model of the hematopoietic acute radiation syndrome: G-colony stimulating factor stimulates hematopoiesis and enhances survival from lethal total-body γ-irradiation..
      Enhanced survival, stimulated recovery from neutropenia, induced mobilization of hematopoietic progenitor cells
      • Moroni M.
      • et al.
      The Gottingen minipig is a model of the hematopoietic acute radiation syndrome: G-colony stimulating factor stimulates hematopoiesis and enhances survival from lethal total-body γ-irradiation..
      FDA approved for medical countermeasure to increase survival in patients exposed to myelosuppressive doses of radiation. FDA approved based on animal studies

      FDA approves Neupogen for treatment of patients with radiation-induced myelosuppression following a radiological/nuclear incident. [cited 2020 10/26/2020]; Available from: https://www.fda.gov/emergency-preparedness-and-response/about-mcmi/fda-approves-radiation-medical-countermeasure.

      Neulasta (Radio mitigator) A pegylated form of granulocyte-colony stimulating factor (PEG-G-CSF).Mouse and nonhuman primates
      • Chua H.L.
      • et al.
      Survival efficacy of the PEGylated G-CSFs Maxy-G34 and neulasta in a mouse model of lethal H-ARS, and residual bone marrow damage in treated survivors.
      ,
      • Farese A.
      • et al.
      Pegfilgrastim administered in an abbreviated schedule, significantly improved neutrophil recovery after high-dose radiation-induced myelosuppression in rhesus macaques.
      Significant survival efficacy as a single dose in a murine model of the Hematopoietic Acute Radiation Syndrome (H-ARS)
      • Chua H.L.
      • et al.
      Survival efficacy of the PEGylated G-CSFs Maxy-G34 and neulasta in a mouse model of lethal H-ARS, and residual bone marrow damage in treated survivors.
      , Administration at days 1 and 7 was most effective at improving neutrophil recovery compared to daily administration
      • Farese A.
      • et al.
      Pegfilgrastim administered in an abbreviated schedule, significantly improved neutrophil recovery after high-dose radiation-induced myelosuppression in rhesus macaques.
      Phase II clinical trials

      Pegfilgrastim (Neulasta) for Stem Cell Mobilization in Patients With Multiple Myeloma. 2012; Available from: https://clinicaltrials.gov/ct2/show/NCT00067639.

      , FDA approved to increase WBC count after chemotherapy

      Pegfilgrastim (Neulasta) for Stem Cell Mobilization in Patients With Multiple Myeloma. 2012; Available from: https://clinicaltrials.gov/ct2/show/NCT00067639.

      Pentoxifylline (Radiomitigator/Radiotherapeutic)Patients

      Misirlioglu CH. et al., Pentoxifylline and alpha-tocopherol in prevention of radiation-induced lung toxicity in patients with lung cancer. Medical oncology, 2007. 24(3): p. 308-311 DOI: https://doi-org.libproxy.temple.edu/10.1007/s12032-007-0006-z.

      After 6 weeks, patients had lower pulmonary toxicity

      Misirlioglu CH. et al., Pentoxifylline and alpha-tocopherol in prevention of radiation-induced lung toxicity in patients with lung cancer. Medical oncology, 2007. 24(3): p. 308-311 DOI: https://doi-org.libproxy.temple.edu/10.1007/s12032-007-0006-z.

      Clinical trials with vitamin E to reduce fibrosis
      • Haddad P.
      • Kalaghchi B.
      • Amouzegar-Hashemi F.
      Pentoxifylline and vitamin E combination for superficial radiation-induced fibrosis: a phase II clinical trial.
      ,
      • Amano M.
      • et al.
      Increase in tumor oxygenation and potentiation of radiation effects using pentoxifylline, vinpocetine and ticlopidine hydrochloride.
      . FDA approved for the treatment of intermittent claudication on the basis of chronic occlusive arterial disease of the limbs

      FDA-Approved Drugs. Available from: https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=overview.process&ApplNo=075028, https://www.accessdata.fda.gov/drugsatfda_docs/label/2012/018631s039lbl.pdf

      Endogenous tetrapeptide (Ac SDKP) (Radiomitigators/Radiotherapeutic)Rat and in vitro modelInhibited EC loss, reduced coronary fibrosis, restored TJ-assembly. In vitro, localized to EC and inhibited ROS generation
      • Sharma U.C.
      • et al.
      Effects of a novel peptide Ac-SDKP in radiation-induced coronary endothelial damage and resting myocardial blood flow.
      Pravastatin (Radio mitigators/Radiotherapeutic)Mouse and in vitro model
      • Jang H.
      • et al.
      Pravastatin alleviates radiation proctitis by regulating thrombomodulin in irradiated endothelial cells.
      Regulate thrombomodulin (TM) expression, exogenous TM inhibits leukocyte adhesion to EC
      • Jang H.
      • et al.
      Pravastatin alleviates radiation proctitis by regulating thrombomodulin in irradiated endothelial cells.
      Captopril (Radio mitigator/Radiotherapeutic)In vitro model: Human endothelial hybrid cell line EA. HY926

      Wei J. et al., Effect of captopril on radiation-induced TGF-β1 secretion in EA. Hy926 human umbilical vein endothelial cells. Oncotarget, 2017. 8(13): p. 20842 DOI: https://doi.org/10.18632/oncotarget.15356.

      Reduced Ang II and TGF-β1 expression and inhibited NF-κB pathway

      Wei J. et al., Effect of captopril on radiation-induced TGF-β1 secretion in EA. Hy926 human umbilical vein endothelial cells. Oncotarget, 2017. 8(13): p. 20842 DOI: https://doi.org/10.18632/oncotarget.15356.

      FDA approved for the treatment of hypertension

      FDA-Approved Drugs. [cited 2020 06/15]; Available from: https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=overview.process&ApplNo=074505, https://www.accessdata.fda.gov/drugsatfda_docs/label/2012/018343s084lbl.pdf.

      BAY 11-7085 (Radioprotector)In vitro model: HUVEC

      Wang H. et al., Gamma radiation-induced disruption of cellular junctions in HUVECs is mediated through affecting MAPK/NF-κB inflammatory pathways. Oxidative medicine and cellular longevity, 2019. 2019 DOI: https://doi.org/10.1155/2019/1486232.

      Decrease of TEER, partially blocking permeability increase

      Wang H. et al., Gamma radiation-induced disruption of cellular junctions in HUVECs is mediated through affecting MAPK/NF-κB inflammatory pathways. Oxidative medicine and cellular longevity, 2019. 2019 DOI: https://doi.org/10.1155/2019/1486232.

      Geranylgeranylacetone (GGA) (Radioprotector)Mouse
      • Han N.-K.
      • et al.
      Geranylgeranylacetone ameliorates intestinal radiation toxicity by preventing endothelial cell dysfunction.
      Ameliorated intestinal injury, preserved intestinal microvessels
      • Han N.-K.
      • et al.
      Geranylgeranylacetone ameliorates intestinal radiation toxicity by preventing endothelial cell dysfunction.
      Plasminogen activator inhibitor type 1 (PAI-1)WT C57BL/6J (PAI-1 +/+) and PAI-1 −/− mouse
      • Abderrahmani R.
      • et al.
      PAI-1-dependent endothelial cell death determines severity of radiation-induced intestinal injury.
      Increased EC survival, vascular density, mucosal integrity
      • Abderrahmani R.
      • et al.
      PAI-1-dependent endothelial cell death determines severity of radiation-induced intestinal injury.
      Bak and BaxChimeric Tie2Bak/BaxFL/−;WT-EC mouse
      • Doan P.L.
      • et al.
      Tie2+ bone marrow endothelial cells regulate hematopoietic stem cell regeneration following radiation injury.
      Bak and Bax deletion in bone marrow EC, protection of bone marrow vasculature
      • Doan P.L.
      • et al.
      Tie2+ bone marrow endothelial cells regulate hematopoietic stem cell regeneration following radiation injury.
      Rosiglitazone (Radioprotector)In vitro: Human telomerase-immortalized coronary artery EC
      • Baselet B.
      • et al.
      Rosiglitazone protects endothelial cells from irradiation-induced mitochondrial dysfunction.
      Increased oxidative metabolism, redox state, decreased levels of apoptosis
      • Baselet B.
      • et al.
      Rosiglitazone protects endothelial cells from irradiation-induced mitochondrial dysfunction.
      FDA approved for use in management of type 2 diabetes mellitus

      FDA Drug Safety Communication: FDA eliminates the Risk Evaluation and Mitigation Strategy (REMS) for rosiglitazone-containing diabetes medicines. Available from: https://www.fda.gov/drugs/drug-safety-and-availability/fda-drug-safety-communication-fda-eliminates-risk-evaluation-and-mitigation-strategy-rems.

      PKCδ-TAT peptide inhibitor (Radiomitigator/radioprotector)HUVEC in a microfluidic device
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      , mice
      Reduced radiation-induced EC damage, leukocyte migration
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      , reduced mortality in irradiated mice
      Safely tolerated in Phase I/II clinical trials for treating acute myocardial infarction
      • Bates E.
      • et al.
      Intracoronary KAI-9803 as an adjunct to primary percutaneous coronary intervention for acute ST-segment elevation myocardial infarction.
      ,
      • Lincoff A.M.
      • et al.
      Inhibition of delta-protein kinase C by delcasertib as an adjunct to primary percutaneous coronary intervention for acute anterior ST-segment elevation myocardial infarction: results of the PROTECTION AMI Randomized Controlled Trial.

      Lack of selective anti-inflammatory therapeutics complicates radioprotection

      While the use of Neupogen and Neulasta to treat hematopoietic acute radiation syndrome (H-ARS) was recently approved by the FDA, therapeutic approaches to the treatment of radiation-induced vascular/ endothelial injury are largely supportive and there are no specific pharmacologic therapies available that protect from radiation-mediated tissue damage [
      • Satyamitra M.M.
      • DiCarlo A.L.
      • Taliaferro L.
      Understanding the pathophysiology and challenges of development of medical countermeasures for radiation-induced vascular/endothelial cell injuries: report of a NIAID workshop, August 20, 2015.
      ]. Given the fact that radiation damage to EC initiates and is mediated by the immune response, radioprotection is also hampered by a lack of effective anti-inflammatory therapeutics. Most anti-inflammatory therapeutics work primarily through immunosuppression, rather than selective immune modulation. This lack of effective anti-inflammatory agents continues to complicate the treatment of inflammatory pathologies ranging from sepsis and COVID-19 to radiation-induced inflammatory response. Radiotherapies for treatment of IR-induced endothelial damage have been primarily focused in 3 areas, targeting EC pathways activated in response to radiation, reconstitution of bone marrow and reduction in vascular barrier permeability and vascular leak [
      • Satyamitra M.M.
      • DiCarlo A.L.
      • Taliaferro L.
      Understanding the pathophysiology and challenges of development of medical countermeasures for radiation-induced vascular/endothelial cell injuries: report of a NIAID workshop, August 20, 2015.
      ].

      Radioprotectors

      Potential radioprotectors include BAY 11-7085, an inhibitor of NF-κB activation, has been shown to partially inhibit the effects of γ-radiation on permeability and TEER in HUVECS [

      Wang H. et al., Gamma radiation-induced disruption of cellular junctions in HUVECs is mediated through affecting MAPK/NF-κB inflammatory pathways. Oxidative medicine and cellular longevity, 2019. 2019 DOI: https://doi.org/10.1155/2019/1486232.

      ]. Geranylgeranylacetone (GGA), a drug used to treat peptic ulcers and gastritis, has been shown to moderate IR-induced intestinal injury and EC dysfunction [
      • Han N.-K.
      • et al.
      Geranylgeranylacetone ameliorates intestinal radiation toxicity by preventing endothelial cell dysfunction.
      ]. Intestinal microvessels were preserved in GGA-treated mice thereby reducing intestinal injury. GGA plays an important role in the promotion of angiogenic activity in damaged EC via inducing VEGF/eNOS signaling and suppressing inflammatory cytokine expression [
      • Han N.-K.
      • et al.
      Geranylgeranylacetone ameliorates intestinal radiation toxicity by preventing endothelial cell dysfunction.
      ].

      Radiomitigators/radiotherapeutics

      Pentoxifylline is a radiomitigator/radiotherapeutic used to treat vascular disease by inhibiting platelet coagulation, enhancing red blood cell deformability and promoting vessel blood flow. Pentoxifylline also inhibits neutrophil adhesion to EC by decreasing platelet coagulation via platelet activating factor (PAF). Administering pentoxifylline and another alpha tocopherol/vitamin E to patients undergoing radiotherapy lowered their pulmonary toxicity compared to controls after six weeks [

      Misirlioglu CH. et al., Pentoxifylline and alpha-tocopherol in prevention of radiation-induced lung toxicity in patients with lung cancer. Medical oncology, 2007. 24(3): p. 308-311 DOI: https://doi-org.libproxy.temple.edu/10.1007/s12032-007-0006-z.

      ].

      Endothelial specific radiomitigators/radiotherapeutics

      Several potential therapeutics that target IR-induced damage to EC are currently being evaluated in in vitro cell cultures and animal models. A novel tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) has been shown to localize to EC and inhibit IR-induced endothelial ROS generation [
      • Sharma U.C.
      • et al.
      Effects of a novel peptide Ac-SDKP in radiation-induced coronary endothelial damage and resting myocardial blood flow.
      ]. In vivo studies demonstrated that Ac-SDKP administration was able to restore IR-induced endothelial barrier damage by preventing EC cell loss and increasing expression of tight junction proteins. Ac-SDKP also showed strong antifibrotic effects that inhibited IR-induced coronary vascular fibrosis thus preserving myocardial blood flow [
      • Sharma U.C.
      • et al.
      Effects of a novel peptide Ac-SDKP in radiation-induced coronary endothelial damage and resting myocardial blood flow.
      ]. The statin, Pravastatin, has also been shown to exert persistent anti-inflammatory and anti-thrombotic effects on irradiated EC and to inhibit leukocyte-EC interaction [
      • Jang H.
      • et al.
      Pravastatin alleviates radiation proctitis by regulating thrombomodulin in irradiated endothelial cells.
      ]. Pravastatin also regulates thrombomodulin (TM) expression which inhibits the leukocyte adhesion to the irradiated EC. Although the mechanisms of TM transcription activation by this drug is unclear, suppression of IR-induced endothelial interaction with leukocyte may prevent intestinal inflammation post-irradiation [
      • Jang H.
      • et al.
      Pravastatin alleviates radiation proctitis by regulating thrombomodulin in irradiated endothelial cells.
      ]. Angiotensin II (Ang II) is produced by the endothelium in response to IR and induces the generation of the profibrotic cytokine TGF-β. In vitro treatment of EC with the ACE inhibitor, Captopril, following IR was shown to reduce Ang II expression, inhibit the NF-κB pathway, and reduce TGF-β1 expression [

      Wei J. et al., Effect of captopril on radiation-induced TGF-β1 secretion in EA. Hy926 human umbilical vein endothelial cells. Oncotarget, 2017. 8(13): p. 20842 DOI: https://doi.org/10.18632/oncotarget.15356.

      ].
      Genetic approaches employing knockout mice have identified other EC targets to mitigate IR-induced cell damage. For example, endothelial expression of the plasminogen activator inhibitor type 1 (PAI-1) following IR increases and plays a role in activating endothelial apoptosis. In response to IR, PAI-1−/− mice had increased EC survival, preserved vascular density and mucosal integrity as compared to wild type mice [
      • Abderrahmani R.
      • et al.
      PAI-1-dependent endothelial cell death determines severity of radiation-induced intestinal injury.
      ]. Mice with key proapoptotic proteins Bak and Bax deletions in both bone marrow EC and bone marrow hematopoietic stem cells (HSCs) exhibited protection of the BM vasculature and increased survival post IR exposure [
      • Doan P.L.
      • et al.
      Tie2+ bone marrow endothelial cells regulate hematopoietic stem cell regeneration following radiation injury.
      ].

      Emerging endothelium-based radiotherapeutics

      To respond to the urgent need for new therapies, new classes of novel therapeutics are being developed to specifically treat endothelial dysfunction resulting from radiation exposure. For example, the peroxisome agonist, Rosiglitazone, which is used clinically to treat diabetes [
      • Deeks E.D.
      • Keam S.J.
      Rosiglitazone.
      ], was recently shown to preserve mitochondrial function after radiation exposure and to stimulate mitochondria biogenesis in EC. When coronary EC were treated with Rosiglitazone before IR exposure, the cells exhibited enhanced respiratory function used for ATP production and apoptosis was reduced [
      • Baselet B.
      • et al.
      Rosiglitazone protects endothelial cells from irradiation-induced mitochondrial dysfunction.
      ]. The effects of treating EC with Rosiglitazone after radiation exposure have not been reported, so the efficacy of this therapeutic for treating victims of mass/accidental radiation exposure remains unclear.
      We identified Protein Kinase C-delta (PKCδ) as a critical regulator of the inflammatory response controlling leukocyte infiltration across endothelium and loss of barrier function. PKCδ is activated in multiple cell types in response to radiation exposure and is involved in radiation-induced apoptosis [
      • Wie S.M.
      • et al.
      Inhibiting tyrosine phosphorylation of protein kinase Cδ (PKCδ) protects the salivary gland from radiation damage.
      ]. Moreover, PKCδ overexpression enhanced radiation-induced apoptosis indicating a critical signaling role [
      • Lee Y.-J.
      • et al.
      Protein kinase Cdelta overexpression enhances radiation sensitivity via extracellular regulated protein kinase 1/2 activation, abolishing the radiation-induced G (2)-M arrest.
      ]. PKCδ−/− mice were protected from radiation-induced damage to the salivary gland and thymus [
      • Reyland M.E.
      • Jones D.N.
      Multifunctional roles of PKCδ: Opportunities for targeted therapy in human disease.
      ].
      We have shown that delivery of a specific PKCδ-TAT peptide inhibitor to the lung had a dramatic anti-inflammatory and lung protective effect in a rodent model of sepsis [
      • Liverani E.
      • et al.
      Protein kinase C-delta inhibition is organ-protective, enhances pathogen clearance, and improves survival in sepsis.
      ]. Similarly, we have used our novel microfluidic assay (bMFA) to show that PKCδ inhibition dramatically attenuates radiation-induced human EC damage, leukocyte migration (Fig. 4A) as well as vascular EC permeability (Fig. 4B) and preserved integrity of irradiated EC (Fig. 4C–E) even when administered 24 hrs post-IR [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ]. Moreover, neutrophil adhesion to irradiated EC was significantly decreased after PKCδ inhibition in a shear-dependent manner. PKCδ inhibition downregulated radiation-induced P-selectin, ICAM-1 and VCAM-1overexpression [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ].
      Figure thumbnail gr4
      Fig. 4PKCδ inhibition as a novel medical countermeasure for radiation‐induced vascular damage; (A) Neutrophil migration across irradiated human EC increases over time by up to 20-fold at 60 min. PKCδ-TAT inhibitor (PKCδ-i) at 24 hours post-IR significantly reduces neutrophil migration by up to 82% after 60 min; (B) Dextran permeability of EC exposed to irradiation is significantly increased. Treatment of cells with PKCδ-i restores permeability to control levels (0 Gy). Data are normalized with respect to the permeability of EC with no treatment; (Mean ± SEM, n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001). (C) EC are aligned in the direction of flow under control conditions; (D) whereas in response to 5 Gy IR, they are not as well aligned and denuded (solid arrows); (E) PKCδ-i 24 hrs post-IR prevents denuding of EC which align in the direction of flow (open arrow); green: VE-cadherin (adherens junction); red: phalloidin (actin filament); blue: Hoechst 33342 (cell nucleus). [Reproduced with permission from reference
      [
      • Soroush F.
      • et al.
      PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.
      ]
      ]; (F) all control mice whole body irradiated with 7 Gy treated with PBS died between days 11 and 12 post-IR, while 80% of mice whole body irradiated with 7 Gy treated with PKCδ-i lived to days 12–16 post-IR, with one mouse living for >60 days (→) post-IR when it was euthanized as required by our animal protocol (n = 5/group).
      In preliminary studies, treatment of whole body 7 Gy irradiated C57BL/6J mice with the PKCδ inhibitor significantly delayed and reduced mortality (Fig. 4F), supporting the hypothesis that PKCδ is activated in response to radiation and PKCδ inhibition provides a protective effect. Furthermore, administration of the PKCδ-TAT peptide inhibitor used in our study has already been shown to be safely tolerated in Phase I/II clinical trials for treating acute myocardial infarction [
      • Bates E.
      • et al.
      Intracoronary KAI-9803 as an adjunct to primary percutaneous coronary intervention for acute ST-segment elevation myocardial infarction.
      ,
      • Lincoff A.M.
      • et al.
      Inhibition of delta-protein kinase C by delcasertib as an adjunct to primary percutaneous coronary intervention for acute anterior ST-segment elevation myocardial infarction: results of the PROTECTION AMI Randomized Controlled Trial.
      ].
      In summary, radiation-induced EC injury in large part mediates and regulates wider tissue damage. Ionizing radiation directly activates and damages the endothelium via increased adhesion molecule expression, leukocyte-EC interactions, mitochondrial damage, barrier permeability and apoptosis. Emerging in vivo and in vitro models of vascular inflammatory response are providing not only a better understanding of mechanisms underlying the progression of radiation-induced EC damage but also a roadmap for developing highly specific radiotherapeutics for preventing and treating side effects of radiotherapy and/or accidental radiation exposure. Novel therapeutics that specifically focus on common key control points or signaling hubs can more effectively regulate the multiple overlapping and redundant mechanisms that modulate the signaling pathways regulating the vascular endothelial response to radiation-induced damage.

      Conflict of interest statements

      All authors confirm that they have read and approved the final version of the manuscript being submitted. They also certify that the article is not under consideration for publication elsewhere.

      Funding information

      This work was supported by the National Institutes of Health, United States , Grant/Award Numbers: GM114359 , GM134701 and Defense Threat Reduction Agency, United States , Grant/Award Number: HDTRA11910012 .

      Appendix A. Supplementary data

      The following are the Supplementary data to this article:

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