Biomarkers in Pathophysiology of COVID-19 and Radiation Injury

There is a remarkable similarity in the pattern of biomarker response to SARS-CoV-2 infection and radiation exposure. Significant changes in hematological, chemical, inflammatory and immune biomarkers are seen in COVID-19 and in irradiated patients. Although the insult (infection or acute radiation exposure) is vastly different, the ensuing pathologies converge to multiorgan dysfunction (MOD), resulting in multiorgan failure (MOF) and mortality. Biomarkers unique to each insult, such as smell dysfunction for COVID-19, or cytogenetic, genomic or metabolomic approaches for radiation biodosimetry are not discussed here.

Hematology

The classical approach of hematology has emerged as a valuable tool in predicting outcome as well as stratifying and management of COVID-19 patients (54–59). For example, a retrospective analysis compared hemocyte counts (neutrophils, lymphocytes and platelets; or “NLP score”) and found a strong correlation between NLP score and COVID-19 disease progression, while another published study noted lymphocyte decrease associated with COVID-19 severity (60, 61). Furthermore, Chen et al. conducted a retrospective study on 548 patients and noted that the counts of lymphocytes, T-cell subsets, eosinophils and platelets decreased markedly, especially in severe/critical and fatal patients. Increased neutrophil count and neutrophil-to-lymphocyte ratio were predominant in severe/critical cases or non-survivors (61). From separate studies, it was observed that an increase in the neutrophil-to-lymphocyte ratio (NLR) is an early warning signal for severe COVID-19 (59, 62, 63). These data are further supported by findings from a published study of 75 patients from Suzhuo, China with confirmed COVID-19 infection; common COVID-19 abnormal hematological indexes on admission included hyperfibrinogenemia, lymphopenia, elevation of D-dimer, and leukopenia, which were significantly different between the mild/moderate and severe COVID-19 groups. Furthermore, the dynamic change of NLR and D-dimer level can distinguish severe COVID-19 cases from the mild/moderate (64).

Hematological data could be further refined to interrogate a single cell population (such as lymphocytes or platelets) that yielded significant correlation with COVID-19 progression and severity. A meta-analysis of 3,099 patients from 24 different studies showed that patients who did not fare well had low lymphocyte counts (mean difference of – 361.06/µl) compared to patients who had good outcomes (65). Other researchers have also reported lymphopenia as a key biomarker in COVID-19 patients (66–68); Terpos et al. (68) referred to lymphopenia as “a cardinal laboratory finding, with prognostic potential,” urging for the need for longitudinal evaluation of parameters to follow the dynamics of the disease progression. Thrombocytopenia is also commonly noted among patients hospitalized with COVID-19 and a low platelet count is associated with higher mortality (57, 69–74). It is hypothesized that SARS-CoV-2 infects both bone marrow cells and platelets via the CD13 receptor, inducing growth inhibition and apoptosis. This entry disrupts hematopoiesis, resulting in thrombocytopenia. Another cause of thrombocytopenia is attributed to lung injury due to activation, aggregation, and retention of platelets in the lung. The formation of thrombus at the site of lung injury leads to decreased platelet production and increased consumption (75, 76). Interestingly, in most cases the platelet decreases did not reach a level where spontaneous bleeding occurred.

As seen with COVID-19 patients, radiation exposure also results in profound hematological perturbations in humans as well as irradiated animal models, characterized by granulocytopenia, lymphopenia and thrombocytopenia (77–81). Unlike COVID-19, significant decreases in neutrophils, in addition to other cytopenias, is a hallmark of ARS (82). The SEARCH (System for Evaluation and Archiving of Radiation accidents based on Case Histories) (82) database contains 824 clinical cases from 81 radiation accidents in 19 countries and allows detailed analysis of the time course of ARS, with the intention to study all medical aspects of ARS and to derive medical treatment protocols for radiation accident victims (MEdical TREatment ProtocOLs; METREPOL) (83). The hematopoietic syndrome can be characterized by granulocyte count kinetics (84). The Radiation Emergency Medical Management (REMM)⁵ website provides guidance on triaging radiation accident victims based on lymphocyte depletion kinetics. The kinetics of lymphocyte depletion have been shown to be directly related to the absorbed radiation dose from 0.5 to 10 Gy (85–87). Furthermore, the ratio of neutrophil to lymphocyte has been employed to determine the radiation dose exposure (88–90). Interestingly, platelet depletion alone has not been used to determine radiation dose, although thrombocytopenia correlates directly with radiation dose and platelet utilization at sites of active bleeding.

Serum Biomarkers

Patients with moderate and severe COVID-19 showed significant increase in levels of serum amyloid A (SSA), C-reactive protein (CRP) which positively correlated to COVID-19 pneumonia (24), and serum albumin (ALB) levels (P < 0.05) (62). Radiation also induces increases in the serum and urine proteome. CRP and serum amylase are commonly elevated after radiation exposure (91–93). In the criticality accident at Tokaimura, all three patients presented with elevated serum amylase (94). CRP levels were reported to correlate with clinical outcome in patients exposed to radiation during the Chernobyl nuclear accident (95). From a published study that identified 260 radiation-responsive proteins (96), Partridge et al. (97) narrowed the panel to IL-6, IL-1β, TNF-α and TGF-β levels as being strongly correlated to irradiation, with IL-6 emerging as the best marker for COVID-19 and acute radiation exposure.

Electrolytes

Electrolyte imbalance with reduced potassium, calcium, chloride and sodium is observed in patients with COVID-19 (72, 76, 98). Pooled analysis of data on serum electrolytes confirms that hyponatremia, hypokalemia and hypocalcemia are associated with COVID-19 severity; however, the authors cautioned that additional information such as calcium concentrations, serum albumin levels and the patients' fluid status is necessary for accurate interpretation of laboratory findings (99). Interestingly, the authors draw a correlation between electrolyte imbalance and progression of COVID-19 disease and MOF. SARS-CoV-2 binds to its host receptor, ACE2, and reduces ACE2 expression, leading to increased angiotensin II, which can cause increased potassium excretion by the kidneys resulting in hypokalemia, while plasma angiotensin II is purported to be a mediator of ARDS and ensuing MOF seen in a significant number of COVID-19 patients (100). Another cause of electrolyte loss in COVID-19 patients is attributed to GI causes such as diarrhea and emesis (101). The U.S. Centers for Disease Control (CDC) has described a similar electrolyte imbalance, accompanied by diarrhea and emesis, due to damage and disruption of the intestine after radiation exposure (102, 103).

Immune Biomarkers

Lymphocyte counts provide a rapid snapshot of the prognosis for patients with COVID-19. Delving into the lymphocyte subsets provides a clearer understanding of the patient's immune status, with both diagnostic and prognostic value (104). In a 103-patient study, a significant decrease in T-cell populations were reported. In particular, the CD3⁺, CD4⁺, CD8⁺ and NK cell counts dropped, with the CD4⁺/CD8⁺ ratio increased in COVID-19 patients compared to healthy controls (105). In other published studies, a correlation was noted between the severity of disease and reduction in T-cell subunits (61, 66) as well as decreases in B-cell numbers (106).

Persistent changes in the immune system after radiation exposure are manifested as abnormalities in the lymphoid populations and function (107). Radiation studies have documented immediate changes in T-cell subsets (108) and B cells (109) linked to radiation dose. In particular, depletion of CD8⁺ cells has been correlated with absorbed radiation at low doses (110). In atomic bomb (A-bomb) survivors, a decrease in CD4 helper T-cell populations, attenuated T-cell function, as well as an increase in B-cell populations, which could drive long-term inflammation. Radiation dose-dependent reduction in CD4⁺ cell population has also been reported (111, 112). While these metrics are not used to monitor disease progression, they are indications of the continued immune dysfunction observed in patients after radiation exposure (113).

Vascular Dysfunction

Histological analysis of COVID-19 has shown that the presence of SARS-CoV-2 within endothelial cells was associated with clusters of inflammatory cells, suggesting that infection initiates endotheliitis throughout the entire human body, leading to systemic macro and microcirculatory dysfunction in vascular beds (44). VEGF-D was noted as an indirect procoagulant biomarker of COVID-19 progression (114) and angiopoietin-2 (a marker for endothelial activation), was associated with microvascular dysfunction (115). Vascular abnormalities such as vascular thickening, detected by thoracic computed tomography (CT), was reported to be significantly associated with COVID-19, when compared to non–COVID-19 pneumonia (59% versus 22%, P < 0.001) (116). Ultimately, endotheliopathy converges with COVID-19-associated coagulopathies; a recently published study showed significant elevation in markers of endothelial cell and platelet activation with mortality strongly correlated to von Willebrand factor (VWF) antigen and plasma thrombomodulin (117).

Vascular dysfunction resulting from radiation exposure has also been reported. Of the 28 people who died within 98 days of the Chernobyl criticality incident, deaths were attributed to skin, GI and lung reactions, but most deaths were characterized by circulatory problems, with a high incidence of edema and focal hemorrhages (118). After the Tokaimura Nuclear Plant accident in Tokai, Japan, Akashi discussed the possible role of inflammation and hemorrhage in radiation-induced MOF. In a review of 110 cases histories of radiation accidents spanning 1945 through 2000, the authors analyzed MOF after total-body irradiation (TBI) and stated that “. . .symptomatology of organ system involvement could be traced not only to the pathophysiology of the rapidly turning over cell renewal systems but – of equal or more importance – to the vascular system and specifically, to the endothelial components.” (119). The primary target of radiation injury to the vasculature is the endothelial cell. The acute phase of damage occurs within hours to weeks postirradiation, and is characterized by endothelial swelling, vascular permeability and edema, lymphocyte adhesion and infiltration, and apoptosis (52). Radiation-induced vascular biomarkers include inflammatory signals, endothelial activation and adhesion markers, and prothrombic markers (48, 120, 121), similar to those reported for COVID-19.

As of September 10, 2020, the FDA has authorized EUAs for more than 150 individual molecular diagnostic tests for SARS-CoV-2. As a sampling, from February 4, 2020 to June 18, 2020, a total of 85 tests received EUA authorization, which included 37 tests for detection of nucleic acids from SARS-CoV-2 and one antigen diagnostic test, with required conditions for manufacturers and authorized laboratories.⁶ Similarly, as of mid-June 2020, the FDA had authorized more than 20 serology biomarker tests, with the caveat to “always refer to the complete instructions for use to put these estimates into the proper context and to understand how to use and interpret these tests.” On June 2, 2020, the FDA authorized the only in vitro diagnostic test for the management of patients with COVID-19, which is based on measuring the circulating IL-6 levels in these patients. In stark contrast, no radiation biodosimetry test has been cleared/authorized by the FDA.

Growth Factors

COVID-19 is characterized by damage to the lung and vasculature, reducing blood oxygenation. In a review of the effects of erythropoietin (EPO), Ehrenreich et al. (188) noted that EPO acts on tissues beyond erythropoiesis; these effects could be brought to bear in fighting SARS-CoV-2 pathology. EPO is produced in the body in response to low oxygen levels, and in the short term, binds to receptors in the brain stem to improve mechanical ventilation. EPO also acts on airways and lung vasculature to reduce inflammation and promote vascularization and has been shown clinically to be neuroprotective. Although at this writing, no trials have been started, a randomized placebo-controlled trial for proof-of-concept has been proposed. EPO has also been shown to accelerate the expansion of erythroid progenitors in mouse irradiation models (189, 190). Galal et al. (191) described the effects of EPO beyond erythropoiesis, through the reduction of oxidative stress via upregulation of anti-inflammatory receptors. These activities point to possible treatments to reduce inflammation contributing to radiation-induced GI, lung, or kidney injury.

Leukine^® (sargramostim or granulocyte-macrophage colony-stimulating factor, GM-CSF; Partner Therapeutics Inc., Lexington, MA) is one of three leukocyte growth factors approved by the U.S. FDA for treatment of ARS (192). Lang et al. (193) noted that, just as immune system stimulation can either help the body fight a viral infection or produce a deleterious inflammatory response, administration or inhibition of GM-CSF may be useful therapies for COVID-19. Lung macrophages depend on alveolar GM-CSF production for their maintenance, and GM-CSF administration could provide protection against viral infection in the early stages of ARDS and promote tissue repair. At this writing, sargramostim is being proposed as a therapeutic against COVID-19 in three trials⁷ (NCT04400929, NCT04326920, NCT04411680). As for deleterious effects, GM-CSF can exacerbate the inflammatory response, driving lung pathologies such as those resulting from COVID-19. In this case, GM-CSF inhibition could reduce expression of the pro-inflammatory cytokines IL-1, IL-6 and TNF, providing a multi-pronged approach to dampen an overstimulated immune system. Monoclonal antibody treatments targeting GM-CSF or GM-CSF receptor that are ongoing include: otilimab (NCT04376684), gimsilumab (NCT04351243), lenzilumab (NCT04351152), TJM2 (NCT04341116) and mavrilimumab (NCT04397497, NCT04399980, NCT04447469). Namilumab is being used in the clinic in an expanded access program. Patients receiving treatment in these experimental protocols will need to be monitored carefully because of the role that GM-CSF plays in immunological homeostasis. In addition, it would appear that the timing of these kinds of growth factor interventions is critical to their potential efficacy.

Antioxidant Approaches

Radiation-induced damage is characterized by increases in reactive oxygen species (ROS) and oxidative stress (194–196). These increases, and the resulting inflammatory response, can damage other sensitive tissues (197–200). The antioxidant N-acetyl cysteine (NAC) has been shown to mitigate radiation-induced damage to the GI tract and improve 10- and 30-day survival in mice receiving total-abdominal irradiation (201). NAC also decreased out-of-field bone marrow damage and ROS levels, suggesting that bone marrow damage contributes to some of the radiation-induced GI injury. Because of similar patterns of tissue damage, Corry et al. hypothesized that COVID-19-induced damage to the lung could also be ameliorated by NAC treatment (202). This hypothesis was further supported by an earlier finding that NAC treatment of patients with acute lung injury and ARDS resulted in reduced mortality (203). Currently, there are several ongoing national clinical trials to address the possible benefit of NAC treatment in COVID-19 patients (NCT04374461, NCT04419025, NCT04370288 and NCT04279197).

Another compound under investigation for COVID-19 is the histamine H2-receptor antagonist famotidine. Typically used to treat acid reflux and heartburn, the drug is also known to have antioxidant activity (204). Although an unlikely candidate to treat viral disease, famotidine first came to the attention of researchers interested in repurposing already-licensed products (205). Generic and off-patent drugs were of particular interest because of their safety and affordability, supported by extensive data in humans (206). In addition, in silico analysis suggested that the drug could be useful as a therapeutic alternative in COVID-19 (207). Clinicians have noted that hospitalized patients taking the drug for other medical indications appeared to recuperate from COVID-19 better than those who did not take the drug (208). A review of over 6,000 patient records suggested that famotidine use led to a death rate of ∼14%, compared to 27% for those who had not taken famotidine. This finding was further supported by a retrospective analysis of COVID-19 patients who received the drug within 24 h of hospital admission, which showed that its use reduced the risk of intubation or death (209). One suggested mechanism of action of famotidine is a direct action on the receptor, leading to improved mast cell regulation (210). Additional, prospective clinical trials to look at the efficacy of the drug in COVID-19 patients are underway (NCT04370262, NCT04389567). A recently published article has suggested that famotidine is ineffective; however, the authors state that “We're not challenging that famotidine might help. We're saying that the mechanism of action is not antiviral”.⁸ This is consistent with the primary proposed antioxidant mechanism of action.

Radiation researchers have also sought to understand the protective effects of famotidine administration, with regards to limitation of DNA damage and cellular protection. With studies performed in vivo, in both pre-clinical irradiation models as well as in patients undergoing radiotherapy, the potential benefits of the drug have been demonstrated. Famotidine was found to be radioprotective in mice that were administered the drug prior to irradiation, as assessed by micronuclei formation in cells of the bone marrow (211, 212). Pre-clinical work with mouse leukocytes collected from irradiated animals documented consistent outcomes, with reductions in DNA damage in animals treated with famotidine prior to irradiation (213). Famotidine also significantly reduced lymphocytopenia in prostate cancer patients who received the drug a few hours prior to undergoing radiotherapy (214). In another study, prostate cancer patients given twice-daily oral doses of famotidine during their radiotherapy led to a reduction in radiation-induced injury to the normal rectal tissue (215). These clinical findings in cancer patients were based on earlier work using peripheral blood samples taken from normal healthy volunteers (216). In those studies, blood was irradiated ex vivo in the presence or absence of vitamin C and famotidine. Comet assay results suggested that the presence of famotidine was protective for radiation-induced apoptosis, with an estimated dose reduction factor of 1.5. The protective effects of the drug noted above suggest the drug may have an antioxidant effect and ability to scavenge free radicals, a mechanism that would justify clinical use of the drug for COVID-19.

Anti-inflammatory Approaches

Severe COVID-19 is characterized by a cytokine storm, indicative of an overactive immune response to the infection (217). Because elevated levels of pro-inflammatory cytokines are associated with high morbidity and mortality, various approaches to modulate the inflammatory response have been proposed. Specifically, COVID-19 results in elevated levels of serum IL-6 (217). The IL-6 receptor antagonist, tocilizumab (Actemra^®, Genentech, San Francisco, CA), indicated for rheumatoid arthritis, has also been proposed to ameliorate radiation-induced tissue damage, and has shown efficacy in diminishing the cytokine storm resulting from cancer immunotherapy (218). In early published clinical studies to assess the potential impact of tocilizumab treatment in COVID-19, Somers et al. (219) performed a single-site trial of the drug in 154 COVID-19 patients on mechanical ventilation. Treatments resulted in a lower hazard of death, although the rate of superinfections increased. Antinori et al. (220) noted the risk of a secondary Candida infection from tocilizumab treatment, and therefore suggested that the drug only be used in well-designed clinical trials. Currently, a tocilizumab treatment arm has been included as part of the University of Oxford's Randomised Evaluation of COVID-19 Therapy (RECOVERY) trial,⁹ the results of which have not been made available at the time of this writing. In a study looking at the effect of the drug in critically ill patients, treatment led to improved oxygenation and blood counts (221). In addition, another anti-IL-6 receptor antibody, sarilumab (Kevzara^®; Regeneron Pharmaceuticals Inc., Tarrytown, NY and Sanofi, Paris, France) is also under clinical consideration in more than 10 registered trials as a treatment for late-stage COVID-19 patients.

Dexamethasone is a generic corticosteroid drug, which is licensed for a broad range of indications including arthritis, allergic reactions and immune system disorders. Dexamethasone has been shown to reduce multi-organ damage, including lung injury, in rats that have been exposed to localized radiation (222). In non-human primate (NHP) models of radiation-induced lung injury, dexamethasone has been used as a component of the medical management, where it is given when there is an increase in the non-sedated respiratory rate, which suggests respiratory distress (138, 223, 224). This use of the drug is similar to its clinical use for dyspnea (225). In these NHP studies, dexamethasone treatment reduced the elevated respiratory rate, lung density, pleural effusion and pneumonitis, leading to improved outcomes. Dexamethasone treatment has also been included as an arm in the RECOVERY trial (226), where its use has been shown to reduce COVID-19 mortality from 40.7% to 29% among patients who required invasive mechanical ventilation, but did not appear to confer a benefit to hospitalized patients who received only oxygen. These data suggest that corticosteroid treatment may only be effective if the immune system is overstimulated to the extent that it is causing significant harm. The WHO Rapid Evidence Appraisal for COVID-19 Therapies Working Group performed a meta-analysis of seven randomized trials and concluded that systemic corticosteroids reduced 28-day all-cause mortality (227). Another treatment that has been shown to improve survival in a mouse model of radiation-induced lung injury is BIO 300 (Humanetics Corp., Edina, MN), which is a nanosuspension of the soy isoflavonoid, genistein. In mice, BIO 300 administration improved survival and reduced other morbidities caused by lung irradiation (228). Although genistein has antiviral activity (229–231), it may also be useful to prevent radiation-induced lung damage. Genistein is thought to act through inactivation of NF-κB (232), and since NF-κB inhibition has been shown to reduce inflammation in a mouse model of COVID-19, it could be effective in treating lung complications caused by SARS-CoV-2 infection. Humanetics has announced initiation of a clinical trial in discharged COVID-19 patients, to determine if treatment with the oral BIO 300 product can reduce late lung fibrosis and improve quality of life in patients who are recovering from the infection (NCT04482595).

Given that the autoimmune disease, rheumatoid arthritis (RA) is caused by an overactive immune response that targets normal joint tissue, it is not surprising that approaches that have shown benefit in RA are being considered for COVID-19. For example, anakinra (Kineret^®; Swedish Orphan Biovitrum AB, Stockholm, Sweden), a specific IL-1 receptor antagonist, has been proposed as a possible treatment. Similarly, anakinra has been reported to reduce vascular inflammation in a mouse model of radiation exposure (233). In that study, administration of the drug for two weeks postirradiation reduced the expression of inflammatory mediators such as pro-caspase and caspase-1. In an early case report from Italy, clinicians described a critically ill patient who was successfully treated with anakinra (234). Other studies have since followed; in fact, King et al. have described ten ongoing clinical trials that target hyper-inflammation. It is clear that many different dosing regimens are being tested and that most of these smaller studies (<400 patients) should be considered preliminary, but data arising from these studies could lead to larger-scale studies with more uniform treatments. The outcomes of some of these anakinra studies have been published. In the anakinra-COVID study performed in France, 25% of patients who were treated with anakinra required invasive mechanical ventilation or died, compared to 44% of historical controls from the same hospital (235). In another small retrospective cohort study from Italy, part of the COVID-19 Biobank study, anakinra-treated patients showed improvement in respiratory parameters (reduced need of supplemental oxygen, improved PaO₂/FiO₂ ratio) and reductions in the inflammatory marker, C-reactive protein.

Anti-fibrotic Approaches

Lung inflammation caused by radiation can progress to fibrosis in later stages of injury, causing shortness of breath and reduced blood oxygen saturation. Drugs currently approved to treat lung fibrosis include nintedanib (OFEV^®; Boehringer Ingelheim, Ingelheim am Rhein, Germany) and pirfenidone (Esbriet^®; Genentech). Nintedanib is a tyrosine kinase inhibitor approved for idiopathic pulmonary fibrosis. Using a mouse model of localized irradiation, researchers demonstrated that nintedanib protected against long-term fibrosis, as detected microscopically at 39 weeks postirradiation (236). Similarly, studies showed protection in a mouse model of thoracic irradiation by pirfenidone treatment (237). In terms of COVID-19 and its progression, it was noted that patients who experienced severe ARDS often exhibit later lung fibrosis (238). Although anti-inflammatory treatments could prevent late-stage disease, it is not known if this will be the case for COVID-19. For this reason, the authors propose that anti-fibrotics, such as those described above, should be studied in clinical trials. In one clinical study (NCT04338802), patients will be randomized into a placebo-control or nintedanib treatment group, and in another, the safety and efficacy of pirfenidone will be studied in patients with SARS-CoV-2 infection (NCT04282902). Similarly, imatinib (Gleevec^®; Novartis, Basel, Switzerland), licensed for chronic myeloid leukemia,¹⁰ and previously shown to increase the survival time of irradiated mice by delaying lung disease, has been suggested as a COVID-19 treatment (239). Several clinical trials are planned (NCT04357613) or recruiting (NCT04394416) patients to study the drug as a possible treatment for the disease.

Pentoxifylline is another drug that could potentially be repurposed as a treatment for COVID-19. Originally licensed to treat pain in individuals suffering from intermittent claudication (peripheral arterial disease),¹¹ pentoxifylline improves blood flow, thereby increasing tissue oxygenation. It has also been shown to inhibit synthesis of pro-inflammatory cytokines, specifically TNF-α (240). Pentoxifylline has been shown to reduce radiation-induced fibrovascular injury in animal models (241) and in the clinic (242). Because of these anti-fibrotic (and anti-inflammatory) activities, pentoxifylline has been proposed as a possible preventative of COVID-19 complications (243) and will be tested in a clinical trial (NCT04433988). Another driver of pulmonary fibrosis, TGF-β (244), presents a potential target for the prevention of pulmonary fibrosis in COVID-19 patients (245). For example, an anti-sense mRNA product that targets TGF-β2 production, OT-101 (Mateon Therapeutics, San Francisco, CA), is in phase 3 trials for several cancers, and has been proposed as a COVID-19 treatment (246). Given the probable involvement of TGF-β in the progression of COVID-19, a preclinical, anti-TGFβ receptor 1 product, IPW5371 (Innovation Pathways, Palo Alto, CA) may also be a promising candidate to treat COVID-19-induced lung fibrosis, as it has previously been shown to reduce fibrosis and improve survival in a mouse model of radiation exposure (247).

RAS-Targeted Approaches

In the early stages of the pandemic, it became clear that one method by which the SARS-CoV-2 virus gained access to the internal cellular machinery was via the ACE2 receptor, the expression of which is most prevalent on lung alveolar epithelial cells (248). This finding was similar to SARS-CoV, which also used the ACE2 receptor to gain entry into cells (249). Therefore, initial treatments considered for patients were angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II type receptor blockers (ARBs). Because many drugs, such as angiotensin-converting enzyme inhibitors (ACEIs), are generic and widely available, they represent a valuable option in repurposed drugs. Clear benefits include low cost, wealth of clinical experience, established human data and minimal side effects. During the initial stages of the COVID-19 response, it was thought that individuals currently taking hypertensive drugs could be at an increased risk of infection, and therefore, their use should be discontinued in COVID-19 patients (250, 251). In one large retrospective study of over 12,000 patients, the relationship between prior use of ACEIs and patient outcomes after infection was considered, with the finding that there was no correlation between prior hypertension medication use and COVID-19 risk (252).

In addition, it became evident that certain segments of the population were more likely to have more severe forms of disease and a propensity to develop ARDS. These individuals included those with cardiovascular disease, diabetes and hypertension, all of which have associations with dysregulated aspects of RAS (253). Still other studies suggested that these drugs should be considered as a potential treatment due to their multi-prong effects (e.g., anti-inflammatory anti-oxidant and antifibrotic) (254). Therefore, there remains a need to evaluate both angiotensin agonists and antagonists for COVID-19 (255). In one study, COVID-19 patients with hypertension were enrolled, to explore if the use of ACEI or angiotensin receptor antagonist treatments would impact the severity and progression of the infection (256). As a biomarker of efficacy, serum levels of IL-6 and circulating T-cell counts were also evaluated. Both treatments were found to increase T cells, decrease viral load and IL-6 levels, and reduce the severity of the course of the disease. Therefore, the recommendation was made to maintain ACEI and angiotensin receptor blocker treatments in patients with COVID-19.

Similarly, the radiation community has established the role that products targeting RAS can play on the progression of radiation-induced organ injuries, primarily the lung and kidneys. Many studies have demonstrated the ability of ACEI products to address radiation-induced lung injuries. Primarily conducted in rat models of injury, ACEIs were found to increase survival and decrease lung, kidney and vascular damage (257, 258). These findings were consistent across different methods of radiation exposures, such as TBI plus bone marrow transplant (BMT), whole-thorax lung irradiation (WTLI), or partial-body irradiation (PBI) with a percentage of the bone marrow spared using shielding (259). Mitigation of lung and kidney injuries was determined through assessment of circulating markers of renal damage (e.g., blood urea nitrogen and creatinine) and CT imaging of lungs in irradiated animals. In an irradiated rat model, captopril and fosinopril both increased survival after 11 Gy (TBI with BMT) and decreased lung injury (257). Similarly, lisinopril was found to mitigate kidney (260) and lung (259) damage after high-dose PBI in adult rats and improve survival in juvenile and geriatric rats (38). Enalapril mitigated injury and improved survival in a WTLI rat model, even when initiated 35 days postirradiation (261). Captopril, administered in a TBI model, improved survival, although that benefit was diminished when coupled with skin trauma (262). Finally, ramipril mitigated radiation-induced damage to the spinal cord (263). In other preclinical models of radiation injury, angiotensin (1–7) [A(1–7)], a component of the RAS mentioned above, has also been studied for its ability to improve survival in irradiated rodents (264, 265). These peptides, which have been shown to alter activity in many cell types, accelerated recovery of the bone marrow in mice receiving TBI, and also improved the platelet nadir in the animals (264). In later studies, angiotensin peptides, even when administered days post-lethal irradiation, improved mouse survival and reduced bleeding time (265).

Approaches Targeting the Vasculature

The ability of the virus to directly infect and dysregulate endothelial cells (41) is the driving force behind vascular coagulopathies and thromboses observed in COVID-19 patients. In addition to vascular effects resulting from direct viral infection, COVID-19 patients have been found to have higher levels of VEGF as compared to healthy controls (266). Elevated VEGF could further increase vessel permeability, leading to some of the symptoms noted in patients. Furthermore, studies have implicated VEGF as a target for therapeutic intervention in ARDS (267). At the time of this writing, several clinical trials of COVID-19 patients are being planned or are recruiting to assess the potential efficacy of bevacizumab, an anti-VEGF, long-lived, humanized monoclonal antibody, as a treatment for COVID-19-associated ARDS (NCT04275414, NCT04344782, NCT04305106). Also known as Avastin^®, the mechanism of action of the drug is to bind to extracellular VEGF and prevent its interaction with its receptor on endothelial cells (268).

Radiation exposure has long been known to lead to vascular impairment, which is believed to explain the multiorgan dysfunction that it causes (121). Many promising clinical approaches that target the vasculature could have an effect on radiation-induced damage. VEGF also represents a molecule that is involved in radiation exposure, and thus, is a target for reducing the negative effects. For example, VEGF levels have been shown to be increased in mice after irradiation (269, 270), and elevated levels of the growth factor have been implicated in the development of radiation-induced necrosis of normal tissue (271). Anti-VEGF antibodies (bevacizumab) have been shown to mitigate radiation necrosis in mouse brains (272), and in rats that underwent gamma-knife radiosurgery (273), and bevacizumab has been used in the clinic for radiation injury, where it was found to reduce necrosis in nasopharyngeal carcinoma patients who received radiotherapy (274).

As observations continue to be made in COVID-19 patients, it has become apparent that DIC may be responsible for many of the complications that have been seen (275–278). This condition, which is characterized by the development and circulation of small blood clots, can lead to the blockage of small vessels. As a follow-on effect, the abnormal consumption of platelets can, in turn, lead to thrombocytopenia and hemorrhage (279). In several animal models of lethal radiation exposure, including ferrets (280) and Yucatan minipigs (281), there has been evidence of DIC, both in the early days after irradiation and at the time of death. It is believed that DIC could be a contributor to radiation-induced human mortality (50), as hemorrhage at time of death has been seen clinically in irradiated patients. A major finding in autopsies of humans who have died from radiation exposure (282), widespread bleeding in the tissues often occurs as a result of DIC. Coagulation abnormalities have also clearly emerged as a key hallmark of COVID-19 infections (283). In addition to thromboses (284), thrombocytopenia has also been noted in some patients experiencing COVID-19 infection. In a study from China involving over 1,000 patients, 36.2% were thrombocytopenic, a finding that was greater in cases that were more advanced (98). This association of low platelet count with the infection is also supported by a meta-analysis in which data from nine studies were examined, involving nearly 1,800 patients (71). Those researchers found that platelet counts were much lower in patients with severe disease and concluded that these lowered counts could indicate an increased mortality risk. It is postulated that infection with SARS-CoV-2 leads to this dysregulated platelet state via a number of different causative pathways, including a reduction in platelet production due to loss of progenitors and growth inhibition, increased clearance due to evolution of autoantibodies, and enhanced platelet consumption due to lung injury, which leads to platelet activation and formation of microthrombi (284).

Like COVID-19, radiation exposures, especially TBI, are known to lead to a reduction in platelet levels. This thrombocytopenia has been postulated to play a major role in deaths from exposure (282). To address this manifestation of radiation injury, drugs that promote platelet production and are FDA-approved for other indications [e.g., immune thrombocytopenic purpura (ITP)] have been tested to see if they could mitigate damage and improve survival. This has included preclinical and clinical studies of drugs such as Nplate^® (Amgen, Thousand Oaks, CA) (6, 285), and Promacta^® (Novartis) (286, 287). Although there is a case report documenting use of Promacta to treat a COVID-19 patient who presented with ITP symptoms while hospitalized (288) and responded well to the treatment, it does not appear that these therapies have been attempted on a broader scale thus far. This may be due to the delicate balance between thrombocytopenia and thrombosis in these patients; timing of interventions is crucial to their efficacy or detriment. According to case reports, heparin has been administered as a means of countering hypercoagulation (289), and the International Society of Thrombosis and Haemostasis is now recommending use of heparin for all COVID-19 patients (290). Clearly, clinical decisions concerning the use of treatments that either enhance platelet counts or seek to reduce clotting are complex, and the use of these kinds of treatments represents an area of great interest. TP508, a 23 amino acid peptide that is a truncated form of human prothrombin, has also been shown to mitigate radiation normal tissue injury and increase survival in a mouse TBI (LD100/15) model (291). Under study in a number of other preclinical models [e.g., ischemia (292) and musculoskeletal injuries (293), and in clinical trials to address diabetic foot ulcers (294)], TP508 has been shown to enhance tissue repair by targeting endothelial cells (292). This product is in early preclinical testing as a treatment for COVID-19¹² because it targets the vasculature as its primary mechanism of action and has generalized ability to mitigate tissue damage.

Statins as a Common Treatment for Vascular Injury

Statins represent another area of drug treatment overlap between radiation and COVID-19 (295, 296). In addition to an anti-inflammatory effect, statins may modify the entry of viruses into cells, inducing autophagy of infected cells or altering activation of the coagulation cascade (297). In silico studies suggest that statins possess direct antiviral activity through blocking infectivity (298) and have been shown to enhance ACE2 levels (299) and protect against ARDS (300).

In clinical trials of COVID-19 patients, observed benefits were suggested to outweigh their potential risk (301). A retrospective study of patients treated with statins for other indications while hospitalized with COVID-19 showed a lower risk of mortality (302). In animal models of radiation injury, statins have demonstrated damage mitigation to normal tissues. Simvastatin, a HMG-CoA reductase inhibitor with widespread clinical use, has been shown to mitigate lung injury in a high-dose thoracic irradiation mouse model (303), and protects the GI tract, bone marrow (304) and salivary glands (305). Similarly, atorvastatin limits radiation-induced heart damage in a rat model (306) and kidney injury in mice (308).

Perhaps most significant, in terms of overlap with a primary mechanism of action involved in COVID-19, atorvastatin has been shown to induce a protective response in irradiated human umbilical vein endothelial cells (HUVECs) (308). Treatment of HUVECs with atorvastatin decreased radiation-induced cell apoptosis, thought to be driven by upregulation of thrombomodulin and protein C activation. Finally, lovastatin, when administered after high-dose irradiation in a WTLI mouse model, increased survival, reduced levels of macrophages and lymphocytes in the lung and decreased collagen (309). Activated protein C (APC) has been tested for its ability to mitigate radiation injuries (310) and has now been proposed as a therapy for the vascular dysfunction and abnormal thrombosis (e.g., DIC) observed in COVID-19 patients (311). APC's ability to downregulate inflammation and generate thrombin indicates its potential to reduce inflammation and limit ischemic injury throughout the body.

Antibiotic Treatments for Radiation or SARS-CoV-2 Infection, beyond Antibacterial Properties

Given the need to rapidly assess potential treatments, many clinicians have turned to antibiotics, which have the advantage of safety data and clinical experience. Several classes of antibiotics, which have activity against secondary bacterial infections and have anti-inflammatory and antiviral properties, have been suggested as potential treatments for COVID-19. In silico modeling and other predictive studies indicate efficacy of aminoglycoside compounds such as streptomycin (312); tetracyclines, such as doxycycline (313, 314) and eravacycline (312); macrolide antibiotics, such as azithromycin (315); streptogramins, such as quinupristin (316); polyether ionophoric antibiotics, such as salinomycin (317); and glycopeptides, such as teicoplanin. Teicoplanin, a drug used to treat Staphylococcus infections, was found to be effective against MERS and is predicted to also be effective against SARS-CoV-2 (318, 319). There was hope that azithromycin, in combination with hydroxychloroquine (320), might be effective in reducing the severity of SARS-CoV-2 infections (321). The drug, commonly used to treat respiratory and other infections, is thought to strengthen interferon-mediated antiviral responses (315). Unfortunately, clinical studies did not provide evidence of efficacy of the drug (322). In contrast, some studies suggest limited benefit from the use of antibiotics, either alone or in combination (323) in both children and adults with COVID-19 (324), and other research suggests that the use of some antibiotics could actually worsen the progression of the disease (325).

As with COVID-19, the efficacy of antibiotics, outside of their normal antibacterial impact, have been observed in the mitigation of radiation normal tissue injuries. In vitro screening of a broad range of antimicrobial agents was conducted to determine if any of these molecules could be used a mitigators of radiation injury. In one screen that used hematopoietic progenitor cells in a clonogenic survival assay, tetracycline was identified as a significant mitigator (326). In another in vitro screen of mouse lymphocytes, two antibiotic classes, tetracyclines and fluoroquinolones (10 different molecules), were identified as potential radiation mitigators, which the authors attributed to being separate from their antibacterial properties. From these potential mitigators, tetracycline showed efficacy in a TBI mouse model of survival and further data mining confirmed these earlier findings (327). The predicted impact of quinolones on mitigation of radiation injuries was not surprising, given earlier studies in mice that showed ciprofloxacin, sparfloxacin and clinafloxacin could enhance colony-forming units in the bone marrow and white blood cell counts in irradiated mice (328). Similarly, a published study from 1961 showed that the presence of chlortetracycline in rodent feed decreased X-ray killing in mice (329). In more recent studies, fluoroquinolones, such as ciprofloxacin and levofloxacin, as well as doxycycline, and neomycin, were all found to increase the mean survival time in mice exposed to lethal doses of radiation. Doxycycline and neomycin also improved day-30 survival in the animals (330). In other work, ciprofloxacin, an antibiotic notable for its efficacy in treating bacterial pneumonia and other infections, was also shown to enhance survival after radiation exposure in vivo and in vitro. In studies using peripheral blood mononuclear cells, ciprofloxacin protected against radiation exposure by inhibiting p53 phosphorylation and increasing Bcl-2 production (331), and has also been shown to increase survival in a mouse model of TBI (332).

Cellular Therapies

The development and use of cellular therapies, in both the oncology clinic (333) and as a radiation treatment (334, 335), has long been an approach of interest. In particular, mesenchymal stem (or stromal) cells (MSCs) have been shown to mitigate the effects of radiation-induced lung injury (336, 337). Stem cells, as well as extracellular vesicles are also being considered as a means of treating injuries caused by SARS-CoV-2 (338). Specifically, MSCs derived from various sources, are under study to address lung injuries in COVID-19 patients (339). These sources include the bone marrow (340), umbilical cord (341), adipose tissue (342), peripheral blood and placenta. Early reported work indicates that the use of MSCs in patients is safe, and their use is effective in improving lung functional outcomes (343). There are more than 30 clinical trials worldwide in which these cells are being used as COVID-19 treatments (clinicaltrials.gov). In fact, several cellular therapies that are under active investigation for their development as radiation normal tissue injury mitigators are also showing promise as therapies in COVID-19 patient trials. These trials include those for placental expanded (PLX) cells¹³ and multipotent adult progenitor cells (Multi-Stem^®; Athersys^® Inc., Cleveland, OH).¹⁴