The emerging antioxidant paradigm of mesenchymal stem cell therapy

Abstract Mesenchymal stem cells (multipotent stromal cells; MSCs) have been under investigation for the treatment of diverse diseases, with many promising outcomes achieved in animal models and clinical trials. The biological activity of MSC therapies has not been fully resolved which is critical to rationalizing their use and developing strategies to enhance treatment efficacy. Different paradigms have been constructed to explain their mechanism of action, including tissue regeneration, trophic/anti‐inflammatory secretion, and immunomodulation. MSCs rarely engraft and differentiate into other cell types after in vivo administration. Furthermore, it is equivocal whether MSCs function via the secretion of many peptide/protein ligands as their therapeutic properties are observed across xenogeneic barriers, which is suggestive of mechanisms involving mediators conserved between species. Oxidative stress is concomitant with cellular injury, inflammation, and dysregulated metabolism which are involved in many pathologies. Growing evidence supports that MSCs exert antioxidant properties in a variety of animal models of disease, which may explain their cytoprotective and anti‐inflammatory properties. In this review, evidence of the antioxidant effects of MSCs in in vivo and in vitro models is explored and potential mechanisms of these effects are discussed. These include direct scavenging of free radicals, promoting endogenous antioxidant defenses, immunomodulation via reactive oxygen species suppression, altering mitochondrial bioenergetics, and donating functional mitochondria to damaged cells. Modulation of the redox environment and oxidative stress by MSCs can mediate their anti‐inflammatory and cytoprotective properties and may offer an explanation to the diversity in disease models treatable by MSCs and how these mechanisms may be conserved between species.


| INTRODUCTION
Mesenchymal stem cells (multipotent stromal cells; MSCs) have been used as tools to treat a broad range of diseases in animal models due to their unique characteristics such as host immune evasion, rapid expansion, and their affluence in adult bone marrow and adipose tissue. The positive outcomes of these studies have driven hundreds of clinical trials into their application for diabetes, inflammatory disorders and various liver, kidney, lung, cardiovascular, musculoskeletal, neurological, and gastrointestinal diseases. 1 While several trials have demonstrated the therapeutic potential of MSCs, the failure to incorporate MSCs into current treatment regimens can be, in part, attributed to the lack of understanding pertaining to their biological mechanisms of action.
Initially, MSCs were explored as tools of regenerative medicine to replace damaged tissue. 2 However, administered MSCs were rarely observed to differentiate and effectively engraft into host tissues despite demonstrating favorable effects in many disease models. 3 Furthermore, the secretome of MSCs was identified to be therapeutic in many disease models in vitro and in vivo. Together, this resulted in a paradigm shift in recognition of the trophic actions of MSCs. 4 Despite extensive research investigating the anti-inflammatory and trophic constituents of the MSC-derived secretome, the therapeutic mechanisms of MSCs remain incompletely resolved. 5 MSCs demonstrate therapeutic attributes across xenogeneic barriers and, therefore, the therapeutic mechanisms of MSCs may be similar between species.
There is strong evidence that the effects of MSCs are mediated via the secretion of protein/peptide ligands; however, it is equivocal whether these ligands are effective across xenogeneic barriers.
Recently, the role of MSCs in ameliorating oxidative and nitrosative injury has received considerable attention. The reductionoxidation (redox) environment regulates many physiological and pathophysiological mechanisms in cellular biology. Antioxidant effects of MSC therapy have been observed in various disease models such as diabetic injuries to the kidney, retina, sensory neurons, brain, and bone formation; chemotherapy-or radiation-induced injury to the lungs, gonads, aorta, and brain; ischemic injury of the brain, heart, kidney, and liver; and traumatic injury to the spine and testis, cognitive disorders, gastrointestinal inflammation, septic injuries, and aging ( Figure 1; Table 1). MSCs can directly reduce oxidative stress-related injury in vitro in glial cells, neurons, cardiomyocytes, renal cells, endothelial cells, immune cells, hepatocytes, islet cells, fibroblasts, skeletal muscle, and other cells ( Table 2). Oxidative stress is concomitant with cellular injury, inflammation, and dysregulated metabolism and, therefore, is a key pathophysiological mechanism of many diseases. Oxidative stress and redox imbalance are mediated by molecular constituents that are present in all living cells and share similar functions. Thus, the ability of MSCs to regulate these processes may offer an explanation to the diversity of disease models treatable by MSCs and to the effects of MSCs conserved between species.
Oxidative stress refers to a deviation from the physiological redox state and an increase in pro-oxidants, or free radicals, that structurally change lipids, proteins, and DNA in a way that causes pathology or damage to a cell or tissue. 6 The most widely studied free radicals are reactive oxygen species (ROS), which can also include reactive molecules that have a stable charge. The three major endogenous ROS include the superoxide anion (O 2 .− ), hydroxyl radical (•OH), and hydrogen peroxide (H 2 O 2 ). 7,8 O 2 .− is predominantly generated by nicotinamide adenine dinucleotide phosphate reduced (NADPH)-oxidase (NOX) family enzymes or, by the mitochondria, as a by-product of oxidative phosphorylation. 9 The level of mitochondria-derived O 2 .− depends on metabolic substrates, cytosolic Ca 2+ levels, pH, and oxygen tension. 10 O 2 .− generated from complexes of the electron transport chain (ETC) are highly reactive and can damage the mitochondrion. 11 The detoxification of O 2 .− into H 2 O 2 is mediated by superoxide dismutase (SOD). 9 However, H 2 O 2 can also be generated in various metabolic processes and by dual oxidases (DUOX). 12  In addition to wielding constitutive antioxidants, MSCs are also capable of significant adaptions in response to redox stress.
MSCs exposed to lipopolysaccharide (LPS) produce oxidative and nitrosative free radicals. 18

| EFFECT OF MSCs ON OXIDATIVE STRESS BIOMARKERS AND ROS
In disease models, oxidative stress is typically quantified via biomarkers of oxidation to DNA and proteins or lipid peroxidation.
Administration of MSCs has been demonstrated to reduce levels of one or more of these markers in a variety of animal models associated with oxidative stress ( Table 1). Injection of MSCs themselves may not be critical to their antioxidant effects as administration of their conditioned medium (CM) also reduced lipid peroxidation in a model of ureteral obstruction-induced kidney injury. 24 Administration of MSC-derived exosomes was also effective to rescue protein oxidation and lipid peroxidation in animal models of septic and hyperglycemic brain injury and cognitive impairment. 25 MSCs also reduced levels of O 2 .− in colitis and spontaneous stroke. 34,35 Typically, the reduction of oxidative stress markers by MSC treatments is associated with functional recovery and positive outcomes in animal models. The exception to this is the diversity of responses at various stages of hepatocarcinoma whereby antioxidant effects of MSCs reduce tumor burden at the early stages of disease by protecting the integrity of DNA but increase tumor progression at the late stages of the disease possibly by reducing ROS-associated cell death. 36 The timing of treatments may also affect MSCs ability to attenuate oxidative stress as pretreatment with MSCs is more effective to prevent oxidative stress in septic lung injury and acute liver failure. 37,38 Several studies have also investigated the therapeutic proper- MSCs which is thought to be critical to their immunomodulatory function which may also explain these inconsistent effects on nitrosative stress. 42 Although studies in cells and animal models unequivocally demonstrate that MSC treatments reduce levels of oxidative stress, albeit limited data exist from human studies. Nonetheless, favorable outcomes in a case study utilizing MSCs to treat the lungs of a subject previously exposed to sulfur mustard gas were attributed to the antioxidant properties of MSCs as evidenced by reduced lipid peroxidation levels in the sputum. 49 The antioxidant effects of MSC treatments are likely to be a specific property of these cells as they are more efficacious than hematopoietic stem cells and fibroblast at reducing oxidative stress in carbon tetrachloride-induced-liver injury and sepsis, respectively. 50,51 Likewise, fibroblast exosomes have no effect on kidney injury-induced by ischemia and chemotherapy. 27,52 The alleviation of oxidative stress in animal models is associated with decreased pro-inflammatory cytokines and markers of cellular death highlighting the close association between these processes. The

| ANTIOXIDATIVE MECHANISMS OF MSCs
The potential for MSCs to attenuate oxidative injury is unequivocally demonstrated by the reduction in ROS and biomarkers of oxidative stress in many disease models. Evidence form in vitro models suggests that MSCs directly protect cells from oxidative stimuli ( Table 2). This is often associated with a reduction in ROS    The effects of MSCs on GSH levels may be mediated by their ability to upregulate glutathione reductase which was observed in septic lung injury, acute pancreatitis, and I/R injury of the small bowel. 37,67,84 Notably, MSCs have also been shown to upregulate the expression of glutathione S-transferases (GSTs) in cells and tissues which detoxify many damaging molecules that arise from redox imbalance such as peroxidized lipids. 88,91 Glutathione reductase and GSTs are secreted by MSCs via exosomes. 31 While GSH has been the best studied scavenger in MSC treatments, they have also been demonstrated to upregulate the expression of the enzyme NAD(P)H quinone oxidoreductase 1 (NQO1) which detoxify quinones that contribute to the generation of ROS in septic lung injury, small bowel I/R injury in vivo, and cytokine-exposed islet cell in vitro. 78,84,92 The multifunctional antioxidant HO-1 has also been implicated in the therapeutic effects of MSC treatments. HO-1's antioxidant and therapeutic mechanisms have been attributed to its ability to degrade heme, which is pro-oxidative and the scavenging abilities of its products, biliverdin and bilirubin. HO-1 expression is inducible in response to oxidative stress via nuclear factor erythroid 2-related factor 2 (NRF2) which appears to be important in the adaptive responses of MSCs to inflammation and ROS. 18 Downregulation of NRF2 and HO-1 has been observed after MSC treatment of chemotherapyinduced pulmonary fibrosis and diabetic sensory neuropathy which may be explained by a reduction in oxidative stimuli. 40

| Antioxidant effects on inflammation
Immune function is regulated by free radicals and the redox system; leukocytes and pro-inflammatory mediators enhance the formation of free radicals and perturb the redox environment creating a positive feedback cycle. 98 The immunomodulatory action of MSCs is a well-    Neutrophils appear to be key mediators of oxidative stress in inflammation. These cells harbor an abundance of MPO, a major catalyst for hypochlorite and NO-derived oxidants. 105,106 MSCs attenuate the infiltration of neutrophils and reduce MPO levels in several disease models. 50,107,108 MSCs can also directly dampen the respiratory burst in neutrophils and suppress MPO activity required to produce free radical required for their pro-inflammatory function which was dependent on SOD3 and occurs in a paracrine manner. 79,80,109 Likewise, MSCs can also directly decrease ROS and MPO in stimulated monocytes and macrophages which suppress their pro-inflammatory phenotype. 110,111 These data suggest that MSCs not only suppress the immune system to prevent oxidative injury, but also that their mechanism of immunosuppression is reliant on their antioxidant properties. which associates with enhanced respiratory capacity of the mitochondria. 112 The effects of MSCs on the mitochondria appear to occur in a paracrine manner as MSC-CM increases the oxygen consumption rate of hypoxia-exposed neonatal porcine islet cells and LPS-treated macrophages. 113 121 Similarly, MSCs transfer mitochondria to chemotherapy-treated endothelial cells, which appears to occur in a unidirectional manner, unlike in T lymphocytes. 120,122 Miro1 is important to TNT formation and its overexpression in MSCs can enhance mitochondrial transfer. 123 The effects of MSC mitochondrial donation are sufficient to rescue cellular respiration, proliferation, and motility in mitochondria-depleted osteosarcoma cells. 124 These effects can be maintained for 45 passages, which highlights the therapeutic potential of MSC-derived mitochondria. 124 Exogenous application of mitochondria isolated from MSCs may also offer therapeutic benefit and are able to protect dexamethasone-treated muscle cells form oxidative stress in vitro. 125  The therapeutic application of these exosomes is yet to be investigated; nonetheless, this may present a viable tool to restore dysfunctional oxidative phosphorylation and ATP synthesis in damaged cells.

| CONCLUSION
The presented studies evidently demonstrate that MSCs exhibit anti-

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
R.S.: conception and design, manuscript writing, final approval of manuscript. K.N.: conception and design, manuscript writing, financial support, final approval of manuscript.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.