Cellular and molecular roles of reactive oxygen species in wound healing

Wound healing is a highly coordinated spatiotemporal sequence of events involving several cell types and tissues. The process of wound healing requires strict regulation, and its disruption can lead to the formation of chronic wounds, which can have a significant impact on an individual’s health as well as on worldwide healthcare expenditure. One essential aspect within the cellular and molecular regulation of wound healing pathogenesis is that of reactive oxygen species (ROS) and oxidative stress. Wounding significantly elevates levels of ROS, and an array of various reactive species are involved in modulating the wound healing process, such as through antimicrobial activities and signal transduction. However, as in many pathologies, ROS play an antagonistic pleiotropic role in wound healing, and can be a pathogenic factor in the formation of chronic wounds. Whilst advances in targeting ROS and oxidative stress have led to the development of novel pre-clinical therapeutic methods, due to the complex nature of ROS in wound healing, gaps in knowledge remain concerning the specific cellular and molecular functions of ROS in wound healing. In this review, we highlight current knowledge of these functions, and discuss the potential future direction of new studies, and how these pathways may be targeted in future pre-clinical studies.

ROS encompass both free-radical or non-radical derivative (peroxides) oxygen intermediates generated by plasma membrane proteins 26 (Fig. 2 ). Physiologically, as well as in pathologies such as wound healing, H 2 O 2 is recognised as the predominant paracrine ROS secondary messenger involved in signalling cascades 27 , 28 . This is due to the fact that H 2 O 2 can quickly and readily diffuse through cell membranes, primarily through aquaporins (AQPs) 29 , 30 , as well as between neighbouring cells through gap junctions – hemichannels composed of connexins which facilitate the transfer of molecules 1–3 kDa large such as ROS between cells to propagate oxidative signals 31 . Additionally, ROS can directly modulate post-transcriptional gene regulation by interacting and reversibly oxidising thiolate groups and methionine 32 , as well as activating mitosis-related signal transduction pathways 8 , 33 , 34 and electron-rich cysteine residues 35 . Fig. 2 Cellular ROS homeostasis. Schematic diagram depicting the various ROS-generating pathways occurring within cells. At the cell membrane, O 2 .− is converted from O 2 in an NADPH-mediated reaction by NOXs, which is then converted to H 2 O 2 by SOD1. H 2 O 2 can also be produced from O 2 in a Ca 2+ -mediated reaction by DUOXs or by UV radiation or other environmental stressors. Extracellular H 2 O 2 can also be imported into cells through AQPs 3, 8, or 9. Within cells, O 2 .− leaks from the ETC during oxidative phosphorylation (OXPHOS) and is converted into H 2 O 2 by SOD2/MnSOD2 and effluxed out of mitochondria into the cell cytosol. Additionally, H 2 O 2 can be produced in the ER by either ERO1 or NOXs and effluxed into the cytosol, as can H 2 O 2 produced by NOXs within peroxisomes. XO, DAO, DDO, and HAO are also produced in peroxisomes. Within the cell cytoplasm, H 2 O 2 can be detoxified into H 2 O and O 2 as well as .OH by CAT. ER endoplasmic reticulum, SOD superoxide dismutase, AQP aquaporin, CAT catalase, XO xanthine oxidase, DAO D-amino acid oxidase, HAO 2-hydroxy acid oxidase, ERO ER oxidoreductin. The main sources of intracellular H 2 O 2 are NADPH oxidases (NOXs) and dual-oxidases (DUOXs) 36 – 38 , in conjunction with superoxide dismutases (SOD), as well as at the mitochondrial electron transport chain (ETC) 39 , 40 – highlighting an important aspect of mitochondria within wound healing 41 . Collectively, NOXs and the ETC generate roughly 85% of H 2 O 2 , with the remaining production deriving from oxidases in the endoplasmic reticulum (ER) and peroxisomes, as well as from cumulative environmental stressors such as UV or ionising radiation 8 , 40 , 42 , 43 . Additionally, membrane-bound NOXs are also responsible for producing .O 2 .− utilised in antimicrobial activities 44 . Other ROS are produced by cytosolic enzymes such as cyclooxygenase 45 , or during lipid metabolism within peroxisomes 46 . As previously mentioned, whilst low to moderate physiological levels of ROS are beneficial for several processes of wound healing pathophysiology, excess ROS can be deleterious. To counteract these harmful effects, a variety of antioxidant enzymes play vital roles in maintaining ROS levels, termed redox balance. These include peroxiredoxins 47 such as catalase (CAT) 48 , glutathione peroxidases 49 , 50 , and mitochondrial nicotinamide nucleotide transhydrogenase (NNT) 51 , which act as ‘sinks’ to remove H 2 O 2 and maintain non-deleterious physiological levels. In addition, SOD, of which there are four isoforms in humans, are antioxidant metalloproteinases which regulate levels of O 2 .− . In particular, the mitochondrial SOD (MnSOD/SOD2) converts .O 2 .− into H 2 O 2 , which is less reactive than .O 2 .− and so can readily be used for cellular signalling. As SOD2 is induced by hypoxia and subsequent HIF-1α activation, it is highly upregulated after wounding 52 , 53 . Oxidative stress is the state induced by an overbalance in the form of excess ROS, and can be a causative factor in chronic wound formation 15 , as well as in the pathogenesis of other diseases including cancers, cardiovascular diseases, Parkinson’s, obesity, and other clinically-relevant age-related diseases 54 – 57 . In the context of wound healing, oxidative stress can lead to the existence of a prolonged pro-inflammatory environment as well as dysregulated re-epithelialisation, as discussed in the following sections 58 . Finally, another important free radical is nitric oxide (NO), which is a reactive nitrogen species (RNS) involved in vascularisation, inflammation, and antimicrobial activities in wound healing 59 – 61 . Most importantly with regards to wound healing, NO plays an important role in pathogen clearance during the inflammatory stage, and this NO is produced by the inducible nitric oxide synthase (iNOS) isozyme 62 , 63 . Here, NO targets both gram-negative and -positive bacteria through aberrant peroxidation and the production of ONOO - , although this can be hindered by its short half

Macrophages play important roles within the wound healing process, including in antimicrobial activities, as well as inflammation, angiogenesis, anti-inflammation, re-epithelialisation, and tissue resolution 74 . ROS-induced HIF-1α stabilisation leads to the activation of macrophages in the early stages of wound healing, and promotes metabolic reprogramming towards glycolysis 75 , 76 , as well as increased angiogenesis 76 . Importantly, both NOX1- and NOX2-produced ROS are required for the activation and differentiation of monocytes into proinflammatory M1 and anti-inflammatory M2 macrophages 77 . Additionally, in atherosclerotic lesions, NOX4-produced ROS drives monocyte and macrophage cell death 77 – a vital step required to prevent prolonged inflammation in wound healing 78 . Although NOXs are essential for macrophage activation, they have been shown to be dispensable for M1 macrophage-mediated proinflammatory cytokine production 79 . Instead, pro-inflammatory cytokine production and inflammasome activation in macrophages predominantly relies on the regulation of the Nrf2 response 80 , which can be primarily activated by glutathione or thioredoxin systems, as well as to a lesser extent NOXs 81 . Other important pro-inflammatory signalling pathways regulated by ROS – and in particular H 2 O 2 – include p38-MAPK-mediated NF-κB/HIF-1α 82 , 83 , and JAK-STAT pathways 84 . Monoamine oxidases (MAOs) – a mitochondrially-located enzyme responsible for catalysing the oxidative deamination of H 2 O 2 85 – is upregulated by the M2 macrophage-activating IL-13 and IL-4, or LPS signals. This process is mediated through JAK signalling pathways and is thus important in anti-inflammation and re-epithelialisation during wound healing 86 . Indeed, MAO inhibitors significantly reduced H 2 O 2 levels and NF-κB/TNFα activation to impair apical migration and proliferation of junction epithelium in a rat chronic wound model 87 . Alternatively, DUOX-induced ROS stimulated the activation of macrophages and promoted epithelial proliferation in a JNK-dependent manner during Drosophila epithelial disc healing 88 . Of note, whilst many studies have demonstrated the importance of ROS and macrophage function in other pathologies and physiological contexts, few have specifically investigated this link within wound healing pathophysiology 89 . Importantly, the majority of studies in this area used the oversimplistic M1 and M2 macrophage classifications, whereas in recent years advancements have been made to study the more elaborate classifications of macrophages in the general immunology setting 90 , and this should thus be applied to the macrophages in wound healing setting 91 . Single-cell RNA-sequencing (scRNAseq) and other advanced omics-based techniques have in recent years significantly powered the investigation of the roles of specific cell lineages, including macrophage lineages, in different stages of wound healing 76 , 92 . As such, future investigations seeking to elaborate on the roles of ROS dynamics in wound healing would greatly benefit from the utilisation of these techniques.

ROS in platelet aggregation and angiogenesis Moderate amounts of ROS (up to a 40% increase) are required for the reduction in platelet adhesion to collagen surfaces and thus platelet activation 102 , 103 . In light of this, and conversely, whilst ROS is known to accelerate platelet function in wound healing, transfer of platelet-derived mitochondria into diabetic mice improved wound healing in part by preventing the overexpression of ROS 104 . Importantly, H 2 O 2 induces the recruitment of vascular smooth muscle cells to the wound site 11 , 105 . As previously mentioned, NO plays an important role in angiogenesis during wound healing. Here, elevated NO production – as a result of increased activation of NOXs, in particular NOX4 – leads to the stabilisation of HIF-1α and thus promotion of endothelial cell (EC) survival, migration, differentiation, and therefore neovascularisation 64 , 106 – 108 . Highlighting this, near-infrared (NIR)-triggered NO production supressed the proteasomal degradation of HIF-1α. Here, by preventing the interaction of HIF1-α with E3 ubiquitin ligases, both VEGF and CD31 expression was enhanced in ECs, coinciding with increased cell proliferation and migration – collectively accelerating wound healing in diabetic mice 64 . H 2 O 2 produced by NOX4 also activated both the TRPM2 109 , and SERCA2 channels 110 to promote Ca 2+ uptake and thus improve EC activity (Fig. 4 ). In wound healing and other hypoxic-state pathologies such as brain ischemia, these ROS-derived effects on ECs are associated with phosphorylation-dependent activation of various signalling molecules including those of ERK, c-JUN, MAPK, AKT, SMAD, and JNK 111 . Fig. 4 ROS and endothelial cell function. Schematic depicting how H 2 O 2 produced by NOX4 increases Ca 2+ uptake into endothelial cells through elevated SERCA2 and TRPM2 channel activity, subsequently leading to increased endothelial cell division and migration – thereby promoting angiogenesis. Finally, production of O 2 · − by both NOX2 and NOX4 also leads to the upregulation of VEGF 110 , 112 . In particular, NOX2 was demonstrated to stimulate VEGFR2 and angiogenesis in wounds through the activation of NF-κB by 2-deoxy-D-ribofuranose 1-phosphate (dRP) – an intermediate of pyrimidine metabolism. This NOX2-derived ROS was primarily generated by both platelets and macrophages 13 , 113 .

A balanced state of oxidative stress is essential for normal wound healing. While physiological levels of ROS are required in the normal transition between wound healing phases, as previously described, an overproduction of ROS has deleterious effects and can hinder wound healing 159 . Multiple molecular mechanisms can explain this effect. For example, healing can be hindered by increased tissue damage, via opposing effects of cytokines such as VEGF and TNFα 160 , 161 . Excessive ROS can also alter and degrade extracellular matrix proteins and impair the function of keratinocytes and fibroblasts 162 . Diabetic wounds, another category of hard-to-heal wounds, are complex and multifactorial. Their aetiology consists classically of a triad of neuropathy, impaired vascularisation and higher susceptibility to infection. These wounds are also notoriously affected by tissue injury after prolonged hypoxia and excessive oxidative stress 93 . It is thus not surprising that targeting ROS has emerged as a potential therapy for hard-to-heal wounds, similar to other diseases, such as cancer 163 , 164 , neurodegenerative diseases 165 , T cell-mediated autoimmune diseases 166 , inflammatory skin diseases 167 , and others. Specifically regarding cancer, ROS play similar pleiotropic roles as they do in wound healing and chronic wound pathogenesis. Depending on the type of cancer and stage, this can include hypoxia-related ROS functions and signalling pathways such as PI3K/AKT or MAPK/ERK pathways, among others, to promote proliferation, migration, or angiogenesis. Similarly, tight regulation of ROS levels is required for cancer progression and can thus also be potentially targeted therapeutically 164 . Another factor to take into account is that of senescence, which is the phenomenon of cell cycle arrest and the inhibition of cell proliferation, and is a hallmark of several age-related pathologies, including in the pathogenesis of some chronic wounds 168 . ROS accumulation and oxidative stress can accelerate senescence in both fibroblasts 169 and endothelial cells 170 , whilst UV-induced ROS upregulation additionally increases senescence and photoageing in skin 171 . Diverse strategies have emerged to modulate ROS in wound healing, namely the use of antioxidant materials such as N -acetyl- l -cysteine (NAC) 172 or enzymes which either increase local perfusion such as glucose oxidase, or clear free radicals through SODs 173 , 174 . Biocompounds that target ROS have also been implicated in improved wound healing, mediated by their effects on perfusion, cell migration, and ROS suppression. Examples of these molecules are Resolvin E1, PDGF, Galectin-1, Alpha-arbutin and Nicotinamide 175 – 179 . Nanoparticles have also been developed to improve wound healing, mainly via ROS scavenging, anti-inflammatory and antibacterial effects, and applied to several in vitro and in vivo models 180 – 184 , as well as other molecules 185 – 188 . On the other hand, increased angiogenesis has been demonstrated when wounds were treated topically with H 2 O 2 189 and both hyperbaric and topical oxygen led to accelerated wound healing 190 – 192 . The array of both pro- and anti-ROS treatment stratagies that have been applied to the study of wound healing are described in detail in Table 1 . Table 1 Molecules targeting ROS in wound healing Category Material Effect Model Ref Antioxidants NAC Reduces ROS levels and improves cell migration and proliferation Cultured human gingival fibroblasts in a high-glucose environment 172 Enzymes Glucose oxidase Increases perfusion (via NO); collagen formation Diabetic mice with full-thickness wounds, applied in wound dressings 173 Superoxide dismutase Clearance of free radicals Hydrogels used in diabetic rat models with full-thickness wounds 174 Bio compounds Resolvin E1 Promotes intestinal wound repair (via CREB, mTOR, Src-FAK) Murine biopsy-induced colonic mucosal wounds 179 PDGF Increases NO (higher perfusion); increased angiogenesis and cell migration Rat model with excisional wounds, mice lacking PDGF receptors/ligands 175 , 176 Galectin-1 Effect on myofibroblast function and signalling with the release of ROS (via NOX) Mice injected with recombinant Galectin-1 protein 177 Alpha-arbutin Promotes healing via upregulation of IFG1R Cultured human dermal fibroblast 178 Nicotinamide Suppresses ROS; increases cell motility Cultured human HaCaT keratinocytes 198 ROS intermediates Topical H 2 O 2 Converts into available O 2 , increase angiogenesis Guinea pigs with ischemic wounds 189 Oxygen Hyperbaric O 2 Reduces wound hypoxia; increases fibroblast proliferation, angiogenesis and accelerated wound healing Diabetic mouse model, in vitro model, patients (with diabetic and miscellaneous wounds) 190 – 192 Topical O 2 Reduces wound hypoxia; accelerates wound healing Patients, meta-analyses 199 Nanomaterials Copper-based nanoenzymes ROS scavenging at low concentrations; promotes re-epithelialization and granulation Murine di

The online version contains supplementary material available at 10.1038/s42003-024-07219-w.