Redox dysregulation in the pathogenesis of chronic venous ulceration

Journal Pre-proof Redox dysregulation in the pathogenesis of chronic venous ulceration Oliver TA. Lyons, Prakash Saha, Alberto Smith PII:

S0891-5849(19)31174-8

DOI:

https://doi.org/10.1016/j.freeradbiomed.2019.09.018

Reference:

FRB 14418

To appear in:

Free Radical Biology and Medicine

Received Date: 15 July 2019 Revised Date:

4 September 2019

Accepted Date: 20 September 2019

Please cite this article as: O.T. Lyons, P. Saha, A. Smith, Redox dysregulation in the pathogenesis of chronic venous ulceration, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/ j.freeradbiomed.2019.09.018. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Title: Redox Dysregulation in the Pathogenesis of Chronic Venous Ulceration Authors: Oliver T A Lyons (1,2), Prakash Saha (1), Alberto Smith (1) Affiliations: 1. Academic Department of Vascular Surgery, School of Cardiovascular Medicine and Sciences, BHF Centre of Research Excellence, King’s College London, St Thomas’ Hospital, United Kingdom 2. Basildon and Thurrock University Hospitals NHS Foundation Trust, United Kingdom. Conflict of interest: None Funding: None Abstract In chronic venous ulcers (CVUs), which account for up to 75% of leg ulcers, the inflammatory stage of wound healing fails to down-regulate, preventing progression to proliferation, remodeling and eventual epithelialisation. The roles of reactive oxygen species (ROS) in the oxidative burst and pathogen killing are well known, but ROS also have important functions in extra-cellular and intra-cellular signalling. Iron deposition, resulting from venous reflux, primes macrophages towards a persistent inflammatory response, with ongoing stimulation by bacteria potentially playing a role. Generation of excessive ROS by activated inflammatory cells causes tissue destruction and disintegration of the dermis, and then at later stages, a failure to heal. Here, we review the evidence for ROS in CVU formation and in normal and delayed healing. We also discuss how ROS modulation might be used to influence the healing of these complex wounds, which cause long-term morbidity and are associated with a significant financial burden to healthcare systems. Keywords: Venous ulcer, venous hypertension, venous insufficiency, wound healing, ROS, macrophage, iron, haemosiderin, hydrogen peroxide

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Clinical condition Chronic venous ulcers (CVUs) usually develop in the medial gaiter region of the leg and account for around 75% of leg ulcers. They affect approximately 1% of the population, rising to over 3% in people aged over 65. Treatment of this condition is currently estimated to consume ~2% of healthcare budgets in Western countries. Major risk factors for chronic venous disease include older age and obesity, both of which are increasingly prevalent in the Western world.[1-5] Venous drainage of the lower extremities relies on several factors including the normal function of the calf muscle pump (including its venous valves) and an unobstructed return of blood back to the heart.[5] Failure of any component of the calf pump (most commonly vein dilatation and valve dysfunction) results in chronic venous insufficiency (CVI), which consists of venous reflux and ambulatory venous hypertension, in which the pressure in the veins remains high during exercise.[1, 2, 5] CVI is caused by venous reflux, or obstruction, or a combination of both. Reflux is most commonly caused by varicose veins and/or damage to valves from previous thrombosis, but more rarely a primary genetic problem may be to blame.[2, 6] Obstruction is most often the result of a previous deep venous thrombosis leading to post-thrombotic syndrome.[1, 2, 7] CVI manifests as pain, oedema, hyperpigmentation, skin damage and in some patients, chronic intractable ulceration of the lower limb.[1, 2, 8] CVU size correlates with the degree of underlying venous reflux.[5, 9] The mainstay of current treatment for CVUs is compression bandaging to reduce ambulatory venous hypertension.[5, 8, 10-12] This treatment is associated with an increase in the number of nutrient capillaries, while the diameter of the capillaries and the dermal papillae diminishes.[13] In addition to compression bandaging, ablation of an incompetent superficial vein (most commonly the great saphenous) speeds ulcer healing and can prevent recurrence.[14] These treatments are costly but not always effective and patients often require a prolonged time period in compression bandaging to produce ulcer healing (approximately 3months).[15] CVUs also frequently recur after healing, partly because compression therapy does not correct the underlying causes of the disease, and existing hypertensive skin damage is not reversed.[5, 14]

Pathophysiological processes associated with development of CVUs Venous ulceration is frequently precipitated by minor trauma followed by rapid and widely spreading ulceration. The mechanisms of this rapid progression of venous ulceration are not fully understood but they likely overlap with those that lead to poor or dysregulated wound healing. The mechanisms of normal wound healing involve defined stages including: i) haemostasis (several hours); ii) inflammation (typically 1-3days); iii) proliferation (tissue formation, 4-21days); and iv) tissue remodeling (21days-1year).[1620] How these processes become dysregulated and give rise to delayed healing and CVU is poorly understood.[21, 22] In the absence of healing, remaining layers of skin necrose to produce full-thickness skin loss; ulceration may enlarge at an alarming rate, eventually extending circumferentially around the leg. Necrosis extends into the subcutaneous fat, superficial fascia, deep fascia, muscles, and may even reach the 2

underlying periosteum (Figure 1). The pathophysiological processes underlying the progression from ambulatory venous hypertension to skin damage remain unclear, and it is also uncertain why some patients progress on towards ulceration whilst others (with similar reflux and/or obstruction) do not.[23, 24] In addition, some patients tend to heal while in other groups ulcers remain recalcitrant to compression therapy. What appears clear, however, is that in chronic wounds such as CVUs, the inflammatory stage is not down-regulated, and there is a failure of progression to proliferation and remodelling.[22] Venous hypertension is thought to drive sequestration of inflammatory cells (neutrophils and monocytes) into the microcirculation of the leg (termed ‘white cell trapping’), which may be causative of CVU.[25-27] Alternative hypotheses include recurrent ischaemiareperfusion injury, alterations in fluid shear stress, and recurrent endothelial distension due to elevated venous pressure, all of which trigger a proinflammatory state.[28, 29] Microcirculatory arterio-venous shunts develop which, whilst insufficient to raise venous pressure and lead to varicose veins, may reduce the ability of the capillary bed to deliver oxygen to ulcerated skin, but whether this contributes to CVU pathogenesis is yet to be confirmed.[30, 31] Analysis by electron microscopy shows an increased mast cell content around arterioles and post-capillary venules in more severely affected patients, and increased macrophage numbers around post-capillary venules in those with active ulceration.[32] Experiments with a rat mesenteric vein occlusion model suggest that a reduction in venous blood flow is sufficient to trigger leukocyte transmigration, and that this effect is enhanced by venous hypertension.[29] Ligation of several large veins in the rat has been used to produce persistent hindlimb venous hypertension.[29] In this model, leucocyte levels were increased in the tissues (including skin) subjected to venous hypertension.[33, 34] Activated inflammatory cells are prodigious producers of reactive oxygen species (ROS) and the generation of excessive ROS (e.g. superoxide and hydrogen peroxide) by these invading leukocytes could provide the stimulus for a variety of pathological mechanisms that result in tissue destruction, rapid disintegration of the dermis and ulcer formation.[3539] Few treatments targeting the pathophysiology of the ulcer have been developed. A better understanding of the pathophysiology of CVU formation and healing may, however, lead to novel treatments to prevent CVU formation, or enhance healing. Here we review the evidence regarding a role for ROS in the regulation of CVU formation and healing – both its roles in promoting normal healing, and how excessive ROS may lead to chronic nonhealing wounds. We discuss how modulating ROS may influence the healing of these complex wounds. ROS in chronic venous ulcer formation All cells produce ROS, such as superoxide (·O2−), hydrogen peroxide (H2O2), hydroxyl radicals (OH·), peroxynitrite (ONOO−) and other reactive species, during normal metabolic processes.[37] Nitric oxide (NO·), produced by nitric oxide synthase (NOS) metabolism of L-arginine, combines with superoxide to form peroxynitrite, a reactive nitrogen species.[15, 22] Low levels of ROS have important physiological roles, such as cellular signaling in response to stimuli (growth factors, hormones, cytokines, sheer stress) and other cellular events such as adhesion, angiogenesis, cell migration, contraction and proliferation.[40-42] Cells that are pathologically stimulated, however, increase their production of ROS, creating excessive oxidative stress in their local 3

environment that damages proteins, lipids, and DNA, inducing senescence and apoptosis. In a bid to maintain redox homeostasis cells produce anti-oxidants such as glutathione and antioxidant enzymes, such as superoxide dismutase, that prevent excessive oxidative damage.[37, 43] An imbalance in pro- and anti-oxidant mechanisms leads to redox dysregulation. Despite conflicting reports, during the early stages of chronic venous disease serum markers of oxidative stress have been demonstrated to be raised (specifically decreased catalase activity and thiol levels, and increased malondialdehyde-bound protein, a byproduct of lipid peroxidation, and protein carbonyls).[44, 45] Circulating estradiol, homocysteine and vascular endothelial growth factor appear, however, to be the serum markers most consistently associated with primary chronic venous insufficiency.[46] Blood harvested from the site of varicose veins has increased IL-6, IL-8, and MCP-1 concentrations compared to blood taken from the arm.[47] Levels of ROS and ROS byproducts (as a marker of ROS levels) such as lipid peroxides and isoprostane are also significantly raised in the environs of CVUs compared with acute wounds,[39, 48, 49] while both NOS and arginase levels are increased in CVUs compared with normal skin. NO· overexpression in CVUs may be involved directly (or through production of peroxynitrite) in the tissue destruction seen in CVUs[22, 50], and in a study of biopsies taken from patients with a CVU, superoxide and hydroxyl radicals, but not H2O2, were the prevailing ROS species [39] Oxygen is essential for wound healing as the energy needed during biosynthesis, intracellular transportation and cell movement relies on adenosine triphosphate, which is most efficiently synthesised aerobically. During the early, inflammatory phase of wound healing, NADPH-linked oxygenase (Nox) expressed by inflammatory cells produce high amounts of oxidants, consuming large amounts of oxygen.[51] Soon after wound initiation, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species. ROS levels (e.g. H2O2) increase with age and, along with an increasing prevalence of venous insufficiency, may partly explain the increased risk for CVUs with advancing age.[52]

The impact of Iron and the Fenton reaction The mean iron concentration in the skin around CVUs is greatly elevated compared with control regions (non-ulcerated leg skin, and healthy skin from the forearm).[53] It is possible that iron deposition may form part of the mechanism of CVU formation, and also their failed healing.[49, 54] Skin biopsies taken from patients with the early stages of chronic venous insufficiency do not show iron deposition (pigmentation at early stages is mostly attributable to melanin) and most patients at this stage will not go on to develop a CVU.[55, 56] In contrast, patients with advanced stages of insufficiency show increased iron deposits, consistent with the hypothesis that increased iron forms part of the pathogenesis of CVUs.[55] Hereditary haemochromatosis is an iron-storage disease with genetic heterogeneity (most commonly caused by mutations in the HFE gene) but with a final common metabolic pathway that results in inappropriately low production of the hormone hepcidin. Its clinical manifestations relate to iron deposition in organs such as the liver, pancreas, joints, skin (including CVUs), and the heart, involving the production of ROS.[57, 58] The most common HFE gene mutations (C282Y and H63D) are associated with an increased risk of CVU formation and a reduction in the onset of a CVU by approximately 10years.[16] The increased risk of skin lesions and the earlier onset of CVUs in those with haemochromotosis are consistent with a potential role for skin iron deposits in CVU formation. 4

Suggested mechanisms by which iron deposition could lead to (or exacerbate) a CVU include promoting the generation of free radicals (from H2O2, via the Fenton reaction) and activation of proteolytic hyperactivity by metalloproteinases or the down-regulation of tissue inhibitors of matrix metalloproteinases (TIMPs), and the promotion of a proinflammatory macrophage phenotype.[16, 39, 59] Experimental studies of CVU pathogenesis have, until recently, been limited by the lack of an effective model of chronic skin wounds. In a novel murine model, however, iron overloading of macrophages (that was also found to occur in patient CVUs) induced a macrophage population with a proinflammatory ‘M1-like’ activation state.[39, 59] Prolonged venous hypertension resulting in venous dilatation and the passage of red blood cells through the endothelium into the interstitium results in cell breakdown and conversion of haemoglobin to haemosiderin.[60] Macrophages in CVUs (in patients) expressed high levels of the haemoglobin scavenger receptor CD163, which mediates the endocytosis of haemoglobin–haptoglobin complexes, leading to accumulation of intracellular iron.[39] This macrophage population perpetuates inflammation via TNF-α and ROS production. Furthermore, the production of ROS by iron-loaded macrophages correlates with an increased level of the ageing markers phosphorylated histone H2AX and INK4A in fibroblasts harvested from CVUs and skin lesions. Induction of DNA damage and a p16(INK4a)-dependent senescence program in locally resident fibroblasts is suggested to eventually lead to impaired wound healing and could also contribute to chronic venous ulceration.[39, 59] Bacterial colonisation and ROS drive inflammation in chronic wounds LIGHT (or Tumor Necrosis Factor Superfamily Member 14) knockout mice exhibit impaired wound healing, with some similar characteristics to those seen in man.[61] These mice show elevated levels of genes involved in oxidative and nitrosative stress.[52] Since LIGHT is also involved in the resolution of macrophage-induced inflammation, this model may replicate the persistent macrophage activation seen in CVUs.[61] In these mice, application of treatments to inhibit the antioxidants catalase and glutathione peroxidase (GPx), leading to increased oxidative stress, and introduction of biofilm-forming bacteria, leads to chronic wounds that remain unhealed for weeks, potentially replicating the situation seen in CVUs. Inhibition of GPx (using mercaptosuccinic acid), or catalase (using 3-amino-1,2,4-triazole), or inoculation with bacteria alone did not, however, lead to the formation of chronic wounds, suggesting that it is the combination of these factors that leads to a failure to heal in-vivo.[52] Histological examination of chronic wounds in these mice demonstrated abnormalities in the migrating tongue of epidermis at the wound edge, poorly developed granulation tissue, and abnormal extracellular matrix deposition, similar to the features of CVUs.[52] It is possible that bacterial colonisation of CVUs contributes to the observed ongoing proinflammatory state with associated excessive ROS production. In a large series of microbiological cultures from CVUs, there was growth of organisms from all ulcers, but no correlation was found between changes in ulcer size and the species and amounts of microorganisms cultured.[62, 63] The presence of organisms including pseudomonas aeruginosa and staphylococcus aureus might, however, be associated with poor healing.[63-65] Culture of ulcers without clinical signs of infection is therefore not currently recommended.

ROS in the pathophysiology of ulcer healing 5

Immediately after wounding, vascular injury initiates the formation of a platelet plug resulting in the sealing of a wound. ROS are generated by activated platelets (e.g. via Nox1/2) and regulate blood coagulation, thrombosis and platelet functions.[66-68] Soon after wound formation, H2O2, formed by dual oxygenase, acts as a signaling molecule to recruit leucocytes to the wound and initiate healing.[69-71] In a zebrafish model, knockdown of dual oxygenase 1 decreased H2O2 production around the wound and concomitantly impaired the recruitment of leukocytes to the site of injury.[70] In a murine model, ROS can be detected as early as 4hours after wounding.[52] Neutrophils arrive initially and are followed by monocytes, which differentiate into mature tissue macrophages.[21, 72] Neutrophil activation is accompanied by an oxidative burst, degranulation and a delay in spontaneous apoptosis. The major source of ROS in human neutrophils is NADPH oxidase (NOX), although other ROS sources exist.[73, 74] ROS released by the NADPH oxidase complex may induce the formation of neutrophil extracellular traps, which as well as their well-known role in pathogen defense, are also prothrombotic.[74] Neutrophilproduced ROS may, however, cause excessive inflammation and tissue damage.[75-77] The rate of production of toxic radicals, and hence the adequacy of oxidative killing, is directly proportional to local oxygen tensions.[51] ROS in ulcer healing: angiogenesis, migration and proliferation Coverage of the ulcer with a new layer of keratinocytes is an essential part of wound healing. In the proliferation phase of wound healing, a low H2O2 concentration induces proliferation and migration of keratinocytes via sustained ERK1/2 activation. [78] H2O2 triggers the activation of receptors for epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) inducing the production of transforming growth factor α (TGFα) in fibroblasts.[79-81] H2O2 also stimulates keratinocytes to release vascular endothelial growth factor (VEGF), driving angiogenesis.[72] Feedback mechanisms involving other growth factors, for example Platelet derived growth factor (PDGF), stimulates the generation of H2O2 that can induce cell migration and may have important roles.[82] Oxygen delivery from the capillaries to the cells relies on diffusion. The distance between cells involved in CVU repair and their nutrient capillary is of particular importance, and relies on effective angiogenesis.[19] Production of ROS for the inflammatory stage of wound healing requires sufficient tissue oxygenation, as oxygen is the main substrate for ROS-producing enzymes. The role of ROS signaling in angiogenesis has been well studied; whilst low levels of ROS (e.g. H2O2) stimulate angiogenesis, higher levels (such as those found in CVUs) appear detrimental.[42, 83-85] Fluid exudate from CVUs, especially in those ulcers that heal slowly, inhibits in vitro angiogenesis.[86] In nonhealing CVUs there is a consistently high level of expression of VEGF, at both the gene transcript and protein level, but angiogenesis is depressed in poorly healing ulcers, and an increase in VEGF production may be due to an ineffectual angiogenic drive.[87] In normal wound healing H2O2 stimulates the arrival of monocytes, which differentiate into tissue macrophages, and (amongst other functions) release VEGF, the main blood angiogenic growth factor.[88, 89] In addition to activating signal transduction pathways directly, ROS also activates the secretion of growth factors. In addition, ROS inhibits the enzyme PHD2, which leads to the stabilization of HIF-α and the formation of the active transcription factor HIF, leading to a cellular response to hypoxia, including VEGF production.[90] H2O2 also stimulates the VEGF promoter directly via a HIF-1 independent pathway. Activation of Rac1 generates intracellular ROS, and over-expression of Rac1 downstream of various cytokine receptors increases VEGF expression.[51] Moderate 6

levels of H2O2 upregulate the production of VEGF by keratinocytes and macrophages, resulting in accelerated angiogenesis, [72, 84] and H2O2 is required for the response of vascular smooth muscle cells and mesenchymal cells to signals such as PDGF.[82, 91]

Potential ROS-modifying therapies It is likely that at different stages of wound healing, the optimal levels of ROS vary significantly.[22] Optimal ROS levels (and concentration gradients) may also vary within different regions of the same wound. Whilst low-level, coordinated ROS production is necessary for the healing process, high concentrations (or disco-ordinated production) are detrimental. Excess exogenous ROS may be helpful in decolonising the surface of a wound covered by bacterial biofilm (explaining the benefits of topical application of H2O2 or the strong oxidising agent potassium permanganate), but decreased or more carefully regulated ROS is required in tissues for the co-ordination of angiogenesis, cell proliferation and wound remodeling.[52] There are no methods in clinical use for determining whether ROS activity in a CVU is at a level consistent with ongoing tissue destruction, or for stimulating angiogenesis and proliferation, and it is unclear whether there is a need to increase or reduce ROS levels to achieve CVU healing in any given wound.[22] Existing therapies are targeted towards ‘CVUs’ in general, and do not allow for more personalised treatment, which may explain the inability to demonstrate any efficacy of these therapies. Several existing treatments that are currently in use for CVU healing do however interact with ROS. The use of H2O2 and potassium permanganate aims to increase ROS in the ulcer area with a resultant antibacterial action.[92-96] Several randomised controlled trials have examined the use of peroxide-based preparations, with favourable results,[94, 97] possibly through a positive effect on vascular perfusion.[98]

Several types of honey are used clinically to promote wound healing. Glucose oxidase present in honey generates H2O2 from glucose. This ROS acts as a potent antimicrobial, which, together with the ability of honey to reduce the production of biofilm, may explain the well-documented wound healing properties of this natural product.[99, 100] Although CVUs are frequently colonised by bacteria, this often does not represent a process of active infection [94, 101], and it is possible that honey-stimulated H2O2 production may aid wound healing through mechanisms that rely on the cellular signaling activity of H2O2, leading to regulation of transcription factors (such as NFkB and HIF1α that can regulate angiogenesis, inflammatory cell activity and wound remodeling.[41, 102, 103] It remains to be determined whether this occurs in CVUs. Some randomised controlled trials have suggested benefits of honey dressings in the debridement of CVUs, but these data are insufficient to recommend routine use.

Hyperbaric oxygen therapy, used to decrease wound hypoxia in a small multicenter randomised controlled trial, has led to improved CVU healing in those not responding to standard therapy within 4 weeks.[104-107] Whilst a moderate hyperoxic challenge is suggested to stimulate the production of growth factors and angiogenesis, extreme hyperoxia would be expected to induce mitochondrial apoptosis, growth arrest, and oxidative stress via ROS.[107] N-acetyl cysteine decreases ROS levels (favouring the formation of NO) and has also been suggested as a promising agent for the enhancement of wound healing.[108] 7

In the rat mesenteric occlusion model, treatment with hydroxyl radical scavenger dimethylthiourea (DMTU) or micronized purified flavonoid fraction (MPFF, which reduces leukocyte adhesion and migration after arterial ischemia/reperfusion) attenuated the increases in leukocyte rolling, adherence, and migration, and reduced parenchymal cell death associated with venous occlusion. MPFF treatment also increased the delay between occlusion and the first detection of tissue microhaemorrhages.[29] In a metaanalysis of randomised controlled trials controls, MPFF (Daflon) was found to have accelerated CVU healing.[109] The methodological quality of these trials has, however, been criticised as being generally ‘low’, with risk of bias.[110] TNFα blockade rescues the delayed healing in a murine model of skin wounding on a background of iron overload, which appears to reproduce the phenotype of CVUs.[39, 111] Topical application of infliximab (a monoclonal antibody that binds TNFα, and is in clinical use for this indication), resulted in significant improvement in healing in 12 of 14 previously therapy-resistant CVUs.[112] TNFα is a potent proinflammatory cytokine and may drive the inflammatory macrophage phenotype by an autocrine feedback loop. These findings have yet to be validated in a larger cohort or a randomised controlled trial, but these data show some promise. Generally, most therapies have insufficient evidence of efficacy, safety and cost effectiveness to support their routine use alongside compression bandaging and the treatment of venous reflux.[5, 8, 10, 14, 17, 101, 110] The recent development of murine models of chronic wounds, which replicate to some degree the features of CVUs may, however, facilitate the development of better therapies.[39, 52] Conclusions ROS have important roles in CVU healing, not only in disinfection during the inflammatory phase, but also in migration, proliferation and remodeling during wound healing. Our understanding of the roles of ROS is limited, and further studies are required to elucidate the regulation and dysregulation of ROS in CVUs. Whilst our knowledge of the initiation and amplification of the inflammatory response, a major contributor to ROS, has progressed, we know relatively little about how inflammation is controlled in order to enable progression into the proliferative phase of wound healing. Iron overloading in macrophages appears to be a major environmental cue responsible for the persistence of an unrestrained proinflammatory M1-like activation state, leading to ongoing secretion of ROS and local tissue damage. A better understanding of the pathophysiology of CVU formation and healing might, however, lead to novel treatments that could prevent CVU formation, or enhance healing, by modulating these processes. Diagnostic tests that allow assessment of inadequate or excessive ROS levels would also be helpful.

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Please note that reference to E.Murphy et al 2019 is an epub ahead of print and therefore not provided in full journal stye. DOI is 10.1016/j.jaad.2019.05.030 and PMID is 31103570

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Figure 1 title: Chronic venous ulceration Figure 1 legend: A large venous ulcer is shown on the right medial gaiter area of the leg. Note the background of brown skin discolouration indicating haemosiderin and iron deposition. Characteristically, small islands of skin remain within the ulcerated area, but maybe become undermined and peel away.

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Figure 2 title: Stages and major events in wound healing after minor skin trauma Figure 2 legend: H2O2 is used here to indicate several ROS species; O2–• and OH• may be the predominant ROS species in chronic venous ulcers.[39] See main text for references. The blue line indicates the course of the great saphenous vein; reflux in this vein is a common cause of chronic venous insufficiency, but may be accompanied by reflux in perforating veins, deep veins, the short saphenous vein, and unnamed varicose veins.

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Chronic venous ulcers account for up to 75% of all leg ulcers Pathophysiology poorly understood but likely to be influenced by inflammatory cell activity Generation of excessive reactive oxygen species by activated inflammatory cells can lead to tissue destruction and delayed healing Modulation of excessive ROS generation may prove a useful treatment to promote chronic venous ulcer healing