Xanthine Oxidoreductase Function Contributes to Normal Wound Healing
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Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H2O2, 0.15%) or allopurinol (30 µg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H2O2 and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H2O2 restored wound healing in tungsten-fed mice. In vitro, nitrite and H2O2 both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.
Chronic, nonhealing wounds arise from diverse etiologies such as diabetes, venous stasis and pressure. Wounds occur in 12–25% of diabetic patients and contribute to a high incidence of limb loss and death (1). Despite improvements in glycemic control, antibiotics, and wound care adjuvants, nonhealing wounds continue to present a formidable problem. The underlying molecular mechanisms of impaired wound healing are poorly understood despite extensive knowledge of the normal wound repair process. Efficient wound repair involves many cytokines, chemokines and growth factors that regulate the inflammatory and regenerative phases of wound healing (2). Unfortunately, novel therapies targeting these factors, such as topical platelet-derived growth factor, have had limited success (3).
Inflammation is a necessary response to tissue injury, leading to the production of nitric oxide (NO) and reactive oxygen species (ROS) and reactive nitrogen species (RNS) that mediate oxidative stress (4). Recent evidence demonstrates that physiologic oxidative stress is essential for normal wound healing (5,6). Redox signaling has been reported to play an important role in antibiosis, hemostasis, inflammation, re-epithelialization, angiogenesis and growth factor modulation (7,8). However, an overabundance of inflammatory by-products has deleterious effects on the wound (4). For example, ROS contributed to the failure of wound healing in a rodent ischemic flap model (9) and high doses of topical hydrogen peroxide (H2O2) delayed wound repair by reducing collagen deposition and angiogenesis (10). Achieving the ideal balance of ROS/RNS in damaged tissues may determine wound healing efficiency.
Wound repair also depends on NO. NO synthesis in wounds occurs through the arginine-NO synthase (NOS) pathway (11) and modulates inflammation, chemotaxis, antibacterial defenses, collagen production and angiogenesis (12, 13, 14). NO deficiency has been implicated in the delayed wound healing in diabetic rodent models (15,16), where NOS was downregulated. In addition, arginase I uses L-arginine to produce polyamines and proline, both essential for collagen synthesis and cell proliferation, and is induced and competes for the NOS substrate (16, 17, 18). Inducible NOS (iNOS) or endothelial NOS (eNOS) deficiency both delayed wound closure in normal mice, whereas restoration of NOS activity in these mice improved healing (19,20), supporting an essential role of NO in wound repair.
Xanthine oxidoreductase (XOR) is a homodimeric enzyme that metabolizes xanthine and generates ROS. It is also a potent nitrite reductase, converting nitrite back to NO (21,22). This pathway is especially efficient in hypoxia, a condition that reduces NOS-dependent NO production. We and others have reported that nitrite can serve as a source of NO through XOR in models of vascular injury and pulmonary hypertension (23,24). XOR has been detected in skin, where it is involved in ROS production in response to lipopolysaccharide (LPS) (25) and ultraviolet irradiation (26). Thus, we hypothesize that XOR participates in normal wound healing through ROS/RNS and NO production. We used a murine excisional wound-healing model and manipulated XOR activity (27,28) to examine its role in normal wound repair.
Materials and Methods
Excisional Wound Healing Model
All procedures conformed to the Guide for the Care and Use of Laboratory Animals (29) and the policies of the Institutional Animal Use and Care Committee of the University of Pittsburgh (protocol #1104675A). Male C57BL/6 mice (8–12 wks old; The Jackson Laboratory, Bar Harbor, ME, USA) were anesthetized with Nembutal (70 mg/kg, Abbott Labs, Chicago, IL, USA) and isoflurane. After shaving, a 1.5 × 1.5-cm excisional wound was created on the back of each mouse and then covered with bio-occlusive dressings (Systagenix, Quincy, MA, USA). Wound area was measured by acetate tracings every other day until wound closure. The areas were calculated using MetaMorph® (Version 126.96.36.199; Molecular Devices, Inc., Sunnyvale, CA, USA). Wounds were also collected at earlier time points for protein and immunohistochemical analyses.
Dietary and Topical Wound Treatments
Tungsten-enriched diet (#960350; MP Biomedicals, Irvine, CA, USA) was started 2 wks before wounding to optimize molybdenum replacement in XOR and maintained thereafter. Allopurinol (100 mg/kg/day; Sigma-Aldrich, St. Louis, MO, USA) in drinking water, sodium nitrite (300 mg/L in deionized water; Sigma-Aldrich) or nitrite-free diet (Harlan Teklad amino acid diet, TD 99366; Harlan, Indianapolis, IN, USA) was initiated 1 wk before wounding and continued.
Topical H2O2 was applied to the wound as a 0.15% H2O2 solution (Thermo Fisher Scientific Inc., Waltham, MA, USA) in normal saline, and the wound was covered. Topical allopurinol (30 µg/wound) was similarly applied to each wound. Treatment was initiated immediately after wounding and continued every other day.
Western Blot Analysis
Wound samples were collected and divided into the granulation tissue and the wound edge. Skin adjacent to the wound was also collected. Samples were homogenized in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) and quantified using a Pierce® BCA Protein assay (Thermo Fisher Scientific). Western blot analysis for XOR (rabbit monoclonal, 1:5,000; ab109235; Abcam, Cambridge, MA, USA), iNOS (rabbit polyclonal, 1:200; ab15323; Abcam) or arginase I (mouse monoclonal, 1:2,000; BD Biosciences, San Jose, CA, USA) was performed using horseradish peroxidase-linked goat anti-rabbit or antimouse secondary antibody (1:10,000; Thermo Fisher Scientific). The membranes were developed by using SuperSignal® West Pico Chemiluminescent #34080 (Thermo Fisher Scientific).
Wounds were collected on d 7 or at wound closure and fixed in 2% paraformaldehyde, cryoprotected in 30% sucrose, embedded in OCT (Tissue Tek®; Sakura Finetek, Torrance, CA, USA) and sectioned (7 µm). Sections were treated with rabbit polyclonal anti-XOR (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-collagen I (1:200; Abcam), monoclonal anti-Ki67 (1:200; Abcam) or rat monoclonal anti-CD31 (1:50; BD Biosciences) antibody followed by goat anti-rabbit 488 or goat anti-rat Cy5 at 1:1,000 (Invitrogen [Thermo Fisher Scientific]). Nuclei were counterstained with Hoechst 33325 (2 µg/mL, Sigma-Aldrich). Images were collected using the Fluoview® FV1000 confocal microscope (Olympus, Center Valley, PA, USA).
Wound sections were stained with CD31, and two confocal images of the wound granulation tissue were obtained for each section. Wound angiogenesis was calculated as the number of CD31-stained lumens with ImageJ (version 1.45s; National Institutes of Health, Bethesda, MD, USA) and as the percent area of CD31 staining using MetaMorph®.
XOR and Aldehyde Oxidase Activity
XOR activity was quantified as described (23) via HPLC with electrochemical detection. Briefly, endogenous uric acid (UA) was removed by using a Sephadex G-25 column (GE Healthcare, Waukesha, WI, USA). Samples were then treated with oxonic acid (2 mmol/L) to inhibit uricase. XOR activity was quantified by UA production after addition of xanthine (75 µmol/L). Total XDH activity was assessed by exposure to NAD+ (0.5 mmol/L) and pyruvic acid (5 mmol/L). The specificity for XOR activity was verified by allopurinol inhibitable UA formation. Aldehyde oxidase (AO) activity was measured by incubating tissue homogenates with the AO substrate 4-(dimethylamino)cinnamaldehyde (DMAC) (25 µmol/L in potassium phosphate [KPi], pH 7.8, and at 25°C) and monitored for a decrease in absorbance at 398 nm.
Wound ROS/RNS Measurement
Total ROS/RNS was quantified by using the OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) per instructions. Homogenized wound tissues (50 µg) were assayed for ROS/RNS by the conversion of dichlorodihydrofluorescein to 2′,7′-dichlorodihydrofluorescein diacetate.
In Vitro Studies in Keratinocyte Cells and Endothelial Cells
Human epidermal keratinocyte cells (KCs) (PCS-200-011; ATCC, Manassas, VA, USA) were cultured in Dermal Cell Basal Media (PCS-200-030, ATCC) plus Keratinocyte Growth Kit (PCS-200-040, ATCC). KCs were differentiated with 1.2 mmol/L of calcium. Whole cell lysates were used for Western blot analysis for XOR (1:100; Santa Cruz). For immunocytochemistry, KCs were cultured on glass slides, fixed with 2% paraformaldehyde, blocked in 2% BSA in PBS and then incubated with rabbit polyclonal anti-XOR (1:100; Santa Cruz) and mouse monoclonal anti-K10 (1:100; Abcam) followed by secondary goat anti-rabbit or antimouse antibody (1:1,000). Nuclei were counterstained with Hoechst 33325 (2 µg/mL) (Sigma-Aldrich). Images were acquired on the Fluoview® FV1000 confocal microscope (Olympus).
Human dermal microvascular endothelial cells (ECs) (VEC Technologies; Rensselaer, NY, USA) were cultured as described (30) and low passage was used. Briefly, cells were grown in a 1:1 mix of MCDB131 (VEC Technologies) and Dulbecco modified Eagle medium (DMEM) with 5% fetal bovine serum (FBS). In proliferation assays, ECs were cultured in DMEM with 1% FBS overnight. Proliferation was measured by using 3H-thymidine as described (31) in 1% FBS medium with nitrite, H2O2, catalase or allopurinol (Sigma-Aldrich). Migration was measured using the scratch assay at 6 h (32) and quantified with ImageJ. For in vitro angiogenesis, ECs were cultured on Matrigel™ (BD Biosciences) as described (30) with and without nitrite, allopurinol, H2O2 or catalase. Tube formation was examined at 6 h, quantifying boxes and number of long tubes (>110 pixels in length) as a marker of healthy tubes.
Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed with a Student t test or analysis of variance by using the SigmaStat 11.0 software (Systat Software Inc., San Jose, CA), and significance was determined at P < 0.05. All pairwise multiple comparisons were performed by using the Holm-Sidak method.
XOR, iNOS and Arginase I Expression in Wound Tissue
Tungsten-Inhibited XOR Activity and Delayed Wound Healing in C57BL/6 Mice
Because oral allopurinol was much less effective in blocking XOR activity in wounds and skin than tungsten, we examined the effect of locally applied allopurinol on wound healing in mice. Topical allopurinol significantly impaired wound healing at nearly all time points compared with wounds from control mice (Figure 2C). Measurement of XOR activity showed that allopurinol applied to the wounds markedly reduced XOR activity by over 70% compared with control wound tissues (156.2 ± 90.5 µU/mg versus 547.4 ± 15.9, respectively; n = 3/group; P = 0.013).
Minimal Expression of Other Mb-Containing Oxidases Was Detected in Wound Tissue
AO and sulfite oxidase (SO) both contain Mb and can be inhibited by tungsten. They can also function as nitrite reductases. Very low levels of AO were detected in the skin and wound tissues by Western blot (Figure 2D). SO, which is primarily mitochondrial, was not detectible in the wounds as well (data not shown). Assay of wound tissues for AO activity showed no detectable oxidation of DMAC (n = 3), supporting the absence of AO activity in wounds. These findings support that the predominant effect of tungsten on wound healing is mediated through XOR.
XOR Inhibition with Tungsten Reduced ROS/RNS in Granulation Tissue
XOR Inhibition with Tungsten Reduced Wound Angiogenesis and KC Proliferation
Tissue sections from healed wounds were stained for CD31 as a marker for ECs (Figure 3B). Wounds from tungsten-treated mice had 25.3% fewer CD31-positive luminal structures versus wounds from control mice (Figure 3C; P < 0.05; n = 6). Wound proliferation was measured by Ki67 staining at d 7. Ki67 was most prominent in the wound edge basilar KCs in control mice (30.7 ± 5.3%, Figure 3D). However, tungsten-treated mice (Figure 3D) had significantly fewer proliferating basilar KCs (9.1 ± 4.8%, P < 0.05).
Topical H2O2 Reversed Tungsten-Induced Delay in Wound Healing and Improved Angiogenesis
To determine if the role of XOR in wound healing was mediated through H2O2 production, wounds were treated topically with 0.15% H2O2 or saline every other day until complete wound closure. H2O2 did not alter wound healing rates in control mice (Figure 3E), but it significantly improved healing in tungsten-fed mice versus tungsten diet alone (18.5 ± 0.7 versus 21.0 ± 0.7 d; P < 0.01), approaching the rates in control mice. H2O2 treatment of wounds in tungsten-treated mice showed a trend toward improved angiogenesis (Figure 3F; P = 0.067).
Dietary Nitrite Manipulations Did Not Alter Systemic Levels of Nitrite or Wound Healing
Mice receiving supplemental sodium nitrite showed no improvement in wound healing compared with controls (17.4 ± 0.3 versus 16.7 ± 0.3 d, P = nonsignificant [NS], n = 5). Similarly, mice on a nitritefree diet showed no change in wound healing versus controls (17.2 ± 0.3 versus 16.7 ± 0.3 d, respectively; P = NS). However, serum nitrite levels remained unchanged in mice receiving nitrite supplementation or depletion compared with regular chow (282.5 ± 35.5 versus 254.8 ± 64.0 versus 199.3 ± 53.9 nmol/L, respectively; P = 0.54, n = 4). The inability to change serum nitrite levels with diet may explain the lack of effect observed.
XOR Mediates KC and EC Function
Because tungsten also reduced wound angiogenesis, the role of XOR in EC function was also examined. By Western blot, cultured human ECs express XOR at baseline (data not shown). There was a trend toward reduced proliferation when ECs were treated with allopurinol (P = 0.064) and catalase (P = 0.073) (Figure 4B), suggesting XOR and H2O2 contribute to baseline EC proliferation. Nitrite significantly increased EC proliferation, and this was reversed with allopurinol, indicating that nitrite effects were mediated by XOR. Treatment with H2O2 also increased proliferation. Similarly, nitrite and H2O2 both increased cell migration (Figure 4C). Allopurinol did not alter migration but nitrite could enhance XOR-mediated NO production to increase migration, suggesting that XOR did not regulate baseline migration but could be used to improve migration with nitrite. EC tubing, a measure of angiogenic activity, was inhibited by allopurinol, which reduced box formation and reduced the number of long tubes (Figures 4D, E). Nitrite significantly increased tubing complexity and tube lengths in an allopurinol reversible fashion.
Our investigations identified a novel role for XOR in the normal wound healing process. High levels of XOR expression were detected in the skin and wound edge of normal mice, with most of the expression located within the basilar KCs. This expression was upregulated shortly after wounding and likely coincides with the proliferative activity of the KCs at the wound edge. This enhanced XOR expression is observed throughout the healing process and likely subsides as the KCs stop proliferating and the epidermis matures. We also observed XOR expression in the early inflammatory infiltrates in the wound. XOR inhibition with tungsten significantly delayed wound closure. Local wound inhibition of XOR with topical allopurinol also significantly delayed wound healing. Both of these findings support an important role for XOR in wound repair. The ability to reverse effect of tungsten with topical H2O2 suggests that XOR contributes to wound H2O2 production and is required for normal healing. In vitro studies demonstrated the ability of XOR to mediate pro-angiogenic functions in ECs and proliferation in KCs. These findings together strongly support the contribution of XOR in the normal wound healing process.
XOR represents a mix of xanthine oxidase (XO) and dehydrogenase (XDH) (both are involved in purine metabolism ) and is clinically notable for its role in gout, where metabolism of xanthine to uric acid (UA) results in joint deposition and inflammation. This pathway also yields H2O2 and superoxide, both of which are associated with tissue injury during ischemia/reperfusion (I/R) (33,34). Inhibition of XOR reduces ROSmediated I/R injury (28). In the setting of ischemia and hypoxia, XO predominates and more efficiently generates superoxide, although XDH also possesses partial oxidase activity (35). While detrimental in excess, physiologic levels of ROS are essential in cell signaling (36) and tissue homeostasis and repair (6,7). Most tissue production of ROS has been attributed to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the role of XOR in the generation of physiologic ROS has been underappreciated. More recently, XOR gained attention for its ability to convert nitrite to NO to mediate cytoprotection (23,37). We and others have shown that XOR is required for the beneficial actions of supplemental nitrite in the inhibition of intimal hyperplasia after vascular injury (23) and in reversing pulmonary hypertension (24). XOR expression in skin has been previously reported, linked to the pathogenesis of sunburn and skin inflammation (24,25). Beyond that, little is known about the role of XOR in the skin.
XOR inhibition with dietary tungsten significantly delayed wound closure. Tungsten replaces the Mb within XOR, irreversibly inhibiting its function (21). However, Mb is also essential for other oxidoreductases such as AO and SO. We found low levels of these enzymes in wound tissues. The other inhibitor of XOR that we used was allopurinol, a drug used to clinically treat gout and as an investigative tool in other experiments (23,24). In our studies, dietary allopurinol did not alter wound healing rates and raised concerns that tungsten effects may be mediated by actions on AO or SO. However, we identified essentially no AO or SO expression and no measurable AO activity as well in untreated wounds. We did find that tungsten dramatically inhibited skin and wound XOR activity, while dietary allopurinol only reduced it modestly in the wound edge and granulation tissue. Allopurinol has been used to block XOR activity in other tissues, such as in carotid and pulmonary arteries, but was much less effective in our model. The reduced ability of allopurinol to inhibit XOR in wounds may be due to poor biodistribution in the relatively hypoperfused wound bed and skin. Its bioavailability may also be diminished by renal clearance and the requirement for conversion to its active form, oxypurinol (38,39). In addition, the binding of XOR to endothelial glycosaminoglycans results in resistance to allopurinol (38,39). In contrast, topical allopurinol significantly delayed wound closure and reduced XOR activity and wound ROS/RNS levels. These findings confirm the role of wound XOR function in mediating normal wound repair. They also illustrate the potential ability to target wound XOR by topical treatments.
NO is required for normal tissue repair (12,14), and its deficiency contributes to delayed wound healing, whereas NOS gene transfer restores healing rates to normal (19). Diabetic wounds have reduced NOS expression and NO production (16,18), resulting in delayed wound healing. Arginase I is also upregulated during wound healing (16) for the production of polyamines that are essential for cell proliferation and collagen synthesis but competes with NOS for arginine and can reduce NO production (17). Dietary nitrite can serve as an alternate source of NO through XOR (21,22). This pathway has been shown to be biologically important in the setting of vascular injury and pulmonary hypertension where dietary nitrite reduced the adverse vascular remodeling (23,24,37). On the basis of these reports and the abundance of XOR in skin, we proposed that wound NO production may be increased through XOR-mediated nitrite conversion to NO. Harnessing this local nitrite reductase activity may be an attractive mechanism to increase wound NO production in settings where NOS activity is reduced, such as diabetic wounds (15, 16, 17, 18). In our study, neither dietary nitrite depletion nor supplementation altered healing rates. Measurements of serum nitrite levels revealed that these dietary manipulations did not change serum nitrite levels. A potential explanation for these findings is that systemic nitrite production may contribute to significant serum stores, making it difficult to reduce or elevate systemic levels with diet alone. The poor vascularity of the wounds may also reduce tissue nitrite bioavailability. Thus, no conclusion can yet be drawn about the ability to manipulate XOR-mediated NO production in wound healing. In addition, normal wound healing is efficient and difficult to improve. Future studies will examine XOR in models of impaired wound healing such as diabetes.
Other products of XOR that may contribute to wound healing are ROS. While high levels of ROS in the skin are linked to injury and disease (40,41), physiologic levels of ROS, particularly H2O2, are essential for wound repair (6,8). Overexpression of catalase, which breaks down H2O2, delayed wound healing, whereas physiologic levels of H2O2 applied to a wound-enhanced healing (6). In zebrafish, H2O2 production is upregulated immediately after wounding and promotes the inflammatory phase by rapid leukocyte recruitment (8). The decomposition of H2O2 stalled vascular endothelial growth factor (VEGF)-VEGF receptor signaling and impaired angiogenesis and wound healing (42). In all of these studies, NADPH oxidase was determined to be the source of the H2O2 and other ROS. In the tungsten-fed mice and in topical allopurinol-treated wounds, ROS/RNS levels were significantly reduced, suggesting that a deficiency in oxidants contributes to poor wound healing. In a hypoxic environment, such as the wound bed, the predominant ROS generated by XOR is H2O2 (43). Thus, we applied dilute H2O2 topically on wounds in tungsten-fed mice and restored healing to near normal and improved wound angiogenesis. These findings support that XOR production of ROS/RNS, likely H2O2, mediates normal wound healing that was previously attributed to NADPH oxidase. Our studies do not quantify or discount the role of NADPH oxidase-derived ROS/H2O2 in wound healing but strongly support the important contribution of XOR in the production of oxidants necessary for normal biologic processes. Interestingly, Nam et al. (44) showed that NADPH oxidase inhibition reduced KC H2O2 production at early time points (30 min) but did not alter production at 20 h, supporting an alternate source of H2O2.
Another product of XOR function is UA. UA has been associated with inflammation and has been detected in wound fluids, where its concentrations correlated with degree of impaired healing (45). UA may have direct injurious actions independent of XOR-derived ROS, but this has yet to be determined. We hypothesize that, in the setting of abnormal wound repair, such as in diabetes, XOR may be upregulated and leads to the increased oxidative stress that results in protracted inflammation and poor healing. NO and H2O2 have been implicated in KC proliferation and in the modulation of epidermal growth factor receptor in lung and human foreskin KC (46,47). Similarly, they have also been implicated in wound angiogenesis (5). We found prominent proliferation in the basilar KC lining the wound edge, and tungsten reduced the number of proliferating KCs in the wound. We also observed reduced angiogenesis in wounds from tungsten-treated mice, supporting a role for XOR in wound angiogenesis. Our in vitro studies confirm an important role for XOR in mediating EC proliferation, migration and angiogenic activity. Allopurinol blocked proliferation and tube formation in vitro, indicating that endogenous XOR activity contributes to these key EC behaviors. The ability of nitrite and H2O2 to enhance EC proliferation and migration suggests that both NO and H2O2 production likely mediate the wound-healing properties of XOR in vivo. Kou et al. (48) reported the role of endogenous XO in maintaining VEGF-induced EC survival. It was also reported that NO derived from XOR regulates hypoxia-inducible factor 1-α (HIF1α)- and VEGF-dependent angiogenesis (49). The impaired angiogenesis resulting from XOR inhibition may be due to loss of H2O2-mediated induction of angiogenic VEGF (42). It was also shown that VEGF downstream signaling depends on ROS production (42), again presumably through NADPH oxidase. Future studies will isolate the contribution of XOR-derived ROS from that of NADPH oxidase.
Isolated XOR deficiency in humans is rare and is only documented in a few case reports (50,51). However, Mb cofactor deficiency has been described and is associated with severe refractory seizure activity with childhood fatality (reviewed in ref. ). Global XOR deficiency is fatal, and, thus, no XOR-deficient mouse is currently available to assist in the investigation of XOR function in wound healing. The generation of tissue-specific or conditional XOR knockouts will be extremely helpful and is currently underway.
We provide the first evidence that XOR contributes to normal wound healing. Our data showed that XOR stimulates KC proliferation and wound angiogenesis through the production of ROS. The in vitro effects of XOR on EC behavior are mediated by both NO and H2O2. Future studies are necessary to better define the role of XOR-mediated NO generation in these effects. In addition, it is important to determine how XOR activity is regulated in impaired wound healing such as in diabetes, venous stasis and ischemia. Targeting wound XOR may provide a way to manipulate local ROS and NO production, possibly achieved through topical routes that are extremely attractive in the management of patients with difficult wounds.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We gratefully acknowledge the excellent technical assistance provided by NJ Hundley, SI Zharikov and N Cantu-Medellin. This material is based on work supported in part by the Department of Veterans Affairs, Veterans Health Administration and Office of Biomedical Laboratory Research and Development (ET), through funding from the National Institutes of Health (NIH) (NIH T32 HL098036 to MC Madigan, RM McEnaney and AJ Shukla and NIH R01 HL058115 to MM Tarpey). M Gladwin received research support from NIH grants R01HL098032, R01HL096973 and PO1HL103455 as well as from the Institute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania.
The contents of this manuscript do not represent the views of the Department of Veterans Affairs or the United States Government.
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