Published online before print July 19, 2004, doi: 10.1161/01.CIR.0000137969.87365.05
(Circulation. 2004;110:2484-2493.)
© 2004 American Heart Association, Inc.
Molecular Cardiology |
Gene Therapy of Endothelial Nitric Oxide Synthase and Manganese Superoxide Dismutase Restores Delayed Wound Healing in Type 1 Diabetic Mice
From the Departments of Pharmacology and Neurology and the Neuroscience Program (J.-D.L., Y.-Y.W., A.F.C.), Michigan State University, East Lansing; Department of Pharmacology, Guangzhou Medical College, Guangzhou, China (J.-D.L.); and Southwestern Hospital, Third Military Medical University, Chongqing, China (Y.-Y.W., W.-L.F., J.W., A.F.C.).
Correspondence to Alex F. Chen, MD, PhD, Departments of Pharmacology and Neurology and Neuroscience Program, College of Human Medicine, B403 Life Sciences Bldg, Michigan State University, East Lansing, MI 48824-1317. E-mail chenal{at}msu.edu
Received December 11, 2002; de novo received June 3, 2004; accepted June 14, 2004.
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Abstract |
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Background— Nitric oxide (NO) deficiency contributes to diabetic wound healing impairment. The present study tested the hypothesis that increased cutaneous superoxide (O2–) levels in type 1 diabetic mice cause NO deficiency and delayed wound healing.
Methods and Results— Wound healing was markedly delayed in streptozotocin-induced type 1 diabetic mice compared with the normal controls. There were significantly reduced levels of endothelial NO synthase (eNOS) protein and constitutive NOS activity in diabetic wounds, whereas O2– levels were markedly increased. A single regimen of cutaneous gene therapy of eNOS or manganese superoxide dismutase (MnSOD) restored such healing delay, with a concomitant suppression of wound O2– levels and augmentation of both eNOS protein and constitutive NOS activity. Gene therapy of MnSOD also increased cutaneous MnSOD activity. Cutaneous O2– levels were also increased in Ins2Akita diabetic mice. In vitro glucose treatment of cutaneous tissues from normal mice for 24 hours increased O2– levels in a concentration-dependent manner. The enhanced cutaneous O2– levels induced by high glucose in both normal and diabetic mice were abolished by the NADPH oxidase inhibitor apocynin and the protein kinase C inhibitor chelerythrine. Furthermore, ex vivo gene transfer of dominant-negative HA-tagged N17Rac1, which inhibits NADPH oxidase subunit Rac1, significantly inhibited cutaneous O2– formation induced by high glucose in both normal and Ins2Akita diabetic mice.
Conclusions— These results indicate that hyperglycemia augments cutaneous O2– levels, at least in part, via NADPH oxidase and protein kinase C pathways, resulting in impaired wound healing in type 1 diabetic mice. Gene therapy strategies aimed at restoring cutaneous NO bioavailability may provide an effective means to ameliorate delayed diabetic wound healing.
Key Words: wound healing • nitric oxide • superoxides • diabetes mellitus • gene therapy
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Introduction |
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Wound healing impairment in diabetic patients represents a particularly challenging clinical problem for which there is currently no efficacious treatment regimen.1 The etiology of delayed diabetic wound healing is multifactorial, and vasculopathy and neuropathy are major contributors.1 Diabetes is characterized by chronic hyperglycemia associated with significant vasculopathy.2 Sustained hyperglycemia is known to increase vascular superoxide (O2–) production, which inactivates nitric oxide (NO) and causes vascular dysfunction in diabetes.3–5
Compelling evidence indicates that NO plays an integral role in normal wound repairs.6,7 NO promotes processes central to wound healing, including angiogenesis8–10 and migration and proliferation of fibroblasts,11 epithelial cells,12 endothelial cells,8,9 and keratinocytes.10 Consequently, a NO deficiency contributes to wound healing impairment. The NO synthase (NOS) inhibitors, when applied to the wound surface13 or given systemically,14 retard wound healing. In contrast, a systemic administration of the NO precursor L-arginine improves wound repair in normal individuals15 and in aged individuals with impaired healing.16 In agreement with these findings, recent studies with targeted disruption of NOS genes have revealed that the excisional wound closure is delayed by 30% in both endothelial NOS (eNOS) and inducible NOS (iNOS)–knockout mice compared with the wild-type littermates.17,18 Conversely, adenoviral vector– mediated gene transfer of iNOS to the wound site of iNOS-knockout mice completely reversed the delayed healing.18
In streptozotocin-induced mice (a model of type 1 diabetes), impaired wound healing is paralleled by both decreased wound NOS expression and NO levels,19 and L-arginine improves such healing delay,20 suggesting that impaired wound NO synthesis is a key factor for the delayed healing. However, the underlying mechanisms responsible for NO deficiency–induced wound healing impairment remain to be delineated. In the present study, we tested the hypothesis that hyperglycemia impairs cutaneous NO function and wound repair through O2– generation, which may be reversed by gene therapy of eNOS or manganese superoxide dismutase (MnSOD) in type 1 diabetic mice. Our results indicate (1) that cutaneous O2– levels were significantly increased in chemical substance streptozotocin-induced and autosomal dominant mutation in Ins2 gene–induced Ins2Akita diabetic mice, and (2) that decreased eNOS protein and activity contribute to wound healing delay and in vivo gene therapy of eNOS or MnSOD promotes the healing of full-thickness excisional wounds in streptozotocin-induced diabetic mice.
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Methods |
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Induction of Type 1 Diabetes
This study was approved by the animal research committees of Michigan State University and Chinese Third Military Medical University. Male C57/B6 mice (Jackson Laboratories, Bar Harbor, Maine) aged 8 to 12 weeks were injected intraperitoneally with streptozotocin (Sigma Chemical Co) in dissolved sterile citrate buffer (0.05 mol/L sodium citrate, pH 4.5, 45 mg/kg). Streptozotocin or citrate buffer (control) was administered for 5 consecutive days during the first week of the study.21 Blood was collected from the dorsal vein of the mouse hind foot with the use of Microvette blood collection tubes (Microvette CB 300 LH, Sarstedt). Whole-blood glucose levels were measured by a glucose analyzer (2300 Stat Plus analyzer, YSI Incorporated). Mice with a blood glucose level >280 mg/dL were considered diabetic and used for wound experiments.21 Ins2Akita diabetic mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Ins2Akita diabetic mouse is an autosomal dominant mutation in Ins2 gene and is well-known animal model of type 1 diabetes.
Excisional Wound Model
Ten weeks after streptozotocin or citrate buffer injection, mice were anesthetized with ketamine (100 mg/kg IP; Sigma Chemical Co) and xylazine (10 mg/kg IP; Sigma Chemical Co), and the dorsum was clipped free of hair. A full-thickness excisional wound (1.5x1.5 cm), including the panniculus carnosus, was created with fine scissors.18 Wounds were dressed with a Bioclusive transparent dressing (Johnson & Johnson), and wound closure rates were measured by tracing the wound area every other day onto the Bioclusive dressing. The tracings were digitized, and the areas were calculated in blinded fashion with the use of a computerized algorithm (Sigma Scan; Jandel Scientific).18
Adenoviral Vector–Mediated Gene Therapy
Replication-incompetent adenoviral vectors, driven by the cytomegalovirus immediate-early promoter encoding a cDNA sequence for MnSOD, eNOS, dominant-negative HA-tagged N17Rac1, which inhibits Rac1, the small GTPase component of NADPH oxidase (N17Rac1, a kind gift from Dr John Engelhardt, University of Iowa, Iowa City), or ß-galactosidase (ß-gal) reporter gene, were propagated, purified, and titrated as previously described.22,23 The prepared vectors were stored at –80°C in 0.01 mol/L Tris, 0.01 mol/L MgCl2, and 10% glycerol before use. In vivo gene transfer was performed as described.18 Briefly, after the wound was created on the dorsum of mice, gene transfer of eNOS, MnSOD, or ß-gal was immediately performed in some animals by placing 200 µL of viral solution (1010 plaque-forming units [pfu]/mL) directly onto the wound surface for 30 minutes. The solution was then removed with sterile gauze, and the wounds were covered with a Bioclusive dressing to maintain sterility. For ex vivo gene transfer, skin tissues from Ins2Akita diabetic mice were randomly exposed to eNOS, MnSOD (1010 pfu/mL), dominant-negative HA-tagged N17Rac1 (N17Rac1), or ß-gal (1011 pfu/mL) in Eagle’s minimal essential medium (EMEM) for 4 hours followed by incubation in fresh EMEM for 20 hours, or skin tissues from normal mice were randomly exposed to either N17Rac1 or ß-gal (1011 pfu/mL) in EMEM for 4 hours followed by incubation in fresh EMEM with 300 mg/dL glucose for 20 hours. Some skin tissues were incubated in EMEM for 24 hours without gene transfer. All skin tissues were used for O2– measurement.
Immunoblotting of eNOS
Western blot analysis for eNOS protein was performed as described.23 Briefly, skin tissues were homogenized in lysis buffer (0.5 mol/L Tris HCl [pH 6.8], 10% SDS, 10% glycerol) with protease inhibitors (0.5 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin (all from Sigma Chemical Co). Homogenates were centrifuged (11 000g for 10 minutes, 4°C), and the protein supernatants were measured. Equal amounts of protein (6.8 µg) were separated on SDS-polyacrylamide gels (7.5%) and electrotransferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories) and blotted with the primary antibody against eNOS (1:2500; BD Transduction Laboratories). Human endothelial lysate (BD Transduction Laboratories) was used as a positive control for eNOS. The secondary antibody used was mouse anti-goat antibody (1:2000; BD Transduction Laboratories). Blots were developed with the use of Supersignal West Pico chemiluminescent substrate (Pierce) and reprobed with actin (Santa Cruz Biotechnology, Inc). Molecular band intensity was determined by densitometry (Bio-Rad image software).
Radioimmunoassay of NOS Activity
Skin tissues were homogenized and centrifuged, and supernatants were subjected to NOS activity assay with a commercial NOS radioimmunoassay (RIA) kit (Calbiochem). Aliquots of supernatants were incubated with [3H]arginine (10 µmol/L final arginine, 62 Ci/mmol, Amersham) in the presence of 1 mmol/L NADPH, 3 µmol/L tetrahydrobiopterin, 600 µmol/L CaCl2, 1 µmol/L flavin-adenine dinucleotide, and 1 µmol/L flavin mononucleotide in a final volume of 50 µL for 60 minutes at room temperature.24 The reaction was quenched with 400 µL of stop buffer (50 mmol/L HEPES and 5 mmol/L EDTA, pH 5.5). Experiments were also performed in the presence of either EGTA (5.0 mmol/L) or N
-L-arginine methyl ester (L-NAME) (1 mmol/L).24 After resin was added, the reaction mixtures were transferred to spin cups and centrifuged. L-[3H]citrulline content in eluate was determined by liquid scintillation counting. Samples of buffer containing L-[3H]arginine in the absence of skin tissue served as background counts, which were subtracted from all measurements. Constitutive NOS (eNOS and neuronal NOS) activity was determined by subtracting total counts from both L-NAME– and calcium chelator EGTA–blocked counts and normalized for protein content (measured by the Bradford assay [Bio-Rad]).
Nitrite Measurement
When the wound was completely healed, the wound tissue was cut into small pieces that were used to assay the nitrite level with Griess reagents (Sigma Chemical Co). After incubation with EMEM in a CO2 incubator for 24 hours, the wound tissue was weighed, and the NO stable metabolite nitrite concentration in EMEM was determined.
SOD Activity Measurement
Skin tissues were homogenized in cold buffer (20 mmol/L HEPES, 1 mmol/L EGTA, 210 mmol/L mannitol, and 70 mmol/L sucrose), and homogenates were centrifuged at 13 000g for 15 minutes at 4°C.25,26 The resulting supernatant that contained cytosolic SOD25,26 was subjected to an SOD activity assay with a commercial SOD kit (Cayman Chemical). Briefly, the addition of 1 mmol/L potassium cyanide was used to inhibit both Cu/Zn-SOD and extracellular SOD, resulting in the detection of only MnSOD activity.27 Hypoxanthine/xanthine oxidase was used to generate O2–, which was detected by tetrazolium salt through reading the absorbance at 450 nm.
Superoxide Measurement
Cutaneous O2– levels were quantified by lucigenin-enhanced chemiluminescence as described.23 Skin tissues clipped free of hair were cut into small pieces (2x2 mm) and placed in polypropylene tubes containing 5 µmol/L lucigenin in 1 mL modified Krebs’ solution. Tubes were read in a luminometer (TD-20/20, Turner Designs). The luminometer reports relative light units emitted, which were integrated over 5 minutes. The superoxide-sensitive fluorescent dye dihydroethidium was used to evaluate in situ production of O2– as described.23 Unfixed frozen skin tissues were cut into 30-µm-thick sections and placed on a glass slide. Slides were incubated with dihydroethidium (1x10–6 mol/L) in a light-protected, humidified chamber at 37°C for 30 minutes and coverslipped. Tissue sections were then visualized with a Zeiss 210 confocal microscope, fluorescence was detected with a 590-nm long-pass filter, and images were collected and stored digitally.
To investigate the enzymatic sources of cutaneous O2–, skin tissues were treated with chelerythrine (a protein kinase C inhibitor; 3x10–6 mol/L),28,29 apocynin (a NADPH oxidase inhibitor; 10–4 mol/L),30,31 diphenyleneiodonium (a flavoprotein inhibitor; 10–4 mol/L),32 allopurinol (a xanthine oxidase inhibitor; 10–4 mol/L),33 or L-NAME (a NOS inhibitor; 10–4 mol/L)34 for 30 minutes in vitro before the assay.
Statistical Analysis
Data are presented as mean±SEM. Experimental means were subjected to unpaired Student t test and 1-way ANOVA with Newman-Keuls multiple comparison test. A probability value of <0.05 was considered statistically significant.
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Streptozotocin-Induced and Ins2Akita Diabetic Mice
To test the role of hyperglycemia on wound repair without the influence of insulin, a 5-day low-dose streptozotocin injection regimen was used to ensure 13 weeks of sustained hyperglycemia, which was required to impair wound healing.21 Under such a regimen, streptozotocin-treated mice maintained hyperglycemia and experienced body weight loss throughout the study (Figure 1). The glucose levels were significantly elevated (308±30 versus 98±10 mg/dL; P<0.05), and body weights were markedly decreased (24.1±0.67 versus 29.5±0.30 g; P<0.05) in 16-week-old Ins2Akita diabetic mice compared with control mice.
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Cutaneous Gene Therapy of eNOS or MnSOD on Wound Healing in Diabetic Mice
The rate of wound closure in streptozotocin-induced diabetic mice to a 100% closure point was delayed by 23.6% compared with that of the control mice. In vivo gene transfer of eNOS or MnSOD accelerated the closure rates by an average of 15.7% and 13.8% to a 100% closure point, respectively, compared with the nontransduced diabetic mice or mice transduced with ß-gal reporter gene (Figure 2).
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Impaired Wound-Induced Endogenous eNOS Expression and NOS Activity in Diabetic Mice
Endogenous eNOS protein levels in skin tissues were not significantly different between normal and diabetic mice (Table and Figure 3A through 3C). However, constitutive NOS activity was markedly decreased in diabetic mice compared with normal mice (Figure 4A). Specially, there was significant impairment of wound-induced endogenous eNOS expression during the wound healing process in diabetic mice compared with normal mice (Table and Figure 3A through 3C). Similarly, wound-induced activation of cutaneous NOS was absent in diabetic mice (Figure 4A).
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Gene Therapy of eNOS or MnSOD on Cutaneous eNOS Protein Expression, NOS Activity, and NO Levels in Diabetic Mice
In vivo gene transfer of eNOS or MnSOD onto the wounds of diabetic mice resulted in a significant increase of eNOS protein during the wound healing process on days 1, 3, 5, 7, 10, and 14 after wounding compared with nontransduced diabetic mice, whereas gene transfer of ß-gal had no such effect (Table and Figure 3B through 3E). Similarly, constitutive NOS activity was significantly increased on days 1, 7, and 14 after wounding in eNOS- or MnSOD-transduced diabetic mice compared with nontransduced or ß-gal–transduced diabetic mice (Figure 4A). Cutaneous NO levels in eNOS- or MnSOD-transduced diabetic mice were increased by 177.3% and 40.5%, respectively, an effect that was inhibited by NOS inhibitor L-NAME (10–4 mol/L) (Figure 4B).
Gene Therapy of MnSOD on Cutaneous MnSOD Activity in Diabetic Mice
In vivo gene transfer of MnSOD onto the wounds in diabetic mice resulted in a significant increase of cutaneous cytosolic MnSOD activity during the wound healing process on days 1, 7, and 14 after wounding compared with nontransduced diabetic mice, whereas gene transfer of ß-gal had no such effect (Figure 4C).
Gene Therapy of eNOS or MnSOD on Cutaneous Superoxide Levels in Diabetic Mice
In streptozotocin-induced and Ins2Akita diabetic mice, cutaneous O2– levels were markedly higher than that of the control mice. In vivo gene transfer of eNOS or MnSOD impeded O2– levels in streptozotocin-induced diabetic mice when measured on wound closure (Figure 5A). Ex vivo gene transfer of eNOS or MnSOD also reduced the O2– levels in Ins2Akita diabetic mice (Figure 5C).
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Inhibition of PKC and NADPH Oxidase Reduces High Glucose–Induced Cutaneous O2–
The increased cutaneous O2– levels in streptozotocin-induced and Ins2Akita diabetic mice were significantly decreased after the skin tissues were treated for 30 minutes in vitro with protein kinase C inhibitor chelerythrine (3x10–6 mol/L) or NADPH oxidase inhibitor apocynin (10–4 mol/L) or diphenyleneiodonium (10–4 mol/L) (Figure 5B to 5C). In contrast, inhibition of xanthine oxidase by allopurinol (10–4 mol/L) or NOS by L-NAME (10–4 mol/L) failed to produce the same effect in streptozotocin-induced diabetic mice (Figure 5B). Ex vivo gene transfer of dominant-negative HA-tagged N17Rac1 significantly inhibited cutaneous O2– in Ins2Akita diabetic mice, whereas gene transfer of ß-gal failed to produce such an effect (Figure 5C). Similarly, in vitro glucose treatment of skin tissues from normal mice increased O2– levels in a concentration-dependent manner (Figure 6A), an effect that was blunted by chelerythrine, apocynin, or diphenyleneiodonium (10–4 mol/L) but not allopurinol or L-NAME (Figure 6B). Ex vivo gene transfer of dominant-negative HA-tagged N17Rac1 also significantly inhibited cutaneous O2– induced by 300 mg/dL glucose (Figure 6B to 6C).
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Discussion |
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The present study demonstrates, for the first time, (1) that high glucose levels induce cutaneous superoxide via NADPH oxidase and protein kinase C, resulting in impaired NO function and delayed wound healing, and (2) that cutaneous gene therapy of eNOS or MnSOD increases eNOS expression and function, resulting in accelerated wound healing in type 1 diabetic mice.
Reduced Cutaneous NO Bioavailability Contributes to Diabetic Wound Healing Impairment
There is growing evidence that NO plays a pivotal role in normal wound healing. All 3 NOS isoforms, including eNOS, iNOS, and neuronal NOS (nNOS), are expressed in skin tissues.6 Expression of eNOS can be detected in keratinocytes of the basal epidermal layer, dermal fibroblasts, endothelial capillaries, and eccrine glands.35 NO serves as an autocrine and paracrine mediator in maintaining normal skin homeostasis.6,7 It promotes processes central to wound healing, including angiogenesis8–10 and migration and proliferation of fibroblasts,11 epithelial cells,12 endothelial cells,8,9 and keratinocytes.10
However, the direct effects of NO on wound repair under pathological conditions remain unknown. Our results demonstrate that although endogenous eNOS protein level in skin tissues was not different between normal and diabetic mice before wounding, its expression was significantly impaired during the wound healing process only in diabetic mice. More importantly, constitutive NOS activity was markedly decreased in diabetic mice compared with normal mice, with a concomitant reduction of the stable NO metabolite nitrite. These findings suggest that impairment of wound-induced endogenous eNOS expression and NOS activity is responsible for reduced cutaneous NO bioavailability in type 1 diabetic mice. These results are also in agreement with previous studies showing that impaired wound healing was paralleled by decreased wound eNOS expression19 and that treatment with insulin and L-arginine partially restored NO production and improved wound healing in diabetic animals.20
To ascertain that impairment of cutaneous NO function contributes to delayed wound healing, we investigated the direct effect of gene therapy of eNOS or MnSOD on wound healing in type 1 diabetic mice. The results show that gene therapy of eNOS or MnSOD onto the wounds in diabetic mice significantly accelerated wound healing. The observed therapeutic effect is most likely due to enhanced wound NO bioavailability because cutaneous eNOS protein expression and constitutive NOS activity were both significantly increased in eNOS- or MnSOD-transduced diabetic mice compared with nontransduced or ß-gal–transduced diabetic mice. Furthermore, the augmented endogenous eNOS expression observed in normal mice during wound healing was absent in diabetic mice, a process that was reversed by eNOS or MnSOD gene therapy. Because wounding per se significantly induced cutaneous eNOS expression in normal mice, as shown in this study, it would be difficult to distinguish endogenous and exogenous eNOS expression in transduced normal mice. Hence, a comparison of eNOS expression in transduced normal mice and diabetic mice may not yield such desired information. Collectively, our data indicate that reduced eNOS protein and activity contribute to delayed wound healing and demonstrate, for the first time, that increased local NO bioavailability via cutaneous gene therapy is an efficacious means to improve wound healing in diabetic mice.
Although a central role for protein-type growth factors and mitogens during wound repair has been well known for years, the application of these factors in the treatment of wound healing has not provided reliable treatments in clinical studies.6 One possible explanation for the failure of protein-type mitogens to markedly accelerate closure of chronic wounds may be tremendously elevated protease activities in the fluids of chronic wounds. These findings suggest that the molecular environment of chronic wounds may impair the ability of exogenously applied growth factors to stimulate healing. The results of the present study suggest that NO might represent a novel target molecule that circumvents the difficulties that arise because of proteolytic cleavage of therapeutically applied growth factors in chronic wound situations. Because NO is a short-lived gas molecule, maintenance of an effective level of NO at a wound site is difficult. Our results demonstrate proof of the principle that cutaneous gene therapy of eNOS or MnSOD may be an efficacious means to achieve a sustained local NO level at the wound site. However, future studies are warranted to determine the optimal gene dose regimens.
Increased Cutaneous O2– Contributes to Decreased NO Bioavailability
Although impaired NO function contributes to delayed wound healing in diabetes, the mechanisms underlying cutaneous NO dysfunction are not clear. Causative factors for hyperglycemia-induced tissue damage include activation of the polyol pathway, nonenzymatic glycation and protein kinase C pathways, and increased hexosamine pathway flux.2 However, there was no apparent common element linking these known mechanisms. Recent studies suggest that these different mechanisms may be linked to a single cellular process: an overproduction of O2– induced by sustained hyperglycemia.36 Sustained hyperglycemia is known to increase vascular O2– levels, which results in cardiovascular dysfunction.3 O2– produced in the vasculature rapidly inactivates NO and reduces its bioactivity in diabetic blood vessels.28 Independent strategies aimed at reducing O2– formation have been shown to prevent high glucose–induced protein kinase C activation, formation of advanced glycation end products, sorbitol accumulation, and nuclear factor-
B activation, resulting in the improvement of endothelium-dependent NO-mediated vasodilatation as well as wound healing.3,36–38 These studies suggest that the increase in O2– levels is a key factor for NO dysfunction in diabetes. However, the effects of hyperglycemia on cutaneous O2– and NO levels in diabetes remain unknown.
The results of the present study demonstrate that in vitro glucose treatment of skin tissues from normal mice increased O2– levels in a concentration-dependent manner and that there is a marked increase of cutaneous O2– levels in chemical substance streptozotocin-induced type 1 diabetic mice and autosomal dominant mutation in Ins2 gene–induced type 1 diabetic mice. Gene therapy of MnSOD significantly elevated MnSOD activity, resulting in decreased O2– and increased NO levels in streptozotocin-induced type 1 diabetic mice. These findings are consistent with a previous study showing that inhibition of lipid peroxidation restored wound healing to nearly normal levels in diabetes-impaired wounds.38 Because the vector titer used for MnSOD gene therapy was exactly the same as for eNOS and ß-gal, the profile of MnSOD transgene expression should also be similar. The augmented MnSOD activity 14 days after MnSOD transduction supports this stipulation. The increased cutaneous NO levels may be a combined result of reduced O2– inactivation of NO and augmented NOS protein expression and activity after MnSOD gene therapy. Although the mechanism of enhanced NOS protein expression and activity is unclear, it may be related to the formation of hydrogen peroxide after MnSOD gene therapy. This speculation is based on previous experimental observations demonstrating that hydrogen peroxide is an effective stimulus of eNOS expression in endothelial cells of animals and humans.39,40
NADPH Oxidase and Protein Kinase C Are Key Sources for Cutaneous O2– Production
In diabetic vasculature, the main enzymatic sources for O2– production include NADPH oxidase, xanthine oxidase, mitochondrial oxidase, and uncoupled eNOS.3,28,36,41 However, the humoral factors responsible for O2– production in diabetic wounds are unknown. NADPH oxidase is expressed in endothelial cells, fibroblasts, neutrophils, and phagocytes, and it is composed of multiple subunits in which the small G protein Rac1 is required for its activation.42,43 Apocynin is a selective NADPH oxidase inhibitor that prevents the binding of the cytosolic subunits to the membrane-bound subunits, thus preventing oxidase activation and subsequent O2– production.42,43 Overexpression of dominant-negative Rac1 has been shown to inhibit Rac1 activity, resulting in NADPH oxidase inactivation and a lack of O2– generation.44–46 In the present study we observed that both apocynin, a selective inhibitor of NADPH oxidase, and chelerythrine, a selective inhibitor of protein kinase C, suppressed cutaneous O2– formation induced by high glucose in both normal mice and diabetic mice. Moreover, gene transfer of dominant-negative HA-tagged N17Rac1 also significantly inhibited glucose-induced cutaneous O2– production. In contrast, neither NOS inhibitor L-NAME nor xanthine oxidase inhibitor allopurinol produced similar effects. Because cutaneous gene therapy of eNOS increased local NO and reduced O2– levels, these results also suggest that eNOS itself does not play a significant role in glucose-induced cutaneous O2– generation.
MnSOD has been shown to be mainly localized to mitochondria and plays an important role in protecting against oxidative stress–induced tissue damage.47 In bovine endothelial cells, disruption of mitochondrial O2– production was achieved with several different approaches, including the following: (1) treatment with carbonyl cyanide m-chlorophenylhydrazone, a small-molecule uncoupler of mitochondrial oxidative phosphorylation; (2) overexpression of uncoupling protein-1, a protein uncoupler; or (3) overexpression of MnSOD. Each of these approaches blocked the hyperglycemia-induced increase in O2– production. Consequently, the hyperglycemia- induced effects on nuclear factor-B, protein kinase C, advanced glycation end products, and sorbitol were also suppressed.47 The results of the present study demonstrated that in vivo or ex vivo MnSOD gene transfer resulted in decreased O2– in streptozotocin-induced type 1 diabetic mice and autosomal dominant mutation in Ins2 gene–induced type 1 diabetic mice, which indicates that mitochondria may be another important source of cutaneous O2– generation in diabetes. Meanwhile, our results also showed that in vivo MnSOD gene transfer significantly elevated cytosolic MnSOD activity in streptozotocin-induced type 1 diabetic mice, which indicates that gene therapy of MnSOD may be an effective means to improve wound healing delay in diabetes through disruption of cytosolic and mitochondrial O2– production.
Taken together, these findings suggest that NADPH oxidase, protein kinase C, and mitochondria are important sources for cutaneous O2– formation in response to high glucose. Studies are under way to examine the direct effects of in vivo inhibition of NADPH oxidase or protein kinase C on cutaneous O2– levels and wound repair in streptozotocin-induced diabetic mice.
Oxidants and Antioxidants and Wound Healing
Results of a recent study showed that increased oxidants accelerated cutaneous wound healing in normal mice and induced vascular endothelial growth factor expression in human keratinocytes.48 Low concentrations of reactive oxygen species (ROS), including O2–, hydrogen peroxide, and hydroxyl radicals, may serve as signaling messengers and regulate gene expression processes.49 Protein-type growth factors such as platelet-derived growth factor and transforming growth factor regulate cell growth through ROS-dependent signal pathways.49 These results suggest that low concentrations of ROS may be necessary for maintaining normal cell function. However, it is well established that O2– overproduction leads to cell injury under pathological states, including diabetes.3–5 Consistent with this notion, previous studies have shown that antioxidants accelerated diabetic wound healing.39,50 On the basis of these studies and our own findings, it is reasonable to speculate that high glucose–induced cutaneous O2– increase contributes, at least in part, to impaired NO function in diabetic wounds, and antioxidant strategies may be beneficial for improving diabetic wound healing.
In summary, the present study demonstrates, for the first time, (1) that cutaneous O2– levels are markedly increased in streptozotocin-induced and autosomal dominant mutation in Ins2 gene–induced type 1 diabetic mice and in skin tissues from normal mice treated with high glucose in vitro, and (2) that cutaneous gene therapy of eNOS or MnSOD promotes wound healing in streptozotocin-induced type 1 diabetic mice, an effect attributable to decreasing O2– levels induced by NADPH oxidase, protein kinase C, and mitochondria. Gene therapy strategies aimed at reducing O2–-induced wound healing impairment may be an effective means of improving wound healing delay in diabetes.
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Acknowledgments |
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This work was supported in part by the Juvenile Diabetes Research Foundation innovative grant 5-2001-311; American Diabetes Association research award 7-01-RA-10; American Heart Association grants 9806347X, 0130537Z, 0225408Z, and 0455594Z; and the Intramural Research Grants Program grant 41140 of Michigan State University (all to Dr Chen). We thank Drs Jiesheng Zheng, Lixin Li, and Xiangqun Yang for their technical assistance and discussions.
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References |
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1. Jeffcoate WJ, Harding KG. Diabetic foot ulcers. Lancet. 2003; 361: 1545–1551.[CrossRef][Medline] [Order article via Infotrieve]
2. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001; 414: 813–820.[CrossRef][Medline] [Order article via Infotrieve]
3. Kim YK, Lee MS, Son SM, et al. Vascular NADH oxidase is involved in impaired endothelium-dependent vasodilation in OLETF rats, a model of type 2 diabetes. Diabetes. 2002; 51: 522–527.
4. Cosentino F, Eto M, De Paolis P, et al. High glucose causes upregulation of cyclooxygenase-2 and alters prostanoid profile in human endothelial cells: role of protein kinase C and reactive oxygen species. Circulation. 2003; 107: 1017–1023.
5. Guzik TJ, Mussa S, Gastaldi D, et al. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662.
6. Frank S, Kampfer H, Wetzler C, et al. Nitric oxide drives skin repair: novel function of an established mediator. Kidney Int. 2002; 61: 882–888.[CrossRef][Medline] [Order article via Infotrieve]
7. Witte MB, Barbul A. Role of nitric oxide in wound repair. Am J Surg. 2002; 183: 406–412.[CrossRef][Medline] [Order article via Infotrieve]
8. Ziche M, Morbidelli L, Masini E, et al. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994; 94: 2036–2044.[Medline] [Order article via Infotrieve]
9. Papapetropoulos A, Garcia-Cardena G, Madri JA, et al. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest. 1997; 100: 3131–3139.[Medline] [Order article via Infotrieve]
10. Frank S, Stallmeyer B, Kampfer H, et al. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair. FASEB J. 1999; 13: 2002–2014.
11. Schaffer MR, Efron PA, Thornton FJ, et al. Nitric oxide, an autocrine regulator of wound fibroblast synthetic function. J Immunol. 1997; 158: 2375–2381.[Abstract]
12. Noiri E, Peresleni T, Srivastava N, et al. Nitric oxide is necessary for a switch from stationary to locomoting phenotype in epithelial cells. Am J Physiol. 1996; 170: C794–C802.
13. Shabani M, Pulfer SK, Bulgrin JP, et al. Enhancement of wound repair with a topically applied nitric oxide-releasing polymer. Wound Repair Regen. 1996; 4: 353–362.[CrossRef][Medline] [Order article via Infotrieve]
14. Schaffer MR, Tantry U, Gross SS, et al. Nitric oxide regulates wound healing. J Surg Res. 1996; 63: 237–240.[CrossRef][Medline] [Order article via Infotrieve]
15. Barbul A, Lazarou SA, Efron DT, et al. Arginine enhances wound healing and lymphocyte immune responses in humans. Surgery. 1990; 108: 331–337.[Medline] [Order article via Infotrieve]
16. Kirk SJ, Hurson M, Regan MC, et al. Arginine stimulates wound healing and immune function in elderly human beings. Surgery. 1993; 114: 155–160.[Medline] [Order article via Infotrieve]
17. Lee PC, Salyapongse AN, Bragdon GA, et al. Impaired wound healing and angiogenesis in eNOS-deficient mice. Am J Physiol. 1999; 277: H1600–H1608.[Medline] [Order article via Infotrieve]
18. Yamasaki K, Edington HDJ, McClosky C, et al. Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest. 1998; 101: 967–971.[Medline] [Order article via Infotrieve]
19. Stallmeyer B, Anhokd M, Wetzler C, et al. Regulation of eNOS in normal and diabetes-impaired skin repair: implications for tissue regeneration. Nitric Oxide. 2002; 6: 168–177.[CrossRef][Medline] [Order article via Infotrieve]
20. Witte MB, Thornton FJ, Tantry U, et al. L-Arginine supplementation enhances diabetic wound healing: involvement of the nitric oxide synthase and arginase pathways. Metabolism. 2002; 51: 1269–1273.[CrossRef][Medline] [Order article via Infotrieve]
21. Schatteman GC, Hanlon HD, Jiao C, et al. Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice. J Clin Invest. 2000; 106: 571–578.[Medline] [Order article via Infotrieve]
22. Chen AFY, O’Brien T, Tsutsui M, et al. Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery. Circ Res. 1997; 80: 327–335.
23. Li LX, Crockett E, Wang DH, et al. Gene transfer of eNOS and MnSOD on arterial VCAM-1 expression and superoxide production in DOCA-salt hypertension. Arterioscler Thromb Vasc Biol. 2002; 22: 249–255.
24. Paolocci N, Biondi R, Bettini M, et al. Oxygen radical-mediated reduction in basal and agonist-evoked NO release in isolated rat heart. J Mol Cell Cardiol. 2001; 33: 671–679.[CrossRef][Medline] [Order article via Infotrieve]
25. Mattiazzi M, D’Aurelio M, Gajewski CD, et al. Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice. J Biol Chem. 2002; 277: 29626–29633.
26. Allen S, Heath PR, Kirby J, et al. Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. J Biol Chem. 2003; 278: 6371–6383.
27. MacMillan-Crow LA, Crow JP, Kerby JD, et al. Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci U S A. 1996; 93: 11853–11858.
28. Hink U, Li H, Mollnau H, et al. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res. 2001; 88: E14–E22.[Medline] [Order article via Infotrieve]
29. Guzik TJ, Mussa S, Gastaldi D, et al. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662.
30. Kashiwagi A, Shinozaki K, Nishio Y, et al. Endothelium-specific activation of NAD(P)H oxidase in aortas of exogenously hyperinsulinemic rats. Am J Physiol. 1999; 277: E976–E983.[Medline] [Order article via Infotrieve]
31. Oeckler RA, Kaminski PM, Wolin MS. Stretch enhances contraction of bovine coronary arteries via an NAD(P)H oxidase-mediated activation of the extracellular signal–regulated kinase mitogen-activated protein kinase cascade. Circ Res. 2003; 92: 23–31.
32. Li L, Fink GD, Watts SW, et al. Endothelin-1 increases vascular superoxide via endothelin(A)-NADPH oxidase pathway in low-renin hypertension. Circulation. 2003; 107: 1053–1058.
33. Ungvari Z, Csiszar A, Edwards JG, et al. Increased superoxide production in coronary arteries in hyperhomocysteinemia: role of tumor necrosis factor-alpha, NAD(P)H oxidase, and inducible nitric oxide synthase. Arterioscler Thromb Vasc Biol. 2003; 23: 418–424.
34. Landmesser U, Dikalov S, Price SR, et al. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201–1209.[CrossRef][Medline] [Order article via Infotrieve]
35. Shimizu Y, Sakai M, Umemura Y, et al. Immunohistochemical localization of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes. J Dermatol. 1997; 24: 80–87.[Medline] [Order article via Infotrieve]
36. Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemia damage. Nature. 2000; 404: 787–790.[CrossRef][Medline] [Order article via Infotrieve]
37. McFarlane SI, Kumar A, Sowers JR. Mechanisms by which angiotensin-converting enzyme inhibitors prevent diabetes and cardiovascular disease. Am J Cardiol. 2003; 91: 30–37.
38. Altavilla D, Saitta A, Cucinotta D, et al. Inhibition of lipid peroxidation restores impaired vascular endothelial growth factor expression and stimulates wound healing and angiogenesis in the genetically diabetic mouse. Diabetes. 2001; 50: 667–674.
39. Cai H, Davis ME, Drummond GR, et al. Induction of endothelial NO synthase by hydrogen peroxide via a Ca(2+)/calmodulin-dependent protein kinase II/janus kinase 2-dependent pathway. Arterioscler Thromb Vasc Biol. 2001; 21: 1571–1576.
40. Shimizu S, Shiota K, Yamamoto S, et al. Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells. Free Radic Biol Med. 2003; 34: 1343–1352.[CrossRef][Medline] [Order article via Infotrieve]
41. Desco MC, Asensi M, Marquez R, et al. Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol. Diabetes. 2002; 51: 1118–1124.
42. Bokoch GM, Diebold BA. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood. 2002; 100: 2692–2696.
43. Babior BM. NADPH oxidase: an update. Blood. 1999; 93: 1464–1476.
44. Seshiah PN, Weber DS, Rocic P, et al. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91: 406–413.
45. Lopes NH, Vasudevan SS, Gregg D, et al. Rac-dependent monocyte chemoattractant protein-1 production is induced by nutrient deprivation. Circ Res. 2002; 91: 798–805.
46. Sanlioglu S, Williams CM, Samavati L, et al. Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B. J Biol Chem. 2001; 276: 30188–30198.
47. Evans JL, Goldfine ID, Maddux BA, et al. Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev. 2002; 23: 599–622.
48. Sen CK, Khanna S, Babior BM, et al. Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing. J Biol Chem. 2002; 277: 33284–33290.
49. Droge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002; 82: 47–95.
50. Galeano M, Torre V, Deodato B, et al. Raxofelast, a hydrophilic vitamin E-like antioxidant, stimulates wound healing in genetically diabetic mice. Surgery. 2001; 129: 467–477.[Medline] [Order article via Infotrieve]
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