Regulation of Nitric Oxide Synthesis in Wounds by IFN-γ Depends on TNF-α

Macrophage-derived nitric oxide is a critical mediator in wound healing. Its regulation in vivo, however, remains unclear. We hypothesized that interferon (IFN)-γ plays an important role in the regulation of nitric oxide in wounds. Groups of 12 male IFN-γ -knockout mice and wild-type controls underwent dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. Mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Synthesis of nitric oxide (NO), tumor necrosis factor (TNF)-α, and IFN-γ was measured in the wound. Wound-derived macrophages were tested for NO synthesis in the presence or absence of IFN-γ, TNF-α, and anti-TNF-α antibody. In a separate experiment, IFN-γ -knockout mice and wild-type controls were treated with molsidomine, a nitric oxide donor. It was found that wound collagen deposition and wound breaking strength were impaired in IFN-γ-knockout mice (p <. 05). Impaired healing was reflected in diminished synthesis of TNF-α and NO in wounds (p <. 05). In vivo treatment with molsidomine reversed impaired healing in IFN-γ-deficient mice. Ex vivo, addition of IFN-γ stimulated the synthesis of TNF-α and NO in wound-derived macrophages. IFN-γ -induced NO synthesis by wound-derived macrophages was abolished by anti-TNF-α-antibody-treatment, which could be fully reversed by exogenous TNF-α. Thus we conclude that IFN-γ-deficiency impairs wound healing and diminishes NO synthesis in wound-derived macrophages. The stimulatory effect of IFN-γ on macrophage NO production depends on endogenous TNF-α synthesis.     

MHC-class-II-deficiency impairs wound healing

BACKGROUND: MHC-class-II-deficient mice lack T helper cell dependent immune reactions. T cell related immune functions are critical for normal wound healing. We hypothesized that MHC-II-deficiency compromises wound repair by affecting the normal wound immune response. MATERIAL AND METHODS: Groups of 10 male MHC-class II-knockout mice and wild-type controls underwent dorsal skin incision. Polyvinyl alcohol sponges were then inserted subcutaneously. The mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Activity of T cells and macrophages isolated from the spleens and from the healing wounds was investigated. Fibroblasts derived from the wounds were tested ex vivo for proliferative activity and collagen synthesis. RESULTS: Wound collagen deposition and wound breaking strength were impaired in MHC-class-II-knockout mice (P < 0.05). Impaired healing was reflected in diminished mitogen-reactivity of splenic T-cells (P < 0.01), and decreased CD4 expression in wounds. In addition, basal and LPS + IFN-gamma-induced synthesis of TNF-alpha and nitric oxide by wound-derived macrophages was impaired. Exvivo, fibroblast proliferation and fibroblast collagen production from MHC-II-deficient mice was decreased. CONCLUSION: MHC-II-deficiency compromises wound healing. This may be a reflection of impaired wound immune cell function and decreased activity of wound fibroblasts.     

Differential expression of inflammatory mediators in radiation-impaired wound healing

BACKGROUND: Radiation impairs healing, although the underlying mechanisms are not clearly defined. Normal healing requires a fine balance of promoting and inhibiting factors. We hypothesize that there may be a down-regulation of promoting factors (nitric oxide) and, in turn, an up-regulation of healing inhibiting factors (TNF-alpha and IFN-gamma) in the wound after radiation. MATERIAL AND METHODS: Groups of 10 rats were irradiated using single dose 12 or 24 Gy electron radiation at the dorsal skin. Control rats were sham-irradiated. On Day 5 a skin incision in the irradiated area was performed and polyvinyl alcohol sponges were inserted subcutaneously. Rats were sacrificed 10 days later to determine the wound-breaking strength and reparative collagen deposition. Nitrite and nitrate (index of NO synthesis), TNF-alpha, and IFN-gamma were measured within the wound fluid. Expression of the inducible NO-synthase (iNOS) was investigated by immunohistochemistry. Wound-derived fibroblasts were tested in vitro for NO and collagen synthesis. RESULTS: Irradiation significantly reduced wound collagen deposition and wound-breaking strength (P < 0.05). Impaired healing was reflected in diminished wound NO synthesis and iNOS expression (P < 0.01). TNF-alpha and IFN-gamma were increased in irradiated wounds (P < 0.05). Ex vivo, NO synthesis and collagen deposition by fibroblasts from irradiated rats were decreased (P < 0.01). In vitro irradiation of fibroblasts from nonirradiated rats decreased both NO and collagen production (P < 0.01). CONCLUSION: Radiation-impaired healing is reflected in an imbalance of promoting and inhibiting factors, leading to increased levels of TNF-alpha and IFN-gamma and decreased NO expression in the wound.     

Nitric oxide enhances experimental wound healing in diabetes

BACKGROUND: Diabetes is characterized by a nitric oxide deficiency at the wound site. This study investigated whether exogenous nitric oxide supplementation with the nitric oxide donor molsidomine (N-ethoxycarbomyl-3-morpholinyl-sidnonimine) could reverse the impaired healing in diabetes. METHODS: Wound healing was studied by creating a dorsal skin incision with subcutaneous polyvinyl alcohol sponge implantation in diabetic and non-diabetic rats. Half of each group was treated with molsidomine. Collagen metabolism was assessed by wound breaking strength, hydroxyproline (OHP) content, RNA expression for collagen type I and III, and matrix metalloproteinase (MMP) 2 activity in wound sponges. Wound fluid, plasma and urinary nitric oxide metabolite levels, and the number of inflammatory cells were assessed. RESULTS: OHP content and wound breaking strength were significantly increased by molsidomine. MMP-2 activity in wound fluid was decreased in diabetes and upregulated by nitric oxide donors. The impaired inflammatory reaction in diabetes was unaffected by nitric oxide donor treatment and ex vivo nitric oxide synthesis was no different between wound macrophages from control and diabetic animals, suggesting that the nitric oxide deficiency in the wound is due to a smaller inflammatory reaction in diabetes. CONCLUSION: The nitric oxide donor molsidomine can at least partially reverse impaired healing associated with diabetes.     

The role of iNOS in wound healing

BACKGROUND: We have previously shown that the blockade of nitric oxide (NO) synthesis impairs wound healing, in particular collagen synthesis. Conversely, impaired wound healing is accompanied by decreased wound NO synthesis. Fibroblast collagen synthesis, proliferation, and fibroblast-mediated matrix contraction are critical to wound healing. We examined the wound healing-related phenotypic changes that are induced by the loss of inducible nitric oxide synthase (iNOS) gene function in fibroblasts. METHODS: Dermal fibroblasts were obtained from 8- to 12-week-old iNOS--knock out (KO; C57BL/Ai-[KO] Nos2 N5) and wild type mice by an explant technique and used after 1 to 3 passages. Proliferation ([(3)H]-thymidine incorporation) and collagen synthesis ([(3)H]-proline incorporation into collagenase-sensitive protein) were studied after stimulation with 10% fetal bovine serum. Matrix remodeling was assessed by the measurement of the contraction of fibroblast-populated collagen lattices. RESULTS: iNOS-KO fibroblasts proliferated more slowly, synthesized less collagen, and contracted fibroblast-populated collagen lattices more slowly than wild-type fibroblast. Collagen synthesis was restored to normal in KO fibroblasts in response to NO donors (s-nitroso-N-acetylpenicillamine). CONCLUSIONS: iNOS deficiency causes significant impairment in wound healing-related properties of fibroblasts, which suggests that NO plays an important role in wound healing.     

Acceleration of wound repair by curcumin in the excision wound of mice exposed to different doses of fractionated γ radiation

Fractionated irradiation (IR) before or after surgery of malignant tumours causes a high frequency of wound healing complications. Our aim was to investigate the effect of curcumin (CUM) on the healing of deep excision wound of mice exposed to fractionated IR by mimicking clinical conditions. A full-thickness dermal excision wound was created on the shaved dorsum of mice that were orally administered or not with 100 mg of CUM per kilogram body weight before partial body exposure to 10, 20 or 40 Gy given as 2 Gy/day for 5, 10 or 20 days. The wound contraction was determined periodically by capturing video images of the wound from day 1 until complete healing of wounds. Fractionated IR caused a dose-dependent delay in the wound contraction and prolonged wound healing time, whereas CUM administration before fractionated IR caused a significant elevation in the wound contraction and reduced mean wound healing time. Fractionated IR reduced the synthesis of collagen, deoxyribonucleic acid (DNA) and nitric oxide (NO) at different post-IR times and treatment of mice with CUM before IR elevated the synthesis of collagen, DNA and NO significantly. Histological examination showed a reduction in the collagen deposition, fibroblast and vascular densities after fractionated IR, whereas CUM pre-treatment inhibited this decline significantly. Our study shows that CUM pre-treatment accelerated healing of irradiated wound and could be a substantial therapeutic strategy in the management of irradiated wounds.     

Supplemental dietary arginine enhances wound healing in normal but not inducible nitric oxide synthase knockout mice

BACKGROUND: Although generation of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) has been shown to be required for cutaneous wound healing, no differences have been noted in incisional healing between iNOS knockout (iNOS-KO) and wild type (WT) mice. Because supplemental dietary arginine enhances cutaneous healing in normal rodents and is the sole substrate for NO synthesis, we studied whether arginine can enhance cutaneous wound healing in iNOS-KO mice. METHODS: Twenty iNOS-KO and 20 WT mice, all on a C57BL/6 background, were divided into 4 groups of 10 animals each. Ten animals with each trait were randomized to receive either normal food and tap water or food and water each supplemented with 0.5% arginine (w/w). All animals underwent a 2.5-cm dorsal skin incision with implantation of four 20-mg polyvinyl alcohol sponges into subcutaneous pockets. On postoperative day 14 the animals were killed. The dorsal wound was harvested for breaking strength determination and the wound sponges were assayed for hydroxyproline content and total wound fluid nitrite/nitrate concentration. RESULTS: Dietary arginine supplementation enhanced both wound breaking strength and collagen deposition in WT but not iNOS-KO mice. Wound fluid nitrite/nitrate levels were higher in WT than iNOS-KO animals but were not significantly influenced by additional arginine. CONCLUSIONS: These data demonstrate that supplemental dietary arginine enhances wound healing in normal mice. The loss of a functional iNOS gene abrogates the beneficial effect of arginine in wound healing. This suggests that the metabolism of arginine via the NO pathway is one mechanism by which arginine enhances wound healing.     

Role of curcumin, a naturally occurring phenolic compound of turmeric in accelerating the repair of excision wound, in mice whole-body exposed to various doses of gamma-radiation

The healing of irradiated wounds has always been a central consideration in medical practice because radiation disrupts normal response to injury, leading to a protracted recovery period. The quest for clinically effective wound healing agents is important in the medical management of irradiated wounds. Therefore, the present study was conceptualized to investigate the effect of curcumin (natural yellow, diferuloylmethane), a major yellow pigment and an active component of turmeric on wound healing in mice exposed to whole-body gamma-radiation. A full-thickness wound was created on the dorsum of mice whole-body irradiated to 2, 4, 6, or 8 Gy. The progression of wound contraction was monitored periodically by capturing video images of the wound. The collagen, hexosamine, DNA, nitric oxide, and histological profiles were evaluated at various postirradiation days in mice treated and not treated with curcumin before exposure to 0 or 6 Gy. The whole-body exposure resulted in a dose-dependent delay in wound contraction and prolongation of wound healing time. Irradiation caused a significant reduction in collagen, hexosamine, DNA, and nitric oxide synthesis. Pretreatment with curcumin significantly enhanced the rate of wound contraction, decreased mean wound healing time, increased synthesis of collagen, hexosamine, DNA, and nitric oxide and improved fibroblast and vascular densities. This study demonstrates that curcumin pretreatment has a conducive effect on the irradiated wound and could be a substantial therapeutic strategy in initiating and supporting the cascade of tissue repair processes in irradiated wounds.     

Inhibition of nitric oxide synthesis in wounds: pharmacology and effect on accumulation of collagen in wounds in mice.

OBJECTIVE: To investigate the effect of systemic inhibition of nitric oxide (NO) synthesis in wounds on collagen accumulation. DESIGN: Randomised experimental study. SETTING: Teaching hospital, USA. MATERIAL: 240 Balb/C mice divided into groups of 10 animals each. INTERVENTIONS: Polyvinyl alcohol sponges were inserted subcutaneously through a dorsal skin incision. Beginning on the day of wounding, N omega-nitro-L-arginine-methylester (L-NAME), NG-L-monomethyl-arginine (L-NMMA), aminoguanidine hemisulphate (AGU), and S-methyl isothiouronium (MITU) were given orally or intraperitoneally. The mice were killed 10 days later. MAIN OUTCOME MEASURES: Nitrite and nitrate concentrations, both stable end products of NO, were measured in wound fluid. Sponge hydroxyproline content was assayed as an index of reparative collagen deposition. RESULTS: NOS inhibitors given orally in the drinking water or by daily intraperitoneal injection had no effect on wound nitrite/nitrate concentrations or deposition of collagen in wounds. When given continuously through intraperitoneally-placed osmotic pumps, AGU (500 mg/kg/day) (p < 0.001) and MITU (p < 0.01) significantly reduced wound fluid nitrite/nitrate concentrations in a dose dependent manner. Inhibition of wound nitric oxide synthase by 500 mg AGU/kg/day and 100 mg MITU/kg/day was paralleled by lowered accumulation of collagen in wounds (p < 0.01). CONCLUSION: NO is beneficial in wound healing.     

Differential effect of tacrolimus on dermal and intestinal wound healing.

Tacrolimus, used in organ transplantation, inhibits cellular immune function. Little is known about the effect on dermal and colonic healing. Groups of 10 rats underwent dorsal skin incision, and polyvinyl alcohol sponges were implanted subcutaneously. Beginning at the day of wounding, rats were treated intraperitoneal with 1.0 or 2.0 mg tacrolimus/kg/day. Animals were sacrificed 10 d later to determine wound breaking strength and reparative collagen deposition. Expression of transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma was studied in wounds. Groups of 8 rats underwent laparotomy and left colonic anastomosis. These rats were treated by subcutaneous injections with 2.0 or 5.0 mg tacrolimus/kg. Animals were sacrificed 5 d later to test colonic bursting pressure and reparative collagen deposition. Expression of TGF-beta, TNF-alpha, IFN-gamma, and CD4 and CD8 in the anastomosis was investigated. Tacrolimus impaired dermal healing (p < .05). This was paralleled by decreased expression of TGF-beta (stimulates healing) and increased expression of IFN-gamma and TNF-alpha (both inhibit healing) (p < .05). In contrast, tacrolimus did not inhibit healing of colonic anastomoses. No effect was seen on the expression of TGF-beta, TNF-alpha, IFN-gamma, and CD4 and CD8 in colonic anastomoses. We concluded that tacrolimus differentially effects tissue healing and expression of cellular mediators in dermal and intestinal wounds.     

Supplemental L-arginine enhances wound healing in diabetic rats

L-arginine has been shown to enhance wound strength and collagen deposition in rodents and humans. Diabetes mellitus, which impairs wound healing, is accompanied by a reduction in nitric oxide at the wound site. The amino acid L-arginine is the only substrate for nitric oxide synthesis. We sought to determine whether supplemental L-arginine can restore the impaired wound healing of diabetic rats. Fifty-six male Lewis rats were used in this study, of which twenty-nine rats were rendered diabetic 7 days prior to surgery with intraperitoneal streptozotocin. Twenty-seven untreated rats served as controls. Animals underwent a dorsal skin incision with implantation of polyvinyl-alcohol sponges. Sixteen diabetic and 14 normal rats received 1 g/kg/day of L-arginine by injection, while the remainder received saline injections only. Animals were euthanized 10 days postwounding, and their wounds were analyzed for breaking strength. The wound sponges were assayed for total hydroxyproline and nitrite/nitrate content. Plasma and wound fluid concentrations of L-arginine, ornithine, and citrulline were determined. Wound sponge RNA was extracted and subjected to Northern blot analysis for procollagen I and III. Diabetic wounds had greatly decreased breaking strengths compared with controls. L-arginine significantly enhanced wound breaking strengths in both control (+23%) and diabetic animals (+44%), and also increased wound hydroxyproline levels in both diabetic (+40%) and control animals (+24%) as compared to their saline-treated counterparts. mRNA for procollagen I and III were elevated by L-arginine treatment in both diabetic rats and controls. Treatment with L-arginine significantly increased wound fluid nitrite/nitrate levels in diabetic animals. The data show that the impaired healing of diabetic wounds can be partially corrected by L-arginine supplementation, and that this effect is accompanied by enhanced wound nitric oxide synthesis.     

Sirolimus impairs wound healing

Background and aims Clinically, the immunosuppressive drug sirolimus, used in organ transplantation, appears to impair wound healing. Little is known about the mechanisms of action. We investigated the effect of sirolimus on wound healing, and we analyzed the expression of stimulating mediators of angiogenesis (VEGF, vascular endothelial growth factor) and collagen synthesis (nitric oxide) in wounds. Materials and methods Groups of ten rats underwent dorsal skin incision, and polyvinyl alcohol sponges were implanted subcutaneously. Beginning at the day of wounding, rats were treated with 0.5, 2.0, or 5.0 mg sirolimus/kg/day. Animals were killed 10 days later to determine wound breaking strength and reparative collagen deposition. Expression of VEGF and nitric oxide was studied in wounds. Results Splenic lymphocyte proliferative activity was significantly decreased by sirolimus (p < 0.05). Sirolimus levels in wound fluid were found to be approximately two- to fivefold higher than blood levels (p < 0.01). Sirolimus (2.0 and 5.0 mg kg⁻¹ day⁻¹) reduced wound breaking strength (p < 0.01) and wound collagen deposition (p < 0.05). This was paralleled by decreased expression of VEGF and nitric oxide in wounds. Conclusion Experimentally, our data show that sirolimus impairs wound healing, and this is reflected by diminished expression of VEGF and nitric oxide in the wound. Best abstracts — Surgical Forum 2007.     

Role of nitric oxide in wound repair

After injury, wound healing is essential for recovery of the integrity of the body. It is a complex, sequential cascade of events. Nitric oxide (NO) is a small radical, formed from the amino acid L-arginine by three distinct isoforms of nitric oxide synthase. The inducible isoform (iNOS) is synthesized in the early phase of wound healing by inflammatory cells, mainly macrophages. However many cells participate in NO synthesis during the proliferative phase after wounding. NO released through iNOS regulates collagen formation, cell proliferation and wound contraction in distinct ways in animal models of wound healing. Although iNOS gene deletion delays, and arginine and NO administration improve healing, the exact mechanisms of action of NO on wound healing parameters are still unknown. The current review summarizes what is known about the role of NO in wound healing and points out path for further research.     

Characterization of incisional wound healing in inducible nitric oxide synthase knockout mice

BACKGROUND: Excisional wound healing in inducible nitric oxide synthase knockout (iNOS-KO) mice has been previously shown to be impaired compared with their background strain controls. Incisional wounds were created in this experiment in both types of animals and paradoxically were found to heal with the same rapidity and breaking strength in both groups. METHODS: Dorsal 2.5 cm incisional wounds were created in iNOS-KO mice, as well as their parental strain controls (C57BL/6J). Standardized polyvinyl alcohol sponges were implanted in the wounds to allow for measurement of collagen deposition. Animals were harvested on postoperative days (PODs) 3, 5, 7, 10, 14, and 28, and their wounds subjected to tensiometric breaking strength analysis. Nonisotopic in situ hybridization quantitative analysis for iNOS, endothelial NOS (eNOS), basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and interleukin-4 (IL-4) expression in the wounds was performed. Hydroxyproline levels were quantitated in the harvested polyvinyl alcohol sponges. Data were analyzed with the Students t test. RESULTS: No significant differences were found in breaking strengths or levels of hydroxyproline (and thus collagen) in iNOS-KO versus wild-type wounds at all tested time points. Flawed iNOS expression levels in iNOS-KO animals were similar to (functional) iNOS expression in wild-types. eNOS and bFGF expression nearly doubled on POD 7 in iNOS-KO incisions (P =.002, and.002), respectively and remained 200% to 300% elevated thereafter. TGF-beta1 expression was increased approximately 50% to 100% in iNOS-KO wounds on PODs 5 and 7 (P =.006 and.01, respectively). VEGF and IL-4 expression was elevated by 25% to 100% in wild-type compared with iNOS-KO animals at all time points (P <.01). CONCLUSIONS: The overexpression of TGF-beta1 and eNOS may represent mechanisms in iNOS-KO mice to compensate for their loss of functional iNOS, resulting in incisional wound healing equivalent to controls. Their impaired expression of VEGF and IL-4, on the other hand, may partially explain the delayed excisional wound healing noted in these animals.     

Biological fate and clinical implications of arginine metabolism in tissue healing

Since its discovery in 1987, many biological roles (including wound healing) have been identified for nitric oxide (NO). The gas is produced by NO synthase using the dibasic amino acid l ‐arginine as a substrate. It has been established that a lack of dietary l ‐arginine delays experimental wound healing. Arginine can also be metabolized to urea and ornithine by arginase‐1, a pathway that generates l‐ proline, a substrate for collagen synthesis, and polyamines, which stimulate cellular proliferation. Herein, we review subjects of interest in arginine metabolism, with emphasis on the biochemistry of wound NO production, relative NO synthase isoform activity in healing wounds, cellular contributions to NO production, and NO effects and mechanisms of action in wound healing.     

Wound healing in nude mice: a study on the regulatory role of lymphocytes in fibroplasia

In order to understand the role of T cells in postinjury fibroplasia, we have studied wound healing in congenitally athymic nude mice that lack a normally developed T cell system. Healing of incisional wounds, as assessed by wound breaking strength, was significantly stronger in nude mice compared with normal thymus-bearing animals. This was accompanied by a marked increase in the amount of reparative collagen synthesized at the wound site, as assessed by the hydroxyproline content of subcutaneously implanted sponges. Because nude mice have some extrathymic T cell maturation, we used an anti-Thy-1.2 (30H12) monoclonal antibody to selectively deplete T cells in vivo. Although such treatments impaired wound healing in normal mice, they had no effect on any wound healing parameter in nude mice. In a separate experiment, T cell reconstitution of nude mice, sufficient to significantly enhance in vivo delayed hypersensitivity responses, led to a decrease in both wound breaking strength and hydroxyproline deposition in subcutaneously implanted polyvinyl sponges. The data suggest that T cells play a dual role in wound healing: an early stimulatory role on macrophages, endothelial cells, and fibroblasts, and a late counterregulatory role, which may be responsible for the orderly completion of wound repair.     

Wound healing in Mac‐1 deficient mice

Mac‐1 (CD11b/CD18) is a macrophage receptor that plays several critical roles in macrophage recruitment and activation. Because macrophages are essential for proper wound healing, the impact of Mac‐1 deficiency on wound healing is of significant interest. Prior studies have shown that Mac‐1^?/? mice exhibit deficits in healing, including delayed wound closure in scalp and ear wounds. This study examined whether Mac‐1 deficiency influences wound healing in small excisional and incisional skin wounds. Three millimeter diameter full thickness excisional wounds and incisional wounds were prepared on the dorsal skin of Mac‐1 deficient (Mac‐1^?/?) and wild type (WT) mice, and wound healing outcomes were examined. Mac‐1 deficient mice exhibited a normal rate of wound closure, generally normal levels of total collagen, and nearly normal synthesis and distribution of collagens I and III. In incisional wounds, wound breaking strength was similar for Mac‐1^?/? and WT mice. Wounds of Mac‐1 deficient mice displayed normal total macrophage content, although macrophage phenotype markers were skewed as compared to WT. Interestingly, amounts of TGF‐β1 and its downstream signaling molecules, SMAD2 and SMAD3, were significantly decreased in the wounds of Mac‐1 deficient mice compared to WT. The results suggest that Mac‐1 deficiency has little impact on the healing of small excisional and incisional wounds. Moreover, the findings demonstrate that the effect of single genetic deficiencies on wound healing may markedly differ among wound models. These conclusions have implications for the interpretation of the many prior studies that utilize a single model system to examine wound healing outcomes in genetically deficient mice.     

Wound fluid inhibits wound fibroblast nitric oxide synthesis

BACKGROUND: Fibroblast-derived nitric oxide (NO) is an autocrine stimulator of collagen synthesis by wound fibroblasts. Little is known about the in vivo regulation of wound fibroblast NO synthesis. We investigated the net effect of wound environment on wound fibroblast NO production and characterized a soluble factor mediating this effect. MATERIALS AND METHODS: Wound fibroblasts and acellular wound fluid (pool of 100 Lewis rats) were isolated from subcutaneously implanted polyvinyl alcohol sponges harvested 10 days post-wounding. Fibroblasts were incubated in the presence of 10% (v/v) wound fluid. Nitrite, an index of NO synthesis, was measured in supernatants by Griess reagent. RESULTS: Wound fibroblasts spontaneously synthesize large amounts of NO. Spontaneous NO synthesis was further increased by LPS + IFN-gamma (P < 0.001). Wound fluid significantly inhibited both spontaneous and LPS plus IFN-gamma-stimulated NO synthesis (by 88 and 55%, respectively; P < 0.01). Wound fluid from 5- to 35-day-old wounds equally suppressed NO synthesis. Separation by Sephadex G-100 gel filtration identified the active factor in wound fluid to have a molecular weight of about 100 kDa. Characterization of this factor showed it to be a heat-resistant (56 degrees C, 30 min), trypsin-sensitive, and neuraminidase-resistant protein (ammonium sulfate precipitation). The isoelectric point appeared to be 7.0, as determined by ion exchange chromatography. Addition of high arginine did not restore the effect of wound fluid on fibroblast NO synthesis, suggesting that substrate is not a limiting factor. CONCLUSION: Our data demonstrate that following postoperative day 5 the wound environment contains a high molecular weight protein that inhibits NO synthesis by wound fibroblasts.     

In vivo characterization of interleukin-4 as a potential wound healing agent

Interleukin-4 increases the synthesis of extracellular matrix proteins, including types I and III collagen and fibronectin, by both human and rat fibroblasts. Because fibroblasts are the final common effector cells of most phases of tissue repair, this study set out to investigate the effects of interleukin-4 on the healing of three different types of wounds. Acute excisional and chronic granulating wounds inoculated with Escherichia coli and incisional wounds in streptozotocin-induced diabetic Sprague-Dawley rats were used. Recombinant murine or human interleukin-4 was applied topically to the open wounds at doses of 0.1, 1.0, or 10.0 microg/cm(2)/wound for 5 or 10 days. Incisional wounds received the same doses once-at the time of wounding. The time taken to achieve wound closure or wound breaking strength measurements of wounds was recorded and compared with relevant untreated control groups. Wound contraction was impaired in the presence of bacteria, and this was reversed by all doses of recombinant murine interleukin-4. Recombinant murine interleukin-4 had no effect on the wound closure of noncontaminated wounds; it reduced wound breaking strength in acute excisional wounds, except in a contaminated setting when wounds were treated with 1.0 pg/cm(2)/wound. Recombinant interleukin-4 (1.0 microg) improved breaking strength of both diabetic and normal incisional wounds. The apparent pleiotropic effect of interleukin-4 on wound breaking strength under different wound conditions may be related not only to the activity of the fibroblast but also the ratio of cross-linked collagen/total collagen content of wounds. This study suggests that interleukin-4 may be a useful agent for accelerating closure of wounds, particularly where healing is impaired.     

Effect of bFGF on the inhibition of contraction caused by bacteria

Bacterial contamination of open wounds significantly inhibits wound contraction required in the healing process. Basic fibroblast growth factor (bFGF) has been shown to overcome contraction inhibition in wound-healing models impaired by diabetes or steroids. This study was designed to determine the effect of bFGF on wound contraction inhibition in an area contaminated with bacterial overgrowth. The topically applied bFGF reversed inhibition to wound contraction that normally occurs with bacterial contamination. This reversal does not appear to be due to increased collagen synthesis since bFGF has been shown to decrease collagen synthesis and the treated wounds showed no increase in breaking strength. The use of bFGF significantly decreased the number of days required for wound healing (P less than 0.01) despite active bacterial invasion and may be of value in the treatment of human contaminated wounds.