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.

Enhanced collagen accumulation following direct transfection of the inducible nitric oxide synthase gene in cutaneous wounds.

Inducible nitric oxide synthase (iNOS) is expressed during cutaneous wound repair. Mounting evidence suggests that wound nitric oxide (NO) augments collagen accumulation. We hypothesized that in vivo transfection of wound cells with the iNOS gene would increase physiological wound NO levels and thus augment collagen accumulation. Polyvinyl alcohol sponges were instilled with a mammalian expression plasmid (pMP6) containing either the chloramphenicol acetyl transferase (CAT) reporter or murine iNOS gene driven by a CMV immediate-early promoter. Plasmid DNA was injected alone or in complex with cationic liposomes, and the sponges were placed subcutaneously in male Sprague-Dawley rats which had received a longitudinal dorsal midline incision. Animals were sacrificed at different time points post-wounding and the sponges assayed for CAT activity, transfected iNOS mRNA, total nitrate and nitrite concentration (NOx) (as an index of wound NO synthesis), and hydroxyproline content (as an index of sponge collagen accumulation). The results demonstrate that wound cells were more efficiently transfected by naked DNA than by liposome mediated transfection and that maximal expression of both iNOS and CAT occurred at 48 hrs with a rapid decline after this time point. After 7 days, iNOS transfected sponges had accumulated significantly more collagen than those transfected with CAT. We conclude that cutaneous wounds can be successfully transfected by direct injection of naked DNA and that increased iNOS expression precedes an increase in collagen synthesis.

Tacrolimus impairs wound healing: a possible role of decreased nitric oxide synthesis.

BACKGROUND: The effect of the immunosuppressant tacrolimus on wound healing is not known. Tacrolimus has been shown to decrease nitric oxide synthesis. The systemic inhibition of wound nitric oxide synthesis leads to impaired healing. METHODS: We studied the effect of systemic tacrolimus treatment on wound-breaking strength and collagen deposition 10 days after wounding in rats and to correlate the outcome of healing with wound nitric oxide synthesis. Beginning at the day of wounding, rats were treated once daily by intraperitoneal injections with 0.5, 1.0, or 2.0 mg tacrolimus/kg body weight. Nitrite and nitrate were measured in wound fluid as an index of wound nitric oxide synthesis. Expression of inducible nitric oxide synthase in the wound was investigated by immunohistochemistry. Splenic lymphocytes were tested for proliferative activity. Tacrolimus levels in blood and wound fluid were measured by enzyme-linked immunosorbent assay. RESULTS: Systemic tacrolimus treatment was well tolerated by all rats. Tacrolimus accumulated in wound fluid. Tacrolimus levels in wound fluid were found to be approximately 10-fold higher than blood levels (P < 0.001). Tacrolimus (2.0 mg/kg/day) reduced wound-breaking strength (P < 0.01) and collagen deposition (P < 0.05). This was paralleled by decreased wound nitrite + nitrate levels (P < 0.001) and wound-inducible nitric oxide synthase expression. Splenic lymphocyte proliferative activity was significantly decreased by 1.0 and 2.0 mg tacrolimus/kg body weight/day (P < 0.05), indicating that the tacrolimus doses used were immunosuppressive. CONCLUSION: Our data show for the first time that tacrolimus impairs wound healing, and this is reflected by diminished wound nitric oxide synthesis.

Nitric oxide metabolism in wounds.

Arginine can be metabolized in wounds to nitric oxide and citrulline by nitric oxide synthase or to urea and ornithine by arginase. We investigated the expression of these arginine metabolic pathways over a 3-week period. Groups of 8-10 male Balb/C mice underwent a dorsal skin incision and subcutaneous polyvinyl alcohol sponge implantation. The animals were sacrificed at various times, and sponges were harvested to obtain wound fluid and wound cells. Cells or whole sponges were incubated with L-[2,3-(3)H]arginine, with or without N(G)-L-monomethyl-arginine (NMMA, a competitive inhibitor of nitric oxide synthase). Nitrite and nitrate (both stable end products of nitric oxide metabolism) and amino acids were measured in wound fluid and wound cell culture supernatants. Increasing concentrations of nitrite and nitrate were noted in wound fluid and in whole sponge cultures until the second week postwounding, indicating sustained wound nitric oxide synthesis. In wound fluid arginine levels were undetectable at all times, suggesting sustained utilization. Wound fluid citrulline levels showed an early peak and then a gradual decrease, suggesting that recycling for continued nitric oxide production may occur. Wound fluid ornithine levels increased until Day 10 and remained elevated, indicative of continued arginase activity. In vitro production of nitrite/nitrate and citrulline by cells and whole sponges was inhibitable by NMMA. Inducible nitric oxide synthase expression was confirmed by immunoblotting, while immunohistochemistry demonstrated that macrophages are a major source of wound nitric oxide. The data show that nitric oxide synthesis occurs for prolonged periods after injury and macrophages appear to be a major cellular source.

Diabetes-impaired healing and reduced wound nitric oxide synthesis: a possible pathophysiologic correlation.

BACKGROUND: Nitric oxide (NO) is synthesized in wounds, but its role in the healing process is not fully understood. The inhibition of NO production during wound healing is accompanied by decreased wound reparative collagen deposition. To further define the role of NO in reparative collagen accumulation, we studied its production during diabetes-induced wound healing impairment. METHODS: Male Sprague-Dawley rats (290 to 310 gm) were rendered diabetic by intraperitoneal streptozotocin administration. Seven days after induction of diabetes (blood glucose greater than 300 mg/dl), the rats underwent dorsal skin incision and subcutaneous implantation of polyvinyl alcohol sponges. Beginning on the day of wounding, 21 diabetic animals were treated with 3 units/day insulin via intraperitoneally implanted miniosmotic pumps. Ten days after injury, wound breaking strength was determined, and wound collagen accumulation and types I and III collagen gene expression were measured in subcutaneously implanted polyvinyl alcohol sponges. NO-synthesis, as measured by nitrite/nitrate accumulation, was determined in wound fluid and in supernatants of wound cell cultures. RESULTS: Streptozotocin-induced diabetes markedly impaired wound breaking strength and collagen deposition. A parallel decrease occurred in wound NO synthesis as reflected by decreased nitrite/nitrate concentration in wound fluid and in diminished ex vivo NO production by wound cells. Insulin treatment partially but significantly improved wound mechanical strength (p < 0.01) and collagen accumulation (p < 0.001). Decreased wound NO accumulation and ex vivo NO production by wound cells were also partially restored by insulin treatment. CONCLUSIONS: Impaired diabetic wound healing is paralleled by decreased wound NO synthesis, supporting the hypothesis that NO plays a significant role in wound reparative collagen accumulation.

Sepsis impairs anastomotic collagen gene expression and synthesis: a possible role for nitric oxide.

Although intra-abdominal sepsis is known to impair colon healing by inhibiting anastomotic collagen synthesis, the effect of systemic sepsis on this process is unknown. Endotoxins and cytokines associated with sepsis induce nitric oxide synthesis both systemically and locally within colonic tissue. We hypothesized that systemic sepsis impairs colonic healing and examined a possible correlation with nitric oxide expression. Male Sprague-Dawley rats received intraperitoneal injections of either saline (sham group) or Escherichia coli endotoxin (lipopolysaccharide 1 mg/100 g body weight) at Times -24 and -12 hr (LPS group). All animals underwent laparotomy and left colonic anastomosis at Time 0. At 24 and 96 hr postlaparotomy rats were sacrificed, the anastomoses excised, and [3H]-proline incorporation into protein measured as an index of total new protein synthesis (TNP). Digestion with purified collagenase yielded incorporation into the collagen fraction (CDP). Additional sham and LPS-treated rats were sacrificed at 24, 72, and 120 hr, the anastomoses excised, and nitric oxide synthase activity in the tissue measured by the conversion of [3H]-arginine to [3H]citrulline in an ex vivo culture system. Finally, sham and LPS rats were sacrificed at 120 hr for measurement of colon anastomotic bursting pressure. Systemic sepsis significantly impaired new collagen synthesis in anastomotic tissue at 24 hr compared to control samples (P < 0.02). No difference was noted at 96 hr. TNP synthesis was similar in both groups at 24 or 96 hr. Northern blot analysis confirmed a significant decrease in Type I and Type III collagen mRNA expression at 24 hr in septic rats. Anastomotic bursting pressure was also decreased in the septic group (P < 0.003). Sepsis elevated nitric oxide synthase activity in anastomotic tissue 24 hr postanastomosis, when compared to sham tissue (P < 0.0001). These data suggest that systemic endotoxin induces nitric oxide synthesis at the anastomotic site. The simultaneous dysregulation of collagen gene expression and synthesis with decreased anastomotic strength suggests a possible regulatory role for nitric oxide in gastrointestinal healing.

Nitric oxide, an autocrine regulator of wound fibroblast synthetic function.

Nitric oxide (NO) is synthesized in wounds, but its exact role and cellular source are not known. Wound fibroblasts (WF) are phenotypically characterized by increased collagen synthesis and contractility. We hypothesized that WF may be also phenotypically altered during wound healing to synthesize NO. WF were isolated from polyvinyl alcohol sponges implanted in male Lewis rats and harvested 10 days later. Proliferation in response to 10% fetal bovine serum was assessed by [3H]thymidine incorporation in a microculture system. A fibroblast-populated collagen lattice was used for assaying contractility. Collagen synthesis was determined by measuring the collagenase-sensitive fraction of protein-incorporated [3H]proline. Fibroblasts were incubated in the presence or the absence of 0.5 mM S-methyl-isothio-uronium or 0.5 mM N-monomethyl-L-arginine, both competitive inhibitors of NO synthase. WF spontaneously synthesize and release NO (4.60 +/- 0.29 nmol nitrite/microg DNA/48 h). Normal dermal fibroblasts do not synthesize NO. WF NO synthesis was limited to the first and second passages postharvest and was inhibitable by S-methyl-isothio-uronium (96%) and N-monomethyl-L-arginine (84%). In vivo iNOS expression by WF was confirmed by in situ hybridization and immunohistochemistry. Inhibition of endogenous NO synthesis had no effect on fibroblast proliferation. However, fibroblast-mediated collagen contraction was enhanced (p < 0.01), and collagen synthesis was significantly decreased (p < 0.05) by inhibiting NO synthase. The data show that WF are phenotypically altered during the healing process to synthesize NO, which, in turn, regulates their collagen synthetic and contractile activities.

Acute protein-calorie malnutrition impairs wound healing: a possible role of decreased wound nitric oxide synthesis.

BACKGROUND: Nitric oxide is synthesized in wounds. Systemic inhibition of wound nitric oxide synthesis decreases wound collagen accumulation and wound mechanical strength. The role of nitric oxide during impaired healing is not known. In a model of impaired wound healing induced by acute protein-calorie malnutrition, we correlated wound healing parameters with wound nitric oxide synthesis. STUDY DESIGN: One group of Sprague-Dawley rats was rendered acutely malnourished by restricting its food intake to 50 percent of the food intake of an ad libitum-fed control group. Wound collagen accumulation and types I and III collagen gene expression were measured 10 days postwounding in subcutaneously implanted polyvinyl alcohol sponges. Nitric oxide synthesis was determined in wound fluid and in supernatants of wound cell cultures. RESULTS: Animals with acute protein-calorie malnutrition lost 10.4 +/- 0.8 percent, while controls gained 17.5 +/- 1.2 percent of their original body weight. Protein-calorie malnutrition reduced sponge hydroxyproline contents (995 +/- 84 compared with 1,580 +/- 109 micrograms/100 mg sponge, p < .001), indicating diminished wound collagen accumulation. Gene expression of type III, but not type I, collagen was decreased in wounds of protein-calorie malnutrition animals. Nitrite/nitrate and citrulline concentrations in wound fluid (p < .01) and in wound cell supernatants (p < .001) were also lower in protein-calorie malnutrition animals, indicating a net decrease in nitric oxide production. CONCLUSIONS: Impaired wound collagen accumulation caused by protein-calorie malnutrition may be a reflection of reduced nitric oxide synthesis within the wound.

Stimulation of fibroblast proliferation and matrix contraction by wound fluid.

Fibroblast proliferation and fibroblast-mediated matrix contraction are critical to wound healing. Different cytokines have been shown to modulate fibroblast functions but little is known about the physiological role of these soluble factors during wound repair. In these experiments we characterized a fibroblast stimulating factor in wound fluid. Wound fluid was obtained from subcutaneously implanted polyvinyl alcohol sponges harvested 10 days post-wounding (pool of 100 Lewis rats). Normal dermal fibroblasts were obtained from Lewis rats by an explant technique, while wound fibroblasts were isolated from sponges harvested 10 days post-wounding. Proliferation in response to 0.5% and 10% fetal bovine serum was assessed by [3H]-thymidine incorporation. A fibroblast-populated collagen lattice was used for assaying contractile properties. Wound fibroblasts demonstrated markedly diminished proliferative and enhanced contractile properties compared to normal dermal fibroblasts. 10% wound fluid (v/v) stimulated proliferation of normal dermal fibroblasts (119%, p < 0.001) and wound fibroblasts (103%, p < 0.001). Wound fluid also stimulated collagen gel contraction by normal dermal fibroblasts (24% at 24 hr and 16% at 72 hr, p < 0.01), but not by wound fibroblasts. 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 the soluble factor showed it to be a protein (ammonium sulfate precipitation), sensitive to trypsin digestion, heat resistant (56 degrees C, 30 min), and neuraminidase resistant. The isoelectric point appeared to be 7.0, as determined by ion exchange chromatography. Mitogenic proliferation of thymic lymphocytes was not affected by the active factor, suggesting cell target specificity. These data demonstrate that the wound environment contains high molecular weight protein(s) that promote fibroblast functions, essential for the healing process.

Wound healing in sepsis and trauma.

Wound healing represents a dynamic and immediate response of the body to tissue injury with the purpose of restoring anatomical continuity, structure and function. Success or failure of this complex cascade of events is determined largely by competence of the host's immune system. Sepsis represents one of the most formidable threats to successful wound healing. It can present as a local bacterial colonization of the injury site with minimal systemic reaction or the "systemic inflammatory response syndrome," a primary cause of mortality among critically ill patients. Trauma also predisposes patients to wound complications especially as a result of post-traumatic immunosuppression. This phenomenon exposes the patient to the risk of microbial infection and ultimately the sepsis syndrome. The immune system, therefore, represents a vulnerable gateway through which trauma and sepsis exert their deleterious effect on the wound healing process resulting in increased morbidity and mortality for the surgical patient.

Nitric oxide regulates wound healing.

Nitric oxide (NO) synthesis occurs during wound healing, but its role has not been defined. To study the effect of NO on wound repair, S-methyl isothiouronium (MITU, a competitive inhibitor of NO synthase) was administered at a dose of 10, 50, and 100 mg/kg body weight/day, using intraperitoneally implanted miniosmotic pumps. Groups of 10 male Balb/C mice underwent a dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. The animals were sacrificed 10 days postwounding and wound breaking strength and hydroxyproline content of sponges, an index of reparative collagen deposition, were determined. Some sponges were used to harvest wound fluid and infiltrating cells, which were then incubated overnight with or without 1 mM MITU. Nitrite and nitrate, stable end products of NO, were measured in wound fluid and in wound cell culture supernatants. Continuous intraperitoneal infusion of MITU significantly decreased wound fluid nitrite/nitrate concentrations in a dose dependent manner (P < 0.01). Inhibition of wound NO synthesis by 100 mg MITU/kg/day was paralleled by lowered wound collagen accumulation (P < 0.01) and wound breaking strength (P < 0.01). In vitro NO synthesis by wound cells obtained from animals treated with 100 mg MITU/kg/day was not significantly different from controls (12.6 +/- 1.2 vs 10.7 +/- 0.6 nmole NO2 + NO3/microgram DNA), reflecting the reversible inhibition of NO synthase by MITU. However, NO production was equally inhibited in wound infiltrating cells by the in vitro addition of MITU (83% vs 85%, respectively). These data suggest that nitric oxide synthesis is critical to wound collagen accumulation and acquisition of mechanical strength.