Effect of matrix metalloproteinase inhibition on colonic anastomotic healing in rats.

Wound strength depends on the balance between collagen synthesis and degradation; however, the role of collagen breakdown in wound healing is still not well understood. We investigated the role of matrix metalloproteinases in wound healing by using BE16627B, a matrix metalloproteinase inhibitor. Identical surgical procedures consisting of a colonic anastomosis (single-layer, inverted) and implantation of an osmotic pump in the back were performed in male Sprague-Dawley rats weighing 270 to 290 grams. The animals were randomly assigned to receive either BE16627B (n = 10) dissolved in dimethylsulfoxide and diluted with ethylene glycol at a dosage of 2.4 mg/rat/day for 3 days or the vehicle solution alone (n = 11). The solutions were administered through the surgically implanted osmotic pumps. The animals were killed 4 days after surgery, and the colonic bursting pressure (mm Hg) and hydroxyproline concentration (microg/mg wet tissue, index of collagen) were measured. The administration of BE16627B enhanced colonic anastomotic healing, as measured by the increase in the colonic bursting pressure (160 +/- 12 vs. 125 +/- 7 mm Hg; P < 0.05) and the increase in the soluble fraction of collagen (0.27 +/- 0.01 vs. 0.21 +/- 0.01 microg/mg wet tissue; P < 0.01) in the anastomosis. Histologic examination of the tissue revealed that the use of BE16627B resulted in the preservation of the multilayered colonic structure and increased the network of collagen between both ends of the colon in the thickening submucosal layer. These findings demonstrate that the inhibition of matrix metalloproteinase activity influences colonic anastomotic healing, indicating a potential mechanism for enhancing anastomotic healing.

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 arginine in immunonutrition.

Arginine plays an important role in many physiologic and biologic processes beyond its role as a protein-incorporated amino acid. Dietary supplementation of arginine can enhance wound healing, regulate endocrine activity and potentiate immune activity. Under normal unstressed conditions the arginine requirement of adult humans is fulfilled by endogenous sources, however this is compromised during times of stress, especially in critical illness. These finding have led to use of arginine supplementation as part of an immune-enhancing dietary regimen to help combat the immune suppression seen in such patients. Though the results from studies examining the use of this type of immunonutrition in critically ill patients are far from definitive, they are promising that this mode of therapy may be of some advantage. A better understanding of the in vivo biology of arginine and its metabolism is necessary to truly define a benefit from arginine supplementation.

Arginine and nutrition in renal disease.

The physiological significance of arginine metabolism extends far beyond its incorporation as an amino acid into proteins. In addition to its effects when administered as a dietary supplement, the end-products of arginine metabolism by the enzymes arginase, arginine decarboxylase (ADC), and nitric oxide synthase (NOS) have been shown to play roles in wound healing, immune response, tumor biology, and the regulation of inflammation. These properties make arginine metabolism a significant concern in defining and, likely, treating renal disease.

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.

Modulation of inflammation and immunity by arginine supplements.

Arginine holds a key position in the cellular functions and interactions that occur during inflammation and immune responses. The competition for arginine as a substrate between nitric oxide synthase and arginase appears to be at the core of the regulation of the inflammatory process. This review examines some of the recently defined effects of arginine on various inflammatory processes and immune cell functions.

Metabolic effects of arginine in a healthy elderly population.

Recently there has been much interest in the use of arginine to stimulate immune responses and to promote wound healing. In the present study, the effect of an oral supplementation with arginine on the metabolism of 45 healthy, nonsmoking, elderly volunteers was investigated. Subjects were divided into two groups that received either arginine aspartate (17 g free arginine) (n = 30) or a placebo (n = 15). The supplements were taken for a period of 14 days. Dietary intake of food was not controlled. Blood chemistry, lipid profiles, and as an index of nutritional status, serum insulin-like growth factor-1 levels and nitrogen balance were compared before and after supplementation. Two weeks of arginine supplementation led to a significant elevation of serum insulin-like growth factor concentrations and an improved and positive nitrogen balance (2.0 +/- 0.41 g N) when compared with controls (0.11 +/- 0.47 g N; p = 0.0114). In addition the arginine-supplemented group demonstrated a decreased total serum cholesterol with a reduction in the low-density lipoprotein but not the high-density lipoprotein fraction resulting in a increase in the ratio of low- to high-density lipoprotein fraction. No adverse effects were observed at this dosage of arginine. The data suggest that oral arginine supplementation may be used safely in elderly humans.

Arginine stimulates wound healing and immune function in elderly human beings.

BACKGROUND: Experimentally, arginine enhances immune function and promotes wound healing. In this randomized double-blind study we investigated the effect of oral arginine supplementation on wound healing and T-cell function in elderly human beings (more than 65 years of age). METHODS: Thirty elderly, healthy, human volunteers (15 men and 15 women) received daily supplements of 30 gm arginine aspartate (17 gm free arginine). Fifteen volunteers (nine men and six women) received a placebo syrup. Fibroplastic wound responses were assessed by inserting a polytetrafluoroethylene catheter subcutaneously into the right deltoid region. Epithelialization was examined by creating a 2 x 2 cm split thickness wound on the lateral aspect of the upper thigh. Mitogenic response of peripheral blood lymphocytes to concanavalin A, phytohemagglutinin, pokeweed mitogen, and allogeneic stimuli was assayed at the beginning and end of supplementation. Polytetrafluoroethylene catheters were analyzed for alpha-amino nitrogen (assessment of total protein accumulation), hydroxyproline (index of reparative collagen synthesis), and DNA accumulation (index of cellular infiltration). RESULTS: Arginine supplementation for 2 weeks significantly enhanced wound catheter hydroxyproline accumulation (26.49 +/- 2.39 nmol/cm vs 17.41 +/- 2.04 nmol/cm) and total protein content (43.47 +/- 3.85 micrograms/cm vs 21.95 +/- 2.5 micrograms/cm). Arginine did not influence the DNA content of the catheters or the rate of epithelialization of the skin defect. Peripheral blood lymphocyte responses to mitogenic and allogenic stimulation were greater in the arginine supplemented group. Serum insulin-like growth factor-1 levels were significantly elevated in the arginine group. CONCLUSIONS: The data suggest that arginine supplementation may improve wound healing and immune responses in the elderly.

Arginine enhances T-cell responses in athymic nude mice.

Supplemental L-arginine has been shown to enhance thymic and T-cell responses in rodents. We examined the ability of supplemental dietary L-arginine to induce T-cell function in athymic nude mice that lack a normally developed T-cell system. Groups of male nude (nu/nu) mice (Balb/c background) 7 to 8 weeks old were given for 2 weeks 1.2% arginine hydrochloride solution for drinking, whereas controls received acidified tap water. All mice ingested a standard laboratory chow. In the first experiment, the arginine-supplemented animals had significantly greater number of T cells in the spleen (assessed by the number of Thy 1.2-positive lymphocytes) and these cells had enhanced mitogenic responses to mitogenic stimulation (phytohemagglutinin and concanavalin A). In vivo delayed-type hypersensitivity responses to 2,4-dinitro-1-difluorobenzene were also significantly increased after the 2 weeks of arginine supplementation. In a second experiment, mice maintained under the same conditions were skin grafted with rat tail skin. Animals were observed for 100 days for rejection but no significant difference was noted in skin graft survival. We conclude that dietary arginine can increase extrathymic T-cell maturation and function, but cannot induce in vivo allogeneic graft recognition in athymic nude mice.

Arginine enhances wound healing and lymphocyte immune responses in humans.

Arginine has been shown to enhance wound healing and T-cell-mediated immune function in rodents. In this study the effect of oral arginine supplementation on human collagen synthesis and T-cell function was studied in 36 healthy, nonsmoking human volunteers. While volunteers were under local anesthesia, a 5 cm segment of expanded polytetrafluoroethylene tubing (1 mm outer diameter, 90 mu pore size) was inserted subcutaneously into the right deltoid region. The volunteers were then randomized into three groups that were given the following substances: (1) daily supplements of 30 gm arginine hydrochloride (24.8 gm free arginine); (2) 30 gm arginine aspartate (17 gm free arginine) daily; or (3) placebo. The supplements were given orally for 2 weeks; dietary intake was not controlled. Mitogenic responses of peripheral blood lymphocytes to phytohemagglutinin and concanavalin A were assayed at the start of study and at 1 and 2 weeks after supplementation. At 2 weeks the catheters were removed, and the amount of hydroxyproline was determined as an index of new collagen synthesis and deposition. Arginine supplementation significantly enhanced the amount of collagen deposited into a standardized wound as assessed by the amount of hydroxyproline present (10.1 +/- 2.32 nmol/cm graft in controls vs 17.57 +/- 2.16 nmol/cm in the arginine aspartate group, [p = 0.028] and vs 23.85 +/- 2.16 nmol/cm in the arginine hydrochloride group [p less than 0.001]). In parallel, arginine supplementation at both doses increased lymphocyte mitogenesis in response to phytohemagglutinin and concanavalin A. The data suggest that arginine may be of clinical benefit in improving wound healing and immune responses.

Interleukin 2 enhances wound healing in rats.

Antigen-stimulated lymphocytes secrete lymphokines which have been shown to enhance in vitro fibroblast migration, proliferation, and protein synthesis. In the present experiments, the effect of human recombinant interleukin 2 (RIL-2) on wound healing was assessed in vivo. Groups of male Lewis rats, 225-250 g, underwent intraperitoneal insertion of osmotic pumps and a 7-cm dorsal skin incision with subcutaneous placement of polyvinyl alcohol sponges under anesthesia. The dorsal wounds were closed with stainless-steel sutures. The dose of RIL-2 administered was 60,000 u/rat/day for 7 days in experiment I, and 140,000 u/rat/day for 7 days in experiment II. Controls received equal volumes of excipient. Animals were sacrificed 10 days post wounding and wound healing was assessed by fresh breaking strength, fixed breaking strength (following 72 hr of Formalin fixation which maximally crosslinks the collagen present), and sponge hydroxyproline content (an index of reparative collagen accumulation). In vivo RIL-2 administration significantly augmented wound fresh and fixed breaking strength and wound collagen synthesis. Higher doses of RIL-2 (experiment II) did not result in further increases in the parameters studied. The data suggest that lymphocytes participate directly in the process of wound healing.

Wound healing and thymotropic effects of arginine: a pituitary mechanism of action.

Supplemental dietary arginine HCl (ARG-HCl) minimizes immediate post-wounding weight loss, accelerates wound healing, and is thymotropic for uninjured and wounded rats. The present experiments were to determine if arginine-pituitary interactions underlie these effects because arginine is a growth hormone secretagogue. Effects of 1% dietary ARG-HCl supplements (0.5% added to a regular commercial rat diet containing 1.8% ARG, 0.5% in drinking water) were studied in (a) hypophysectomized (hypox) rats supplemented with ACTH, L-thyroxine, testosterone propionate, (b) such hypox rats additionally supplemented with bovine growth (hypox + bGH) hormone, (c) intact rats (Int), and (d) intact rats supplemented with growth hormone (Int. bGH). Group (a) hypox rats healed their wounds as rapidly as intact rats (dorsal skin incision breaking strength, accumulation of reparative collagen in sc polyvinyl alcohol sponges). Group (b) hypox, bGH rats showed increased wound breaking strength and accumulation of reparative collagen in the sc sponges to levels significantly greater than those of intact controls; bGH given to intact controls did not affect these indices of wound healing. Supplemental ARG-HCl given intact rats significantly minimized immediate postoperative weight loss, increased wound breaking strength and sponge reparative collagen accumulation, and increased thymic weight. None of these effects of supplemental ARG-HCl were observed in group (a) hypox rats or group (b) hypox + bGH rats. We conclude that an intact hypothalamic-pituitary axis is necessary for these beneficial effects of supplemental ARG-HCl given wounded rats.

Arginine stimulates thymic immune function and ameliorates the obesity and the hyperglycemia of genetically obese mice.

The effect of 6-day dietary arginine supplementation on the weight gain, blood glucose, thymus weight, thymic lymphocyte content, and in vitro thymic lymphocyte immune reactivity was studied in obese (C57BL/6J-OB/)B) and heterozygous lean mice. Control mice were fed a commercial laboratory chow (1.8% arginine content) and drank tap water, while supplemented mice were given 0.5% arginine in the chow and 0.5% arginine solution for drinking. All mice ate and drank ad libitum. Supplemental arginine significantly decreased the weight gain (1.2 g vs. 2.2 g, p less than 0.01) and blood glucose levels (303 mg% vs 236 mg%, p less than 0.02) of the OB/OB mice; no such effects were noted in the lean heterozygotes, all of which had normal blood glucose levels. OB/OB mice had thymus glands which weighed less and contained significantly fewer lymphocytes than their lean littermates. In vitro mitogen-stimulated thymic lymphocyte protein synthetic rates were equal in chow-fed lean and OB/OB mice. In both groups, supplemental arginine significantly increased thymus weight, the number of thymic lymphocytes per gland, and thymic lymphocyte immunoreactivity in vitro. The hormonal secretagogue activity of arginine on the pituitary may explain its beneficial effects on the rate of weight gain, hyperglycemia, and depressed thymic immune function of OB/OB mice.

Arginine stimulates lymphocyte immune response in healthy human beings.

The effect of daily dietary supplements of 30 gm of arginine HCl for 7 days on peripheral blood lymphocyte (PBL) mitogenic reactivity in vitro was measured in 21 healthy human volunteers. Arginine significantly increased stimulation indices of PBL following concanavalin A (Con A) (57.9 +/- 11.4 versus 216.9 +/- 46.6, P less than 0.01) and phytohemagglutinin (PHA) (84.1 +/- 12.8 versus 307.0 +/- 59.4, P less than 0.001) stimulation in a microculture assay utilizing RPMI 1640 medium supplemented with 10% heat-inactivated autologous serum. Similar enhanced blastogenesis was observed using medium supplemented with 10% heat-inactivated pooled AB normal human serum. In six volunteers studied following 3 days of similar arginine supplementation, blastogenic responses of their peripheral blood lymphocytes were already significantly enhanced, although not as greatly as after 7 days. Arginine had no effect on total peripheral blood lymphocyte counts and on T- and B-cell ratios. The effects of supplemental dietary arginine could not be duplicated in vitro by increasing the arginine concentration in the culture medium. Furthermore, dietary arginine supplementation did not increase cell viability in culture. Minimal side effects were noted, such as nausea or diarrhea, which responded to lowering the dose ingested at one time. No deleterious effects were noted on liver function test results. We conclude that supplemental dietary arginine is a safe nutritional stimulator of lymphocyte immune reactivity in healthy human beings.

Thymic stimulatory actions of arginine.

Various arginine HCl supplements (0.5-3%), half added to a basal commercial rodent chow (1.8% arginine) and half to the drinking water, were given to 8- to 9-week-old male CBA/J mice for 6 days. Control animals were fed the basal chow and drank tap water. All mice ate and drank ad libitum. Weight gain and food intake were similar in all groups. All arginine supplements increased significantly: thymic weight (average 22%), thymic lymphocyte content (average 45%), and the in vitro reactivity of thymic lymphocytes judged by the incorporation of 3H-leucine into the TCA-precipitable protein fraction in response to stimulation by phytohemagglutinin and concanavalin A. All these thymic effects resulted from the 0.5% arginine hydrochloride supplement; further increases in arginine supplementation did not increase these effects. These data suggest that supplemental arginine may improve host defence mechanisms and thereby may play an important role in the care of severely injured or ill patients, since it is well established that their defense mechanisms are reduced.

Supplemental arginine increases thymic cellularity in normal and murine sarcoma virus-inoculated mice and increases the resistance to murine sarcoma virus tumor.

Arginine supplements were given to 6 week old CBA mice beginning 3 days prior to inoculation with a murine sarcoma virus, the Moloney Sarcoma Virus (MSV). Although the basal diet contained 1.8% arginine and was therefore not arginine-deficient, supplementation of the diet and the drinking water with 0.5% arginine HCl reduced tumor incidence, lengthened the latency period, decreased tumor size, and hastened tumor regression. Arginine also increased thymic weight and cellularity in normal and in MSV-inoculated mice. The antitumor action of arginine may be related to its effect on the thymus.

Arginine: an essential amino acid for injured rats.

The influence of arginine supplements on growth and healing of skin incisional wounds was studied in rats fed either a chemically defined diet lacking arginine or a laboratory chow containing 1.8% arginine. Rats fed the arginine-free diet grew more poorly than did arginine-supplemented rats (1.8 vs. 7.0 gm/day) in the preoperative period. After operation arginine-deficient animals grew very poorly (1 gm/day), while arginine-supplemented rats gained 4.3 gm/day. Arginine-deficient animals showed impaired wound healing, as judged by the breaking strengths of their incisions 10 days after wounding (228 vs. 293 gm for the arginine-supplemented rats). Arginine-deficient rats also showed decreased collagen deposition in a specific wound site, as indicated by the decreased content in hydroxyproline in sponge granulomas (2.5 vs. 4.2 mg/100 mg. of sponge for the arginine-supplemented rats). In rats fed commercial chow, 1% arginine decreased the postoperative weight loss associated with injury (0.7 vs. 5.2 gm) in one experiment and improved wound strength in two experiments (312 vs. 188 gm in one experiment and 309 vs. 246 gm in another). Arginine also increased hydroxyproline deposition in a specific wound area (5.5 vs. 4.1 mg in one experiment and 3.1 vs. 1.9 mg. in another). It is concluded that arginine has two roles in wounded animals. It is essential for the synthesis of the increased amounts of reparative collagen required for wound healing, and it decreases some of the negative aspects of the metabolic responses to injury. These are thought to be associated with an arginine-induced growth hormone release.

White cell involvement in the inflammatory, wound healing, and immune actions of vitamin A.

Stress or injury-induced phenomena, such as impaired wound healing and immune depression, may be related to impaired function of certain leukocyte populations. Since vitamin A prevents some aspects of stress, we studied its effect on various white cell populations in normal and injured rats. Supplemental vitamin A (150,000 IU/kg chow) to normal rats resulted in marked increases in thymic weight and lymphocytes without any effct on adrenal weight. The basal chow contains 13,700 IU vitamin A per kg. In rats subjected to moderately severe injury (dorsal wounding or unilateral femoral fracture), supplemental vitamin A greatly diminished the thymic involution observed in chow-fed controls and delayed or minimized the accompanying adrenal hypertrophy. In uninjured rats, supplemental vitamin A induced in three to four days a temporary circulatory leukocytosis characterized by lymhocytosis, monocytosis, and a relative neutropenia. These changes in the blood picture persisted one day after femoral fracture. On the second and third day postfracture the lymphocyte and neutrophil values returned to normal while the monocytosis persisted. Polyvinyl alcohol sponges implanted next to the fracture site demonstrated that supplemental vitamin A consistently increased the number of white blood cells migrating into the wound area and showed significantly larger numbers of monocytes/macrophages. These data suggest that vitamin A influences the numbers and nature of white cells involved in immune, inflammatory, and wound healing processes. In addition to the known antiglucocorticoid activity of vitamin A, these effects may represent a direct beneficial action of dietary vitamin A supplements for stressed and injured animals.