Nitric oxide drives skin repair: Novel functions of an established mediator
2002; Elsevier BV; Volume: 61; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1755.2002.00237.x
ISSN1523-1755
AutoresStefan L. Frank, Heiko Kämpfer, Christian Wetzler, Josef Pfeilschifter,
Tópico(s)Laser Applications in Dentistry and Medicine
ResumoNitric oxide drives skin repair: Novel functions of an established mediator. Wound healing of the skin represents a highly ordered process of important tissue movements that aims for a rapid closure of the wound site and a subsequent regeneration of the injured tissue. The factors ensuring the intercellular communication during repair are only known in part. However, although protein-type mediators are well-established players in this process, it has become evident that the diffusible, gaseous molecule nitric oxide (NO) participates in the orchestration of wound healing. The role of wound-derived NO that critically influences macrophage, fibroblast, and keratinocyte behaviour within the intercellular communication network during repair is subject of this review. Thus, cutaneous wound healing prototypically reflects processes that generally occur also in kidney injury and regeneration. Nitric oxide drives skin repair: Novel functions of an established mediator. Wound healing of the skin represents a highly ordered process of important tissue movements that aims for a rapid closure of the wound site and a subsequent regeneration of the injured tissue. The factors ensuring the intercellular communication during repair are only known in part. However, although protein-type mediators are well-established players in this process, it has become evident that the diffusible, gaseous molecule nitric oxide (NO) participates in the orchestration of wound healing. The role of wound-derived NO that critically influences macrophage, fibroblast, and keratinocyte behaviour within the intercellular communication network during repair is subject of this review. Thus, cutaneous wound healing prototypically reflects processes that generally occur also in kidney injury and regeneration. To overcome tissue damage, the process of cutaneous wound repair represents a highly ordered process that is characterized by temporally and spatially overlapping phases of tissue movements comprising hemorrhage, inflammation, re-epithelialization, granulation tissue formation, and the late remodeling phase of repair. As a result, the integrity of the body's protective layer is maintained, although repair fails to perfectly replace the original skin tissue. Loss of a functional healing process might lead to severe disabilities. Accordingly, chronic, non-healing wound conditions represent a situation of major clinical importance. A series of pathological changes accompanied with several diseases finally leads to severely disturbed wound healing conditions. Among those, the most prominent chronic wound situations are known as decubitus or pressure ulcers, venous ulcers, and diabetic ulcers1.Falanga V. Chronic wounds: Pathophysiologic and experimental considerations.J Invest Dermatol. 1993; 100: 721-725Abstract Full Text PDF PubMed Google Scholar. Molecular and cellular biology and the use of different model systems, especially including genetically engineered animals, have greatly extended our knowledge on skin repair. Nevertheless, the most promising discoveries of growth factor actions for repair to date could not be successfully translated with respect to clinical outcome. To this end, our search for novel mediators has to proceed with the intent to develop novel concepts to improve healing of chronic skin wounds. In most cases, skin injury results in the extravasation of blood constituents from damaged vessels. Blood coagulation then results from a cascade of events that are closely associated to platelet aggregation at the wound site. As a result, a clot is generated that covers the wounded area, plugs the injured vessels, represents a provisional matrix for infiltrating cells, and serves as a reservoir of chemotactic mediators, cytokines, and growth factors that initiate and promote early tissue movements2.Clark R.A.F. Wound repair: Overview and general considerations.in: Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 3-50Google Scholar, 3.Martin P. Wound healing — Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar, 4.Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4308) Google Scholar. Polymorphonuclear neutrophils (PMN) and monocytes represent the first cells that arrive at the wounded area. Both leukocytic cell types are attracted from the circulation by a variety of chemotactic factors. Among those, the CXC-chemokines and CC-chemokines represent potent attractors for both PMN and monocytes, respectively, within the process of skin repair in mice and humans5.Gillitzer R. Goebeler M. Chemokines in cutaneous wound healing.J Leukoc Biol. 2001; 69: 513-521PubMed Google Scholar. Once they arrive at the site of injury, PMN process extracellular matrix (ECM) components, decontaminate bacteria, and produce cytokines. Arriving monocytes differentiate into mononuclear phagocytes at the wound site. Activated macrophages are central to wound repair, as they amplify the initial inflammatory response by release of a variety of signaling molecules, and, moreover, provide growth factors for fibroblast and endothelial cell proliferation and contribute to ECM degradation2.Clark R.A.F. Wound repair: Overview and general considerations.in: Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 3-50Google Scholar, 3.Martin P. Wound healing — Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar, 4.Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4308) Google Scholar, 6.DiPietro L.A. Wound healing: The role of the macrophage and other immune cells.Shock. 1995; 4: 233-240Crossref PubMed Scopus (248) Google Scholar. After a short lag period of several hours after injury, the opening phase of re-epithelialization is marked by the first keratinocytes of the cut edges starting to migrate. Keratinocytes were guided into the wound by fibronectin and fibrin bundles in the provisional clot, which are sensed by the cells by expression of highly controlled integrin receptor subsets2.Clark R.A.F. Wound repair: Overview and general considerations.in: Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 3-50Google Scholar. Migrating keratinocytes do not differentiate, and the leading edge keratinocytes express constituents of a very active fibrinolytic machinery such as plasminogen activators and matrix metalloproteinases (MMP)3.Martin P. Wound healing — Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar. Keratinocytes at the wound margins start to proliferate one or two days after injury. Re-epithelialization is closely controlled by growth factors, predominantly by members of the epidermal growth factor (EGF)-3.Martin P. Wound healing — Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar, and fibroblast growth factor (FGF)-7.Werner S. Keratinocyte growth factor: A unique player in epithelial repair processes.Cytokine Growth Factor Rev. 1998; 9: 153-165Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar family of growth factors. However, unexpected mediators such as the cytokine leptin8.Frank S. Stallmeyer B. Kampfer H. et al.Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (235) Google Scholar or the small gaseous molecule nitric oxide (NO)9.Stallmeyer B. Kampfer H. Kolb N. et al.The function of nitric oxide in wound repair: Inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098https://doi.org/10.1046/j.1523-1747.1999.00784.xAbstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, as reviewed below, crucially contribute to cutaneous re-epithelialization also. Formation of the granulation tissue starts about three to four days after injury Figure 1 and 22.Clark R.A.F. Wound repair: Overview and general considerations.in: Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 3-50Google Scholar, 3.Martin P. Wound healing — Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar, 4.Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4308) Google Scholar. The main cell types driving the generation of new stroma are macrophages, fibroblasts, and endothelial cells. Interaction of these cell types leads to deposition of newly synthesized connective tissue and ingrowth of new blood vessels. Wound fibroblasts own three main capacities: proliferation, migration and production of ECM. Additionally, fibroblasts contribute to the “growth factor cocktail” by secreting important mediators such as keratinocyte growth factor (KGF), connective tissue growth factor (CTGF), or activin. To make their way into the wound site, migratory fibroblasts express a series of proteolytic enzymes (MMP-1, -2, -3). As repair progresses, the migratory phenotype is subsequently changed into a profibrotic one, and fibroblasts start to deposit collagen, a process that is potently regulated by the action of transforming growth factor-β1 (TGF-β1). A proportion of wound fibroblasts specializes into myofibroblasts, which finally decrease the overall wound size by contracting the granulation tissue. Neovascularization has to accompany granulation tissue formation, as the newly forming cellular complex must be supplied with oxygen and nutrients. Outgrowth of endothelial cells from pre-existing vessels at the wound margins is triggered by a series of angiogenic factors that activate endothelial cell proteases that are crucial for the cells to transmigrate the basement membrane and infiltrate the wound. Especially vascular endothelial growth factor (VEGF), which is expressed in large amounts by epidermal cells during repair, regulates endothelial cell action at the wound site. Finally, the late phase of granulation arises when wound fibroblasts undergo apoptosis, and, thus, mark the transition from a cell-rich granulation tissue to a more or less acellular scar.Figure 2Nitric oxide (NO) as a signaling molecule at the wound site. The gaseous molecule NO serves as a mediator that regulates gene expression and proliferation in keratinocytes, and collagen synthesis in fibroblasts. Abbreviations are: NO, nitric oxide; MCP-1, macrophage chemoattractant protein-1; RANTES, regulated upon activation, normal T-cell expressed and secreted; VEGF, vascular endothelial growth factor.View Large Image Figure ViewerDownload (PPT) In mammals, the synthesis of nitric oxide (NO) is catalyzed by NO synthase (NOS) that exists in three distinct isoforms10.Alderton W.K. Cooper C.E. Knowles R.G. Nitric oxide synthases: Structure, function and inhibition.Biochem J. 2001; 357: 593-615https://doi.org/10.1042/0264-6021:3570593Crossref PubMed Scopus (3073) Google Scholar. NOS catalyzes the oxidation of the amino acid L-arginine to finally release citrulline and NO. Molecular cloning has revealed the existence of three different NOS isoforms: the constitutively expressed neuronal (nNOS) and endothelial (eNOS) NOS isoforms, and the inducible NOS (iNOS) isoenzyme, respectively10.Alderton W.K. Cooper C.E. Knowles R.G. Nitric oxide synthases: Structure, function and inhibition.Biochem J. 2001; 357: 593-615https://doi.org/10.1042/0264-6021:3570593Crossref PubMed Scopus (3073) Google Scholar,11.Moncada S. Palmer R.M. Higgs E.A. Nitric oxide: Physiology, pathophysiology, and pharmacology.Pharmacol Rev. 1991; 43: 109-142PubMed Google Scholar. All three NOS isoenzymes are homodimeric proteins that depend on nicotinamide adenine dinucleotide phosphate (NADPH), reduced flavins, heme-bound iron, and 6(R) 5,6,7,8-tetrahydrobiopterin as essential cofactors. The constitutively expressed NOS isoforms are activated by increasing Ca2+ levels via calmodulin. It is well established that all three NOS isoforms are expressed in skin tissue. Expression of nNOS has been observed in keratinocytes and melanocytes12.Baudouin J.E. Tachon P. Constitutive nitric oxide synthase is present in normal human keratinocytes.J Invest Dermatol. 1996; 106: 428-431Abstract Full Text PDF PubMed Scopus (52) Google Scholar,13.Romero-Graillet C. Aberdam E. Biagoli N. et al.Ultraviolet B radiation acts through the nitric oxide and cGMP signal transduction pathway to stimulate melanogenesis in human melanocytes.J Biol Chem. 1996; 271: 28052-28056Crossref PubMed Scopus (155) Google Scholar, expression of eNOS could be detected in keratinocytes of the basal epidermal layer, dermal fibroblasts, endothelial capillaries and eccrine glands14.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: 80-87Crossref PubMed Scopus (89) Google Scholar,15.Wang R. Ghahary A. Shen Y.J. et al.Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible nitric oxide synthase isoforms.J Invest Dermatol. 1996; 106: 419-427Abstract Full Text PDF PubMed Scopus (143) Google Scholar, whereas iNOS can be induced in keratinocytes16.Bruch-Gerharz D. Fehsel K. Suschek C. et al.A proinflammatory activity of interleukin 8 in human skin: expression of the inducible nitric oxide synthase in psoriatic lesions and cultured keratinocytes.J Exp Med. 1996; 184: 2007-2012Crossref PubMed Scopus (155) Google Scholar,17.Sirsjo A. Karlsson M. Gidlof A. et al.Increased expression of inducible nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes.Br J Dermatol. 1996; 134: 643-648Crossref PubMed Scopus (110) Google Scholar, fibroblasts15.Wang R. Ghahary A. Shen Y.J. et al.Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible nitric oxide synthase isoforms.J Invest Dermatol. 1996; 106: 419-427Abstract Full Text PDF PubMed Scopus (143) Google Scholar, Langerhans18.Qureshi A.A. Hosoi J. Xu S. et al.Langerhans cells express inducible nitric oxide synthase and produce nitric oxide.J Invest Dermatol. 1996; 107: 815-821Abstract Full Text PDF PubMed Scopus (73) Google Scholar and endothelial cells19.Kuhn A. Fehsel K. Lehmann P. et al.Aberrant timing in epidermal expression of inducible nitric oxide synthase after UV irradiation in cutaneous lupus erythematosus.J Invest Dermatol. 1998; 111: 149-153https://doi.org/10.1046/j.1523-1747.1998.00253.xAbstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar. It is now reasonable to assume that NO participates in the regulation of skin homeostatic functions such as circulation, UVB-mediated melanogenesis, sunburn erythema, and the maintenance of the protective barrier against microorgansims20.Bruch-Gerharz D. Ruzicka T. Kolb-Bachofen V. Nitric oxide and its implications in skin homeostasis and disease – A review.Arch Dermatol Res. 1998; 290: 643-651https://doi.org/10.1007/s004030050367Crossref PubMed Scopus (82) Google Scholar. In line, NO restricts pathogen growth after cutaneous infection21.Stenger S. Donhauser N. Thuring H. et al.Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase.J Exp Med. 1996; 183: 1501-1514Crossref PubMed Scopus (269) Google Scholar. In general, the presence of iNOS is associated with pathological conditions, a situation that also is true of skin tissue. Accordingly, the expression of iNOS has been described for inflammatory diseases of the skin, including psoriasis, lupus erythematosus, inflammatory dermatoses, and hypersensitivity reactions20.Bruch-Gerharz D. Ruzicka T. Kolb-Bachofen V. Nitric oxide and its implications in skin homeostasis and disease – A review.Arch Dermatol Res. 1998; 290: 643-651https://doi.org/10.1007/s004030050367Crossref PubMed Scopus (82) Google Scholar. Wound healing of the skin is a highly coordinated process, finally leading to an at least partial reconstruction of the injured tissue. The factors mediating the intercellular communication during wound repair are known in part, but their number is still increasing. Proinflammatory cytokines and various peptide growth factors are known to be key players in this process3.Martin P. Wound healing — Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3430) Google Scholar. However, these protein-type factors are not unique in regulating cellular behavior in wound repair, and evidence is emerging for an important role of small diffusible molecules in wound repair. One of them is NO, a free radical gas, which has become one of the most studied molecules in biomedical sciences during the past years. It has long been known that the amino acid L-arginine, representing the only substrate for NOS enzymatic activity, is implicated in wound healing. An initial study from nearly 25 years ago demonstrated the influence of L-arginine supplementation on healing of wounds in a rat model of incisional repair22.Seifter E. Rettura G. Barbul A. et al.Arginine: An essential amino acid for injured rats.Surgery. 1978; 84: 224-230PubMed Google Scholar. Arginine-deficient animals showed an impaired healing, as assessed by markedly decreased wound breaking strength and collagen deposition, which was thought to be associated with an L-arginine–induced growth hormone release22.Seifter E. Rettura G. Barbul A. et al.Arginine: An essential amino acid for injured rats.Surgery. 1978; 84: 224-230PubMed Google Scholar. A few years later, Albina and colleagues determined enzymatic activities of the oxidative L-arginine deiminase (OAD) and arginase in wound fluids isolated from implanted sponges in the rat23.Albina J.E. Mills C.D. Henry Jr., W.L. et al.Temporal expression of different pathways of L-arginine metabolism in healing wounds.J Immunol. 1990; 144: 3877-3880PubMed Google Scholar. OAD catalyzes the reaction of L-arginine into citrulline and nitrogen intermediates and, thus, clearly resembles the more recently identified NOS. In this model of repair, wound fluids were characterized by low arginine levels, which directly implicate a consumption of L-arginine at the wound site. Moreover, citrulline and nitrite, products of the OAD or NOS activity, respectively, peaked within the early, inflammatory phase of healing23.Albina J.E. Mills C.D. Henry Jr., W.L. et al.Temporal expression of different pathways of L-arginine metabolism in healing wounds.J Immunol. 1990; 144: 3877-3880PubMed Google Scholar,24.Schaffer M.R. Tantry U. van Wesep R.A. et al.Nitric oxide metabolism in wounds.J Surg Res. 1997; 71: 25-31Abstract Full Text PDF PubMed Scopus (93) Google Scholar. L-Arginine levels remained low, as the amino acid is subject to the catalytic activity of arginase later in repair, releasing measurable amounts of ornithine into the wound fluid23.Albina J.E. Mills C.D. Henry Jr., W.L. et al.Temporal expression of different pathways of L-arginine metabolism in healing wounds.J Immunol. 1990; 144: 3877-3880PubMed Google Scholar. However, the potency of L-arginine to improve wound healing must be tightly linked to both the NOS and arginase enzyme systems, as the loss of a functional iNOS gene in iNOS-deficient mice abrogates the beneficial effects of L-arginine in wound healing25.Shi H.P. Efron D.T. Most D. et al.Supplemental dietary arginine enhances wound healing in normal but not inducible nitric oxide synthase knockout mice.Surgery. 2000; 128: 374-378Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar. Consistently, the beneficial effects of L-arginine observed in animal models also were confirmed in humans. Clinical studies demonstrated an enhanced wound healing in L-arginine supplemented patients that was characterized by a markedly increased wound collagen deposition26.Barbul A. Lazarou S.A. Efron D.T. et al.Arginine enhances wound healing and lymphocyte immune responses in humans.Surgery. 1990; 108: 331-336PubMed Google Scholar,27.Kirk S.J. Hurson M. Regan M.C. et al.Arginine stimulates wound healing and immune function in elderly human beings.Surgery. 1993; 114: 155-159PubMed Google Scholar. Although an important role for NO in skin repair has been well defined within the last decade, the specific roles of distinct NOS isoenzymes for wound-derived NO production could only be addressed when NOS-deficient knockout mice or NOS isoenzyme-specific inhibitors became available. Nevertheless, utilization of wound healing models in either normal or transgenic animals, or of specific iNOS inhibitors, respectively, predominantly revealed the first insights in the regulation and possible roles of the iNOS isoform during repair. Data obtained from iNOS-deficient mice clearly indicated an important role for iNOS-derived NO in wound healing. The average time of wound closure was significantly delayed in the knockout animals in a model of full-thickness excisional wounding. The disparity between normal and knockout mice upon injury became greater with time finally resulting in an average delay of 31% in iNOS-deficient animals28.Yamasaki K. Edington H.D. 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: 967-971Crossref PubMed Scopus (359) Google Scholar. Inhibition of iNOS enzymatic activity during repair in wild-type mice using N6-(iminoethyl)-L-lysine (L-NIL) resulted in a similar delay in complete wound closure as compared to iNOS kockout mice. More important, functional evidence for iNOS-derived NO in skin repair was evidenced by an adenoviral-mediated expression of a human iNOS cDNA at wound sites in iNOS-deficient mice, which led to an accelerated wound closure that was comparable to healing in control animals28.Yamasaki K. Edington H.D. 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: 967-971Crossref PubMed Scopus (359) Google Scholar. These data were consistent with the induction of iNOS expression upon cutaneous injury in a murine model of normal healing29.Frank S. Madlener M. Pfeilschifter J. et al.Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.J Invest Dermatol. 1998; 111: 1058-1064https://doi.org/10.1046/j.1523-1747.1998.00434.xAbstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar. iNOS levels remained elevated during the inflammatory phase of repair with infiltrating PMN and macrophages as one major source of iNOS expression29.Frank S. Madlener M. Pfeilschifter J. et al.Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.J Invest Dermatol. 1998; 111: 1058-1064https://doi.org/10.1046/j.1523-1747.1998.00434.xAbstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,30.Reichner J.S. Meszaros A.J. Louis C.A. et al.Molecular and metabolic evidence for the restricted expression of inducible nitric oxide synthase in healing wounds.Am J Pathol. 1999; 154: 1097-1104Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar. Interestingly, iNOS expression was not restricted to immune cells, as proliferating keratinocytes of the wound margins, and to a smaller extent also wound fibroblasts expressed iNOS29.Frank S. Madlener M. Pfeilschifter J. et al.Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.J Invest Dermatol. 1998; 111: 1058-1064https://doi.org/10.1046/j.1523-1747.1998.00434.xAbstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar. In line with data obtained from iNOS knockout mice28.Yamasaki K. Edington H.D. 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: 967-971Crossref PubMed Scopus (359) Google Scholar, a glucocorticoid-mediated delay in wound closure in wild-type animals was associated with a markedly reduced presence of iNOS at the wound site29.Frank S. Madlener M. Pfeilschifter J. et al.Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.J Invest Dermatol. 1998; 111: 1058-1064https://doi.org/10.1046/j.1523-1747.1998.00434.xAbstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar. For the first time, application of an iNOS inhibitory substance, L-NIL, during wound healing revealed an important role of iNOS in regulation of epithelial tissue movements9.Stallmeyer B. Kampfer H. Kolb N. et al.The function of nitric oxide in wound repair: Inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098https://doi.org/10.1046/j.1523-1747.1999.00784.xAbstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar. L-NIL is now established as a partially selective inhibitor of iNOS, with a 50-fold selectivity for iNOS versus eNOS, or 20-fold selectivity versus nNOS, respectively10.Alderton W.K. Cooper C.E. Knowles R.G. Nitric oxide synthases: Structure, function and inhibition.Biochem J. 2001; 357: 593-615https://doi.org/10.1042/0264-6021:3570593Crossref PubMed Scopus (3073) Google Scholar. Compared with control mice, L-NIL-treated animals were characterized by a severely impaired re-epithelialization process, as the hyperproliferative epithelia at the wound edges appeared to be delayed and characterized by an atrophied morphology. Moreover, proliferating keratinocyte cell numbers were strongly reduced during re-epithelialization after inhibition of iNOS during repair9.Stallmeyer B. Kampfer H. Kolb N. et al.The function of nitric oxide in wound repair: Inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098https://doi.org/10.1046/j.1523-1747.1999.00784.xAbstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar. To date, information dealing with an in vivo function of eNOS-derived NO in skin repair is clearly restricted. However, eNOS also contributes to wound-related NO production, as eNOS knockout mice were characterized by an even more pronounced delay in wound closure compared to iNOS-deficient animals as assessed by the same model of full-thickness skin wounding31.Lee P.C. Salyapongse A.N. Bragdon G.A. et al.Impaired wound healing and angiogenesis in eNOS-deficient mice.Am J Physiol. 1999; 277: H1600-H1608PubMed Google Scholar. In parallel to the delayed wound closure, the tensile strength of the regenerating tissue was markedly reduced in eNOS knockout mice31.Lee P.C. Salyapongse A.N. Bragdon G.A. et al.Impaired wound healing and angiogenesis in eNOS-deficient mice.Am J Physiol. 1999; 277: H1600-H1608PubMed Google Scholar, indicating an important role for eNOS-derived NO for ECM production (see the section, Nitric oxide and wound fibroblasts). The study suggests defects in angiogenic processes during repair that are associated with the absence of eNOS. Thus, in vitro and in vivo experiments implicated that eNOS contributes to granulation tissue formation by triggering endothelial migration, proliferation and differentiation and, thus, it participates in capillary ingrowth into the wound site during repair31.Lee P.C. Salyapongse A.N. Bragdon G.A. et al.Impaired wound healing and angiogenesis in eNOS-deficient mice.Am J Physiol. 1999; 277: H1600-H1608PubMed Google Scholar. Interestingly, the observed wound-related OAD/NOS activity was exclusively connected to viable macrophages at the wound site in early studies23.Albina J.E. Mills C.D. Henry Jr., W.L. et al.Temporal expression of different pathways of L-arginine metabolism in healing wounds.J Immunol. 1990; 144: 3877-3880PubMed Google Scholar. More recent data confirmed the wound macrophage as a prominent source of iNOS expression in the early inflammatory phase of repair30.Reichner J.S. Meszaros A.J. Louis C.A. et al.Molecular and metabolic evidence for the restricted expression of inducible nitric oxide synthase in healing wounds.Am J Pathol. 1999; 154: 1097-1104Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar. This observation might relate to findings that define sites of inflammation with prominent macrophage infiltration as uniquely deficient in L-arginine. Consistently, the L-arginine metabolism via NOS and arginase appears to be closely counter-regulated in macrophages. Factors increasing macrophage iNOS activity, such as interferon-γ (IFN-γ) or bacterial lipopolysaccharide (LPS), decrease arginase activity in the cells, whereas transforming growth factor-β1 (TGF-β1)–mediated iNOS inhibition resulted in increasing ornithine levels32.Shearer J.D. Richards J.R. Mills C.D. et al.Differential regulation of macrophage arginine metabolism: a proposed role in wound healing.Am J Physiol. 1997; 272: E181-E190PubMed Google Scholar. This hypothesis is supported by yet unpublished observations from our laboratory, where chronic, non-healing wound situations in diabetic db/db mice were characterized by tremendous numbers of macrophages at the wound site that were associated with a lack of iNOS expression in the presence of an overshooting expression of arginase (S. Fra
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