Acta Pharmacologica Sinica 2005 Mar; 26 (3): 259–264 Invited review Nitric oxide: a newly discovered function on wound healing Jian-dong LUO1,2, Alex F CHEN1,3 1Departments of Pharmacology and Neurology and the Neuroscience Program, Michigan State University, East Lansing, MI 48824-1317, USA; 2Department of Pharmacology, Guangzhou Medical College, Guangzhou 510182, China Key words Abstract angiogenesis; inflammation; nitric oxide; Wound healing impairment represents a particularly challenging clinical problem proliferation; wound healing to which no efficacious treatment regimens currently exist. The factors ensuring appropriate intercellular communication during wound repair are not completely 3 Correspondence to Alex F CHEN. understood. Although protein-type mediators are well-established players in this Phn 1-517-432-2730. process, emerging evidence from both animal and human studies indicates that Fax 1-517-353-8915. E-mail [email protected] nitric oxide (NO) plays a key role in wound repair. The beneficial effects of NO on wound repair may be attributed to its functional influences on angiogenesis, Received 2004-08-14 inflammation, cell proliferation, matrix deposition, and remodeling. Recent find- Accepted 2004-12-14 ings from in vitro and in vivo studies of NO on wound repair are summarized in doi: 10.1111/j.1745-7254.2005.00058.x this review. The unveiled novel mechanisms support the use of NO-containing agents and/or NO synthase gene therapy as new therapeutic regimens for im- paired wound healing. Introduction oxide (NO) in wound repair[3]. In this review, we summarize Wound repair is a well orchestrated and highly coordi- the current knowledge of the modulating functions of NO on nated process that includes a series of overlapping phases: wound repair. inflammation, cell proliferation, matrix deposition, and tis- Chemistry and biosynthesis of nitric oxide sue remodeling. This involves a complex, dynamic series of events including clotting, inflammation, granulation tissue NO is a highly diffusible intercellular signaling molecule formation, epithelialization, neovascularization, collagen implicated in a wide range of biological effects. It is gener- synthesis, and wound contraction[1]. Loss of a functional ated by the enzyme nitric oxide synthase (NOS), which cata- healing process could lead to severe disabilities. Accord- lyzes the conversion of L-arginine to L-citrulline[4]. Three ingly, chronic, non-healing wound conditions represent a NOS isoforms have been characterized, each encoded by situation of major clinical importance. The series of patho- different chromosomes. Two enzyme isoforms are constitu- logical changes associated with several diseases ultimately tively expressed (endothelial and neuronal NOS), whereas leads to severely disturbed wound healing conditions[2]. one isoform is an inducible enzyme (iNOS), initially found Among those, the most prominent chronic wound impair- in macrophages. All three NOS isoforms exist in their ac- ments include decubitus or pressure ulcers, venous ulcers, tive form of homodimers of two domains: a C-terminal re- and diabetic ulcers. The advent of molecular and cellular ductase domain, and an N-terminal oxygenase domain with biology and the use of different modeling systems, most no- molecular masses of approximately 135 kDa (eNOS), 150– tably genetically engineered animals, have greatly extended 160 kDa (nNOS), and 130 kDa (iNOS)[4]. The reductase do- our knowledge of wound repair. Inflammation, re-epitheliali- main contains binding sites for one molecule each of nicoti- zation, and granulation tissue formation are driven in part by namide adenine dinucleotide phosphate (NADPH), flavin a complex mixture of growth factors and cytokines, which mononucleotide (FMN), and flavin dinucleotide (FAD), in are released coordinately into the wounds[1,2]. Besides these close homology with cytochrome P-450 reductase, whereas protein-type factors and mitogens, evidence is emerging for the oxygen domain binds heme, the essential cofactor the important role of small diffusible molecules such as nitric tetrahydrobiopterin (BH4), and the substrate L-arginine[4]. ©2005 CPS and SIMM 259 Luo JD et al Acta Pharmacologica Sinica ISSN 1671-4083 Between these two regions lies the calmodulin (CaM) bind- onstrate that there is a significant increase of cutaneous ing site, which plays a key role in both the structure and eNOS protein expression as well as constitutive NOS enzy- function of the enzyme. The constitutive isoforms (eNOS matic activity after excisional wounding in normal mice[17]. and nNOS) are permanently active, generating low concen- Consequently, an NO deficiency directly contributes to trations of NO (in nmol/L range). Their enzymatic activities wound healing impairment. Inhibition of NOS by competi- are regulated by intracellular calcium fluxes or exogenous tive inhibitors, either applied to the wound surface[18] or given calmodulin. The expression, transcription, and function of systemically[11], decreases collagen deposition and breaking the iNOS is induced by a variety of cytokines, growth factors, strength of incisional wounds and impairs the healing. and inflammatory stimuli on target cells which lead to the Consistent with these findings, studies with targeted dis- release of much higher levels of NO (in µmol/L range), which ruption of NOS genes have revealed that the excisional is involved in host immune response. wound closure is delayed by 30% in both eNOS and iNOS All three NOS isoforms are expressed in skin tissue[3]. knockout mice compared to their wild-type littermates[19,20]. Expression of nNOS has been observed in keratinocytes and Conversely, adenoviral vector-mediated gene transfer of melanocytes; eNOS can be detected in keratinocytes of the iNOS to the wound site of iNOS knockout mice completely basal epidermal layer, dermal fibroblasts, endothelial reversed the delayed healing[20]. capillaries, and eccrine glands; and iNOS can be induced in Finally, there are strong correlations between reduced keratinocytes, fibroblasts, Langerhans, and endothelial cells. cutaneous NO levels and impaired wound healing under dis- Accordingly, NO participates in the regulation of skin ho- ease conditions such as diabetes[11,14,17,21], malnutrition[13], meostatic functions such as circulation, sunburn erythema, and chronic steroid treatment[18]. Diabetic wound healing and maintenance of the protective barrier against micro- impairment is one of the most well-known chronic wound organisms. situations. Studies of ours and others demonstrate that cu- taneous eNOS expression, constitutive NOS activity, and/or Nitric oxide and wound healing NO levels are significantly decreased in streptozotocin (STZ)- induced type 1 diabetic animals[11,14,17,21]. In fact, our find- L-Arginine, the substrate for NOS, was first noted to en- ings indicate that the augmented cutaneous eNOS protein [5] hance wound healing in 1978 . Subsequently, dietary expression and constitutive NOS activity observed in nor- L-arginine intake has been shown to improve collagen depo- mal animals in the healing process are absent in the type 1 [6–8] sition and wound strength in both animals and humans . diabetic mice[17]. These findings suggest that impairment of This effect of L-arginine may be due in part to its conversion wound-induced endogenous NOS expression and NOS ac- to L-ornithine through the action of arginase, an enzyme tivity is responsible for reduced cutaneous NO bioavail-abil- that may compete with NOS for L-arginine and thereby help ity in type 1 diabetic animals (Figure 1). In agreement with [9] regulate NO production during wound healing . However, the above notion, cutaneous gene therapy of eNOS or man- the finding that L-arginine intake does not improve collagen ganese superoxide dismutase (MnSOD) restored eNOS pro- deposition in iNOS-deficient mice to the same extent as in tein and NO levels and accelerated the wound healing rate in wild-type littermates implicates that part of L-arginine’s ef- STZ-induced diabetic mice[17]. Similarly, the NO donor [10] fect involves NO directly . molsidomine (N-ethoxycarbomyl-3-morpholinyl- Accumulating evidence indicates that NO plays a key sidnonimine) or NO releasing poly (vinyl alcohol) hydrogel [11–18] role in normal wound repair (Figure 1) . Production of dressings are also shown to partially restore such healing nitrite (NO2) and nitrate (NO3), the stable NO metabolites, are impairment in STZ-induced diabetic rats[22,23]. Collectively, [11] elevated early in the fluid of subcutaneous wounds , and impairment of skin NO function represents an important fac- urinary nitrate excretion increases in a sustained manner af- tor for delayed wound healing in diabetes and strategies [12] ter excisional wounding . Furthermore, the presence of aimed at restoring cutaneous NO bioavailability with NO nitrite and nitrate is directly correlated with collagen deposi- donors or NOS gene therapy may serve as effective means tion within the wound and in dermal fibroblasts, suggesting for diabetic wound healing. that NO synthesis is critical for wound collagen accumula- tion and acquisition of mechanical strength[11,13,14]. All three NOS isozymes are involved in the wound healing process. Mechanisms of nitric oxide on wound healing Both iNOS and nNOS mRNA and protein expression are in- NO and angiogenesis Angiogenesis, the process of form- creased in cutaneous wounds[15,16]. Our recent findings dem- ing new microvessels, is an important component of normal 260 Http://www.chinaphar.com