The neglected role of copper ions in wound healing

    The neglected role of copper ions in wound healing Allison Paige Kornblatt, Vincenzo Giuseppe Nicoletti, Alessio Travaglia PII: DOI: Reference:

S0162-0134(16)30038-1 doi: 10.1016/j.jinorgbio.2016.02.012 JIB 9926

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

30 October 2015 19 January 2016 10 February 2016

Please cite this article as: Allison Paige Kornblatt, Vincenzo Giuseppe Nicoletti, Alessio Travaglia, The neglected role of copper ions in wound healing, Journal of Inorganic Biochemistry (2016), doi: 10.1016/j.jinorgbio.2016.02.012

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ACCEPTED MANUSCRIPT The neglected role of copper ions in wound healing

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Allison Paige Kornblatt,1 Vincenzo Giuseppe Nicoletti,2,3* and Alessio Travaglia4*

St. George's University, School of Medicine, Grenada, West Indies

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Department of Biomedical and Biotechnological Science, University of Catania, Viale Andrea Doria 6,

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1

Istituto Nazionale Biostrutture e Biosistemi (INBB) - Sezione Biomolecole, Consorzio Interuniversitario,

Viale Medaglie d'Oro 305, 00136 Rome, Italy

Center for Neural Science, New York University, 4 Washington Place, New York, 10003 NY, USA

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4

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3

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95125 Catania, Italy

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*Correspondence and requests for materials should be addressed to:

and

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Email: [email protected]

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Vincenzo G. Nicoletti

Alessio Travaglia

Email: [email protected]

ACCEPTED MANUSCRIPT Abstract Wound healing is a complex biological process that aims to repair damaged tissue. Even though many biological and biochemical mechanisms associated with the

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steps of physiological wound healing are known, there is still significant morbidity and mortality due to dysregulation of physiological mechanisms. It might be useful to revise

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the activity of old players and their links with new, often neglected, molecular entities.

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This review revises new findings supporting the hypothesis that copper ions regulate the activity and/or the expression of proteins crucially involved in the wound repair process.

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A better understanding of these interactions might suggest potential new targets for

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therapeutic intervention on scars or non-healing wounds.

ACCEPTED MANUSCRIPT 1. Wound Healing Wound healing is a complex, efficient and highly regulated biological process that aims to repair damaged tissues [1-3]. Skin is a labile tissue with regenerative stem cells in

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the basal layer of the epidermis [3]. Wound healing begins as soon as tissue injury occurs and it is usually categorized in four sequential, but overlapping, phases: hemostasis,

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inflammation, proliferation, and remodeling [1, 2]. The process requires a well-

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orchestrated series of responses, resulting in blood clotting, vascular wall repair, protection against infection, cell proliferation and migration, and new blood vessels for

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the provision of nutrients to those cells [4-6]. These phases utilize a series of perfectly coordinated cellular and molecular events involving numerous biological processes such

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as proliferation, differentiation, cell migration, and increased biosynthetic activities [3, 5, 6].

Specific signaling is required to start, modulate and complete the molecular

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mechanisms occurring during the healing process [3]. Extracellular matrix proteins,

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growth factors, and cytokines present in the wound bed are fundamental activators of blood coagulation [7-10]. They also contribute to keratinocyte activation, by modifying

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the function and expression of the adhesion molecules that play a major role in inflammation and subsequent keratinocyte migration [6]. Thus, specific biological markers and biochemical pathways are associated with each step of the physiology of wound healing. However, dysregulation of such mechanisms can occur, resulting in fibrosis and chronic non-healing ulcers, associated with morbidity and mortality due to tissue inflammation and infection [1-4, 11, 12]. Metal ions are essential catalytic and/or structural elements of many proteins, enzymes, and transcription factors [13-15]. Metal ions are also able to modulate expression level and activity of several proteins through the activation, respectively, of metal-responsive transcription factors and conformational changes [13, 15, 16]. Data from old and new literature show that the action of many factors involved in the wound healing machinery is modulated by interaction with copper ions [17-31]. Copper has a complex role in various cells, modulating several cytokines and growth factor mechanisms of action, and it is essentially involved in all stages of the wound healing process.

ACCEPTED MANUSCRIPT This review will first describe the “classical” pathways activated and involved in the healing process and then will explore the roles of copper ions to interact with and modulate the expression and activity of many proteins and growth factors involved in

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wound healing machinery.

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1.1 Hemostasis

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Hemostasis begins from the moment injury occurs, as tissue is disrupted and blood vessels are severed [1, 2]. The processes occurring during hemostasis (from Greek

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αἷ μα «blood» and στάσις «stasis») aim to temporarily seal the damaged tissues and stop associated bleeding, by initiating vasoconstriction, platelet plug formation, and blood

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coagulation. Vasoconstriction is the first response to the injury. Vasoconstrictive factors such as serotonin and thromboxane A2 act to limit the amount of blood flow through the damaged area [1, 32]. Platelets play a major role in hemostasis, undergoing adhesion and

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aggregation to the damaged endothelium. Platelets form a transient plug to prevent local

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blood loss [33, 34]. Then, platelets themselves provide a surface for coagulation factors such as fibrinogen that, in turn, is converted to fibrin (via the extrinsic and intrinsic

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coagulation cascade) forming a sturdy blood clot [33, 35, 36]. The blood clot acts as scaffolding, allowing a temporary extracellular matrix (ECM) to form around the clot in the wound area [8]. The extracellular matrix is rich in fibrous proteins such as collagen, fibronectin and fibrin, as well as liquid proteoglycans like hyaluronic acid [8]. It is important to note that the ECM contributes to wound repair not only structurally but also by signaling, attracting different cells to itself [37]. A major player of the ECM involved in wound healing is fibronectin [38, 39]. Fibronectin is a large ECM adhesion glycoprotein that interacts with integrin receptors to stimulate migration and adhesion of fibroblasts, keratinocytes and endothelial cells as well as serving to promote angiogenesis (the physiological process through which new blood vessels are generated from pre-existing vessels) [39]. The temporary matrix envelops platelets as part of the matrix itself. Platelets in the matrix attract and activate numerous cells such as endothelial cells, fibroblasts, neutrophils and macrophages [35, 36, 39]. Thus, besides acting as a “static” plug, the platelets also importantly pave the way of the next steps of wound healing, by releasing several pro-inflammatory factors (including

ACCEPTED MANUSCRIPT prostaglandins, prostacyclins, thromboxane, and histamine), and several cytokines and growth factors, including platelet-derived growth factor (PDGF), transforming growth

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factor beta (TGF-β) and vascular endothelial growth factor (VEGF) [33, 35, 36].

1.2 Inflammation

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Histamine, locally released, is essentially responsible for capillary vasodilatation,

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which: i) allows increased blood flow, ii) causes blood vessels to become porous and thus iii) increases vascular permeability [1, 40, 41]. All together, histamine release results in

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the arrival of inflammatory cells into the wound site, thereby setting the stage for the inflammatory phase [1, 40, 41].

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Neutrophils and macrophages are the main cells involved in the inflammatory reaction in injured tissues [3, 42, 43]. Their chemotactic attraction to the site of injury is guaranteed by the release of PDGF and TGF-β from activated platelets during the

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hemostasis phase [33, 35, 36]. Neutrophils phagocytize debris and kill bacteria by

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releasing free radicals in what is called a “respiratory burst”. This involves the induction of inducible nitric oxide synthase (iNOS) to produce large amounts of nitric oxide (NO)

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that react with the rapidly released superoxide anion radical to form the highly diffusible peroxynitrite and hydrogen peroxide radicals [44]. Macrophages enter the wound site to remove remaining debris, bacteria, damaged tissue, and apoptotic neutrophils, thus paving the way for the resolution of inflammation [3, 42, 43]. Macrophages release many factors, including plasminogen activator (to produce plasmin and remove the fibrin clot), cytokines to recruit keratinocytes and stimulate angiogenesis, thus further facilitating the next phase in healing [3, 37, 42, 43]. Pathologies with low levels or activity of monocytes or macrophages lead to poor wound debridement, delayed arrival of fibroblasts, and inadequate proliferation phase and angiogenesis [4, 11, 32].

1.3 Proliferation The proliferative phase takes place as soon as the inflammatory phase is over and all debris are cleared. This phase is characterized by granulation tissue, revascularization, and re-epithelialization of the wound [1, 2]. The fibroblasts were recruited to the wound

ACCEPTED MANUSCRIPT site mainly by PDGF and TGF-β secreted by the platelets and macrophages [4, 42]. The fibroblasts synthesize the granulation tissue, which contains collagen (types I and III), elastin, proteoglycans, glycosaminoglycans, fibronectin and non-collagenous proteins [8].

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Fibronectin is particularly important, as it is organized into fibrillar structures within the stroma of the granulation tissue [38]. It forms a dense network around the fibroblasts that

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is vital i) for establishing and maintaining tissue architecture, and ii) for regulating

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cellular processes such as adhesion, spreading, proliferation, migration and apoptosis [3739].

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Angiogenesis also occurs within the proliferative process [45]. Endothelial cells were recruited during hemostasis and the inflammatory phase via multiple chemokines,

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specifically TGF-α and VEGF, as well as local secretion of the ECM [8, 37, 45]. The ECM regulates angiogenesis by providing structural support for new vessels, spreading out signals and growth factors for angiogenesis, and having major receptors to mediate

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interactions needed for angiogenesis [8, 45]. Endothelial cells proliferate and begin to

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differentiate, forming a network of branching vessels in the newly formed granulation tissue formation. This step provides nutrition and oxygen to growing tissues and replaces

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the vessels that were destroyed in the wound [45]. Clinically, at this stage granulation tissue is said to have a “beefy red” appearance, which is a consequence of endothelial cell division to create capillary networks.

1.4 Remodeling

During the final phase of healing, morphological changes occurr on the wounded skin. The capillaries aggregate to form larger blood vessels resulting in less blood flow, and thus reduced nutrients availability [1, 4]. This reflects the decrease in cell density and metabolic activity of the tissue, largely due to apoptosis as a collateral effect of inflammation. In the extracellular matrix an increase of the tensile strength occurs upon increased proportion of collagen type I versus collagen type III that rearrange in a tight organized fashion, with a higher number of covalent cross-links [8, 9, 45]. Although the exact mechanism is not known, fibroblasts are hypothesized to differentiate into myofibroblasts and produce a high contractile force using alpha-smooth muscle actin

ACCEPTED MANUSCRIPT [46]. Expression of alpha-smooth muscle is influenced by TGF-β and ECM-specialized proteins like fibronectin [46]. The final result of tissue repair is a scar, which is less elastic than normal skin,

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does not contain skin appendages such as hair follicles or sweat glands, and has a weaker

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tensile strength that only reaches about 80% that of unwounded skin [1, 2].

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2 Nerve Growth Factor and Wound healing

Nerve Growth Factor (NGF) is a protein belonging to the neurotrophin family that is

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involved in cell growth, maintenance, differentiation, survival, and synaptic plasticity of the central and peripheral nervous systems [47, 48]. Over time, however, it has been

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shown that the role and action of neurotrophins is not restricted to the “classical” view of the nervous system. Namely, NGF has been revealed as a molecule with angiogenic properties, as well as having a role in inflammatory and immune responses in non-

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neuronal tissues, specifically with its function in skin and tissue repair [49, 50].

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NGF is a secreted protein that triggers its biological action through interaction with two distinct classes of cell-surface receptors: TrkA (tropomyosin receptor kinase A)

pathways,

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and 75 kDa neurotrophin receptor (p75). Trk signaling occurs through different including

phosphatidylinositide

the

extracellular

3-kinases/protein

kinase

signal-regulated B

(PI3K/Akt),

kinases

(ERK),

Phosphoinositide

phospholipase C gamma (PLCγ) pathway. These pathways affect neurotrophin signaling in terms of cell survival and cell differentiation, while NGF binding to p75 can trigger neuron apoptosis [51].

NGF and NGF receptors are highly expressed in the skin, where NGF is produced by epidermal cells such as fibroblasts, keratinocytes, and mast cells [52-58]. During cutaneous development, NGF is expressed at the highest levels in the epidermis, and its synthesis is triggered during skin innervation by the first axons [59]. The involvement of NGF in cutaneous wound repair was first supported by the observation that removal of the submandibular glands of mice retards the rate of contraction of skin wounds, and that the licking of wounds enhances contraction [60]. Salivary glands contain a high concentration of NGF [61], and exogenous NGF was then shown to accelerate wound healing in normal and healing-impaired diabetic mice [53,

ACCEPTED MANUSCRIPT 62]. Full-thickness wounds have been associated with a rapid increase in serum NGF levels, which reaches the highest level one day after injury and gradually decreases to baseline levels after 14 days [63]. Moreover, cutaneous wounds result in a rise of NGF

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around the wound site, in newly formed epithelial cells at the wound edge, and in granulation tissue fibroblasts [49, 62]. As a consequence of these observations, and as

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further demonstration of its activity, topical application of NGF to superficial wounds

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increased the rate of healing in mice in size, histology, and breaking strength of the wound [49, 62]. Interestingly, when NGF topical treatment was applied to the pressure

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ulcers of nursing home patients, it was found that the average reduction in pressure ulcer area at 6 weeks was statistically significantly greater in the treatment group than in the

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control group [64].

The surge of NGF after tissue injury is not equal in all periods of life. The postinjury rise in NGF has been reported to be particularly high after birth [65].

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Myofibroblasts of granulation tissue, which in general have very high levels of NGF

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synthesis, have the highest NGF levels in neonates [66]. Considering the great healing efficiency of neonates, compared with adults, these data further highlight the importance

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of NGF in the healing process. Developmental differences in NGF levels, as well as differential mechanisms employed in wound healing processes across different age groups deserve further investigation. Some models have been proposed to explain the mechanism of action of NGF in cutaneous wound healing. A number of studies suggest that the signals from cutaneous nerves may be a major contributing factor to the rate of skin healing [67]. This model seems to be supported by the fact that NGF has a major role in stimulating the growth of sensory nerves, and could explain why topical treatment was helpful when applied to diabetic ulcers, whose pathology begins with sensory neuropathy. In fact, NGF administration has been found to protect against experimental diabetic sensory neuropathy in the first place [68]. Furthermore, endogenous NGF has been found to be lower in diabetic skin than in healthy skin [69]. Further preclinical and clinical studies are needed to test the hypothesis that NGF might have a major role in the prevention and cure of diabetic ulcers.

ACCEPTED MANUSCRIPT Another possible mechanism employed by NGF in the healing process is the activation of TrkA signaling, as well as the interaction with different cells and growth factors. For example, NGF stimulates TrkA phosphorylation in vitro in human

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keratinocytes, and their proliferation through apoptosis inhibition [50, 70, 71]. Interestingly, NGF synthesized and released by keratinocytes can act on melanocytes,

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which show increased structural complexity upon NGF stimulation [71]. In this context,

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it is worth mentioning that NGF treatment rescues melanocytes from UV-induced apoptosis [72-74]. Also, UV irradiation down-regulates NGF and TrkA mRNA (but not

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p75), whereas there is no effect on NGF-transfected cells [50, 71]. Additionally, healing may also be improved due to NGF’s role in cell recruitment

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and cutaneous angiogenesis. It is known that NGF acts via chemotaxis on neutrophils, monocytes, and fibroblasts [75, 76]. In addition to their recruitment to the healing site, NGF function promotes the activity of neutrophils and macrophages, such as

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phagocytosis and superoxide production [77, 78]. Not only does NGF increase fibroblast

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migration, but the addition of NGF to wounds also increases α-smooth muscle actin expression and collagen gel contraction [79]. Finally, mounting studies provide

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fascinating evidence that NGF stimulates angiogenesis and upregulates expression of VEGF [80-83].

ACCEPTED MANUSCRIPT 3. Copper ions and wound healing Metal ions, especially zinc, iron, and copper, are emerging as key players in biological processes such as respiration, photosynthesis, gene regulation, replication and

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repair of DNA, antioxidant defense, and neurotransmission [13-15]. Remarkably, metal ions are essential catalytic and/or structural elements for the function of over one third of

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all proteins, enzymes, and transcription factors [13-15, 20].

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Each tissue, cell, and even subcellular compartment has to maintain the concentration of each metal ion within a narrow and specific range to avoid a detrimental

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alteration of metal ion homeostasis (metallostasis). It is an extremely dynamic process, in which metal chaperones, metal transporters, metalloproteins, small molecules and metal

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transcription factors are able to fine-tune metal ion concentration in response to stimuli such as increased stressors, metabolic activity, or injury [13-15, 20]. Of course, pathological dysregulation of metallostasis can lead to severe consequences, including

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cellular death [20, 84].

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A picture of and copper ions cellular homeostasis is summarized in the following. Cu+ is transported across the plasma membrane by the high-affinity transporter Ctr1

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(Copper transporter 1), after to be reduced by plasma membrane reductases, then Cu+ is buffered by metallothioneins or, through a variety of protein-based pathways, delivered to its ultimate intracellular destinations by specific copper chaperone proteins. [13, 15, 20]. Copper ions can be directed to the mitochondria (for insertion into cytochrome c oxidase) and to cytosolic enzymes (e.g. Cu/Zn superoxide dismutase) and to post Golgi compartments. [13, 15, 20, 85] Through a complex network of molecular interactions, metallostasis regulators can partially correct metal ion dyshomeostasis. For example, if and when the intracellular copper concentration is elevated, the adenosine triphosphatase 7A (ATP7A) moves from the trans-Golgi network (TGN) to the cell membrane to eliminate excess copper from the cell. In the next section, we review old and new findings about the interaction of copper with wound healing machinery.

ACCEPTED MANUSCRIPT 3.1 Platelet-derived growth factor and copper Platelet-derived growth factor (PDGF) is a growth factor that is involved during several steps of the healing process, and recent evidence suggests a strict correlation of

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the activities of these proteins with copper ion availability. First, Ashino et al. showed that PDGF stimulation promoted ATP7A translocation from the trans-Golgi network

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(TGN) to the plasma membrane in vascular smooth muscle cell [17]. The authors showed

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that the ability of PDGF to induce ATP7A translocation is responsible for changes in intracellular copper concentration and biological effects downstream of PDGF signaling.

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Namely, depletion of ATP7A prevented the PDGF-induced decrease in copper levels and inhibited vascular smooth muscle cell migration in response to PDGF or wound scratch.

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ATP7A could prove to be an interesting therapeutic target for vascular remodeling [17]. More recently, Tsai et al. showed that the ability of PDGF to activate its downstream signaling (e.g., ERK and Akt) failed in CTR1−/− cells [30]. The authors

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showed that PDGF enhances the phosphorylation of ERK and Akt in CTR1+/+ cells but

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not in CTR1−/− cells. To confirm that the activation of PDGF signaling is dependent on copper availability, the authors showed that the deficit in ERK phosphorylation can be

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induced in CTR1+/+ cells by treatment with a copper chelator. Also, the deficit in the CTR1−/− cells was rescued by adding copper ions to the medium [30]. Finally, Narayanan et al. have shown in vitro and in vivo that silencing CTR1 with specific siRNA was sufficient to inhibit angiogenesis by limiting copper entry into endothelial cells [86].

3.2 VEGF, Angiogenin and copper Angiogenesis, the physiological process through which new blood vessels generate from pre-existing vessels, plays a vital role during the proliferative phase of wound healing. For more than two decades, copper has been a well-known angiogenic factor in vivo. Recent works have shed light on the copper-dependent molecular and cellular mechanisms underlying angiogenesis [19, 87, 88]. Copper binds and interacts with several growth factors involved in vessel formation. However, copper-dependent stimulation of vessel formation has been mainly attributed to its regulation of vascular endothelial growth factor (VEGF) and, to a lesser extent, angiogenin. VEGF is believed to be the most efficacious mediator of angiogenic

ACCEPTED MANUSCRIPT activity during the proliferative phase of wound healing. Thus, copper availability, uptake, and delivery can have a role in the regulation of the overall process [89]. Sen et al. showed that the angiogenic potential of copper may be harnessed to

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accelerate dermal wound healing. They showed that copper sulfate accelerated the closure of excisional murine dermal wounds [26]. Topical treatment with 25µM of

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copper ions, once a day for five days, significantly increased VEGF expression,

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accelerated the healing process, and also improved the quality of regenerated tissue. Histological analysis of the wound edge in copper sulfate-treated mice revealed more

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hyperproliferative epidermis, increased deposition of connective tissue, and higher density of cells in the granulation layer [26].

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Copper induces VEGF expression through the activation of the transcription factor known as hypoxia-inducible factor-1 (HIF-1) [18, 31]. Namely, copper can bind the subunit alpha of the HIF factor, and may inhibit the activity of FIH-1 (factor

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inhibiting HIF-1) to retain the capacity of HIF-1α binding to other cofactors. This allows

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for stabilization of the HIF-1 transcriptional complex and upregulation of downstream target genes such us VEGF [18]. The regulation of expression of other inherent factors

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also has been found to be affected by copper through the mediatory effect of metal transcription factor (MTF-1) [90]. This notion, together with the complexity of proteins and signaling cytokines involved in cellular processes, highlight a strong linkage between wound healing, angiogenesis factors, and metal homeostasis. Angiogenin (ANG), a member of the secreted ribonuclease family, is a potent angiogenesis stimulator that interacts with endothelial cells and triggers a wide range of responses on endothelial cells [91]. Angiogenin is also expressed and secreted by human mast cells (HuMC) and is then available in the subsequent inflammatory response[91, 92]. Interestingly, Pan et al. revealed that high angiogenin expression co-localizes with high vascularity in human burn wounds [93]. Also, the expression level of ANG in deep partial thickness burns is higher than that in superficial partial thickness burns, supporting the hypothesis that angiogenin is involved in burn wound neovascularization [93]. The link between angiogenin and copper is emerging. Angiogenin binds about 2.4 copper ions per molecule at physiological pH [28], and copper affects the intracellular localization of this protein, decreasing its nuclear translocation, which is required for its

ACCEPTED MANUSCRIPT function [21, 94]. Moreover, the ANG-copper system negatively affects protein-induced angiogenesis, as well as endothelial cell migration. Surprisingly, copper also has the ability to modulate the angiogenin transcription [94]. These data also seem to show

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adverse effects of copper in the angiogenic process. These apparent anti-angiogenic effects highlight the pivotal role of copper homeostasis and its subcellular redistribution

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as a novel modulator of intra- and inter-cellular signaling. In summary, the role of the

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copper-angiogenin interaction to promote/modulate dermal wound healing needs further

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study, both in vitro and in a clinical setting.

3.3 Metalloproteinases and copper

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Matrix metalloproteinases (MMPs, mainly MMP-1, MMP-2, MMP-8, MMP-9) and the serine proteases (human neutrophil elastase, HNE) are the major groups of proteases involved in the wound healing process [95, 96]. These proteases i) eliminate

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damaged proteins, ii) facilitate cellular migration and remodel the granulation tissue, iii)

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regulate the activity of growth factors and inflammatory mediators. A physiological healing process requires a balance of MMPs activation and inhibition, by tissue inhibitors

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of metalloproteinases (TIMPs) [97, 98]. Unbalanced levels of MMPs and their TIMPs are typically associated with chronic wounds [98], as well as diabetic complications [99]. Several extracellular and membrane-bound proteases (e.g. Plasmin, elastase, MMP3, and MMP9) have been recognized as key step in the ‘release’ of matrix-bound growth factors, such as VEGF, and promote cell proliferation in angiogenesis and wound healing. However, under conditions of increased protease activity, unexpected degradation of growth factors, receptors and ECM proteins will occur [100]. In turn, keratinocytes at the wound edges tend to be hyperproliferative and non-migratory, while fibroblasts in the base of the wound become senescent [101, 102]. In addition, during inflammatory diseases cytokines and growth factors induce the up-regulation and release of MMPs by immune and stromal cells [103]. A high proteolitic environment can be responsible for rapid degradation of VEGF and low recruitment of vascular supply in non-healing wounds [104, 105]. Low copper concentrations (0.3–3 μM) have been found to stimulate the activity of MMPs, whereas high concentrations (1–100 μM) stimulate the expression of MMPs in

ACCEPTED MANUSCRIPT fibroblasts [24]. Other studies have reported that both MMP2 and MMP3 can be upregulated by copper, although excess free metal can also inhibit MMP activity [106, 107]. The stimulation of PI3K and ERK signaling pathways appears to be involved in this

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activation of MMP2 and MMP3 [108, 109]. The triggering mechanism could be related to metal-mediated activation of membrane receptors. However, a number of different

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metal ligand complexes, including 8-hydroxyquinoline and phenanthroline derivatives,

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can be effective in inducing MMPs [110, 111].

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3.4 GHK and copper

The tripeptide glycyl-L-histidyl-L-lysine (GHK) gained popularity after a series

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of studies in the 1970s, showing in vitro that human plasma was able to control fibrinogen biosynthesis in liver tissue [112, 113]. Further studies revealed that: i) this effect was due to GHK and ii) that the GHK peptide has a strong affinity for copper

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[113].

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A series of experiments showed significant healing activity of copper peptide GHK, both in vitro using fibroblast cultures and in vivo in experimentally-induced rat

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wounds [23]. Maquart et al. proposed that the GHK peptide is released as a result of partial proteolysis of extracellular matrix macromolecules. Thus, they suggested that the GHK peptide is formed wherever the initial injury of tissue occurs, and then acts as a signaling molecule [23]. It has been shown that the complex of copper ions with GHK (GHK-Cu) upregulates the expression of proteins such as collagen, elastin, metalloproteinases, vascular endothelial growth factor, fibroblast growth factor, and nerve growth factor [112, 113], which then play a role in the proliferation and remodeling phases of healing. Additionally, GHK-Cu induced a persistent expression of MMP-9 in the wound tissue, lasting for at least 20 days after wound creation. GHK-Cu also increased pro-MMP-2 and activated MMP-2 during the later stages of healing (day 18 and/or 22) [27]. The tripeptide complex also has anti-inflammatory actions such as the suppression of free radicals and decreased tumor necrosis factor alpha-dependent interleukin-6 secretion in normal human dermal fibroblasts [112]. Moreover, solutions of GHK-Cu

ACCEPTED MANUSCRIPT decreased insulin-like growth factor-2 (IGF-2)-dependent TGF-β1 secretion in normal human dermal fibroblast cells [114]. Taken together, this information on GHK-Cu activities in the process of wound

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healing, tissue repair, and skin inflammation raises the potential for use of this peptide in cosmetics to treat and prevent the formation of hypertrophic scars [115-120].

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Interestingly, topical treatment with GHK-Cu improves wound healing, and systemic

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injection of GHK-Cu has been found to increase healing in animal models [121, 122]. Even though an exact mechanism remains unclear, the current literature suggests that

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GHK-Cu’s ability to bind copper and to modulate its levels in tissues is an important

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factor determining its biological activity [112].

3.5 Nerve Growth Factor and copper

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Old and new literature shows that the concentration, structure, and activity of

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NGF, like the other actors of the wound healing process, is regulated by copper ions. In turn, copper ions seem to take part in NGF signal transduction.

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Copper ions have been reported to modulate the level of NGF and other neurotrophins. NGF is synthesized as a precursor, named proNGF, which is then cleaved to give the mature form intracellularly through the action of furin or pro-hormone convertases,

or

extracellularly

through

the

action

of

plasmin

and

matrix

metalloproteinases (MMPs) [16, 123]. Interestingly, Hwang et al. showed that copper(II) ions induce MMP activity and pro-neurotrophin processing [124, 125]. In particular, they showed that copper(II) ions increased the activity of MMP2 and MMP9 in cortical neurons. This event, in turn, promotes the cleavage of pro- brain-derived neurotrophic factor (pro-BDNF), thus increasing the secretion of mature brain-derived neurotrophic facor (BDNF) in the media [124, 125]. Addition of MMP inhibitors completely blocked the copper-induced increases in BDNF, indicating that these enzymes act upstream in proBDNF processing [124, 125]. It is not hard to imagine that a similar mechanism could be responsible for pro-NGF maturation, because the same enzymes are involved in the maturation of the neurotrophins.

ACCEPTED MANUSCRIPT An

unbalanced

activity

of

the

different

proteases

involved

in

the

maturation/degradation of NGF can be responsible for deficits and failure of NGF trophic stimulation or loss of protection. A failure in the conversion of proNGF to NGF (lower

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plasminogen/plasmin activity), which is exacerbated by an increased NGF degradation resulting from a rise in MMP-9 activity, has been associated with progressive atrophy of

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the basal forebrain cholinergic system and the consequent cholinergic contribution to

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Alzheimer’s Disease-related learning and memory decline [126].

It is currently unknown whether the increase in NGF level observed at the wound site is

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the result of increased synthesis, decreased degradation of NGF, or increased cleavage of the proNGF.

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The activity and the biological outcomes of NGF have been shown to be modulated by copper ions [25, 123, 127]. It has been shown that high concentrations (100 µM) of copper(II) and zinc(II) ions, but not other metal ions, block the NGF-mediated

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neurite outgrowth in chick dorsal root ganglia. Furthermore, 100 μM of zinc(II) or

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copper(II) ions, but not other metal ions, block NGF binding to p75, attenuating its proapoptotic signaling cascade in chick embryonic cell cultures [25, 127]. Thus, depending

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on which receptor is expressed on a target cell, copper ions are able to modulate or trigger different responses of NGF such us neurite outgrowth and differentiation, prosurvival signals or pro-apoptotic outcome. In addition, in pheochromocytoma cells (PC12) zinc(II) and copper(II) ions inhibit the NGF-mediated survival-promoting activity against oxidative stress, in particular against hydrogen peroxide-induced cell death [127]. The latter information is particularly relevant in the context of the wound healing process. In fact, high metabolic activity is present at the wound site and, as mentioned above, reactive oxygen species (ROS) are produced during the inflammation phase. Thus, a possible feedback loop involving ROS, copper ions and NGF might be worth more complete investigation. At the molecular level, such effects have been attributed to the ability of copper to alter the conformation of NGF, making it unable to bind its receptors and activate its signaling [25]. Recently, we have provided experimental data about the coordination features of copper(II) ion complexes with the N-terminal tail of NGF [123], which has been proved by crystallographic, computational, and biochemical data to be both

ACCEPTED MANUSCRIPT necessary and sufficient for specific activities of NGF through the TrkA receptor [128131]. Finally, copper ions seem to actively take part in NGF signal transduction.

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Birkaya and Aletta ran a series of interesting experiments to show the correlation between copper and NGF signal transduction [132]. They showed that NGF is able to

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increase the cellular level of copper in PC12 cells within 3 days of treatment. As a

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consequence of NGF treatment, the copper content per cell increases up to 14-fold [132]. This effect appears to be functional in the context of NGF activity in neuronal

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differentiation, since copper ions are required for optimum neurite outgrowth, whereas copper chelators reduce NGF-induced neurite outgrowth. Other studies are required to

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shed light on the possibility that NGF could be a copper chaperone.

3.6 Integrins and copper

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Keratinocytes, the predominant cell type in the outer layer of the skin, have a

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distinct pattern of integrins, a family of proteins responsible for intercellular and cellsubstrate adhesion (a crucial factor in tissue repair and wound healing) [133]. During the

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re-epithelialization phase, keratinocytes undergo a significant number of modifications in the expression and distribution of integrins, leading to changes in the adhesion molecule phenotype [133]. What is even more interesting is that copper has been reported to affect the expression of integrins involved during the final healing phase (α2, β1 and α6), in proliferating monolayer cultures of keratinocytes [29].

3.7 Lysyl oxidase and copper Copper-dependent enzymes are necessary for remodeling of the extracellular matrix, as well as in the proliferative phase during healing [9, 98, 134-136]. The activity of the copper-dependent enzyme lysyl oxidase increases during wound healing to catalyze the formation of aldehyde cross-links, primarily on collagen and elastin. Moreover, aldehyde cross-links are required at the end of the proliferative phase, when wounded skin becomes stronger. Deficiency in copper would lead to decreased collagen and elastin synthesis and poor wound healing [8, 134, 135].

ACCEPTED MANUSCRIPT 3.8 Copper as antiseptic and antimicrobial tool Metals such as copper and silver have been a valuable antibacterial tool in a multitude of applications, spanning from agriculture to healthcare [137-139]. Copper

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biocide features were already known to ancient Egyptian medicine (2400 BC), which recommended copper sulfate to sterilize water and treat infections [137-139].

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There is a renewed interest in copper use as an antimicrobial agent, and copper-based

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nanomaterials and nanoparticles have emerged for their potential use in wound dressing [115-120, 140-145]. These polymer/metal composites are designed to have antimicrobial

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activities, and are usually prepared as hydrogels or metal embedded nanofiller [115-120, 140-146]. On one hand, these materials can increase the local delivery of copper [115-

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120]. Besides having various roles in mammalian physiology, it is notable that copperdeficiency is associated with numerous alterations in host defenses, involving the impairment of various mechanisms: phagocytosis, immune response, T-cell activation,

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and cytokine expression [147, 148].

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Thus, increasing copper availability can assist tissue repair through the activation of the above-mentioned copper machinery. Furthermore, the use of metal and metal

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complexes with antiseptic and/or antimicrobial activity in wound dressing is an attractive approach to minimize infection in a wound. Copper toxicity has been attributed mostly to its catalytic role in Fenton-like chemistry, which results in ROS formation in close spatial proximity to copper ions [137]. ROS, in turn, are responsible for damage through oxidation of macromolecules such as lipids and proteins[137]. Moreover, sustained copper activity has been also observed in anoxic conditions, both in vitro and in vivo, in an ROS independent process. This is explained by the thiophilic behavior of copper, which is sufficient to competitively disrupt cytoplasmic iron-sulfur enzymes (e.g. intracellular dehydratases) [149]. Conversely, mammalian cells are partially protected by these mechanisms through cytoplasmic metallothioneins, glutathione, and the Cu/Zn superoxide dismutase[150, 151]. It is worth noting that a linkage between copper resistance and virulence has been observed in infections in human, and this linkage was found to be associated with new copper resistance strategies that promote microbial survival [152, 153].

ACCEPTED MANUSCRIPT The renewed interest in copper use as an antimicrobial poses the need for a better understanding of the mechanisms of copper antibacterial activity. This is particularly relevant to get improved insight into bacterial copper resistance cues for the development

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of new copper complexes bearing optimized copper biocide as well as wound healing

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properties[140, 141]

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4. Conclusions and perspectives

Impaired wound healing still results in significant morbidity and mortality, which

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impacts daily lives as well as health care expenses. Clinical management of wound healing requires a comprehensive knowledge of the molecular mechanisms that, over

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time, have a role around the wound edge. The challenge is to recognize the features of the optimal wound environment.

It might be useful to revise the activity of old players and their links with new,

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often neglected, molecular entities. Copper ions are emerging to play a role in many

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biological process, including wound healing. Old and recent literature shows that the activity of PDGF, VEGF, and Angiogenin is copper-dependent. These proteins represent

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crucial elements of wound healing machinery and angiogenesis, which makes their interaction with copper an extremely interesting target for further study. MMPs can modulate the levels of both NGF and VEGF, and have a critical role during the cell migration process. The observation that the level and the activity of MMPs is modulated by copper ions can provide a new mechanism to improve the efficiency of the wound repair process. Furthermore, it is possible that factors that affect copper homeostasis directly modulate the overall would healing machinery. The activity of the tripeptide GHK could be studied in this context. It is very tempting to propose the fascinating hypothesis that NGF-mediated biological responses are, at least in part, due to increased intracellular copper, either as a result of a direct ionophore effect or after changes on copper homeostasis. Such a hypothesis literally would suggest a plethora of possible mechanisms for NGF’s role in the wound healing process. Innovative methods based on copper release have been recently developed as potential therapeutic tools in wound healing treatments. Copper-based nanomaterials and

ACCEPTED MANUSCRIPT nanoparticles have emerged for potential use in wound dressing. On one hand, such materials can increase the local delivery of copper, thus assisting tissue repair through the activation of the above-mentioned copper machinery. On the other hand, the use of metal

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and metal complexes with antiseptic and/or antimicrobial activity in wound dressing is an attractive approach to minimize infection in a wound. In fact, pathogenic bacteria on the

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improper wound healing or chronic non-healing ulcers.

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wound site represent one factor that can interfere with physiological healing, thus causing

Further studies should focus on the molecular mechanisms of copper homeostasis

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during the healing process. Each tissue, cell, and even subcellular compartment has to maintain the concentration of each metal ion within a narrow and specific range to avoid

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a detrimental alteration of the metal ion homeostasis (metallostasis). It is true that low levels of copper can negatively affect the above-mentioned proteins and enzymes required in the healing process. However, elevated copper levels can dysregulate wound

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healing machinery, and possibly trigger red-ox reactions along with the generation of

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reactive oxygen species, resulting in detrimental effects. Additionally, given the established role of copper in angiogenesis and cancer, it is possible to speculate that

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copper ions play a role in the malignant degeneration of chronic wounds [154-156]. Malignant tumors often develop at sites of chronic injury, and tissue injury has an important role in the pathogenesis of malignant disease, with chronic inflammation being the most important risk factor [154-156]. Further studies are needed to address these critical issues.

In conclusion, this review attempts to provide the basis and stimulus to justify the need for more comprehensive studies to better understand the integrated interactions of secreted factors and metal ions in the wound healing process, as well as to highlight the need to develop new compounds able to modulate wound healing as therapeutic interventions on scars or non-healing wounds.

Funding This work was supported by grants PON (prot. PON01_01078) and FIR 2014 (cod.9DD800). The authors declare no conflict of interest.

ACCEPTED MANUSCRIPT List of acronyms and abbreviations used in the text

Protein kinase B

ANG

Angiogenin

ATP7A

Adenosine triphosphatase 7A

BDNF

brain-derived neurotrophic factor

Ctr1

Copper transporter 1

ECM

Extracellular matrix

ERK

Extracellular signal-regulated kinases

FIH-1

Factor inhibiting HIF-1

GHK

Glycyl-L-histidyl-L-lysine

HIF-1

Hypoxia-inducible factor-1

HNE

Human neutrophil elastase

HuMC

Human mast cells

IGF-2

Insulin-like growth factor-2

iNOS

Inducible nitric oxide synthase

MMP

Matrix metalloproteinase

MTF-1

Metal transcription factor

NGF

Nerve Growth Factor

NO

Nitric oxide

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Akt

PC12

Pheochromocytoma cells

PDGF

Platelet-derived growth factor

PI3K

Phosphatidylinositide 3-kinases/

PLCγ

Phosphoinositide phospholipase C gamma

pro-BDNF

pro- brain-derived neurotrophic factor

ROS

Reactive oxygen species

TGF-β

Transforming growth factor beta

TGN

trans-Golgi network

TIMPs

Tissue inhibitors of metalloproteinases

TrkA

Tropomyosin receptor kinase A

VEGF

Vascular endothelial growth factor

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Acknowledgments The authors wish to thank Ian Gardiner for his invaluable contribution in

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preparing the manuscript.

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Copper ions in wound healing: new insight for future applications

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Highlights: Dysregulation of physiological wound healing leads to morbidity and mortality. Copper ions modulate the activity of proteins involved in wound healing. Control of copper homeostasis might be a target for therapeutic intervention.