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Wound healing in the 21st century
Stephan Schreml, MD,a Rolf-Markus Szeimies, MD, PhD,a Lukas Prantl, MD, PhD,b
Michael Landthaler, MD, PhD,a and Philipp Babilas, MD, PhDa
Regensburg, Germany
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doi:1
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Delayed wound healing is one of the major therapeutic and economic issues in medicine today. Cutaneouswound healing is an extremely well-regulated and complex process basically divided into 3 phases:inflammation, proliferation, and tissue remodeling. Unfortunately, we still do not understand this processprecisely enough to give direction effectively to impaired healing processes. There have been many newdevelopments in wound healing that provide fascinating insights and may improve our ability to manageclinical problems. Our goal is to acquaint the reader with selected major novel findings about cutaneouswound healing that have been published since the beginning of the new millennium. We discuss advancesin areas such as genetics, proteases, cytokines, chemokines, and regulatory peptides, as well as therapeuticstrategies, all set in the framework of the different phases of wound healing. ( J Am Acad Dermatol2010;63:866-81.)
Key words: cellular; molecular; novel findings; signal transduction; pH value; skin wound.
Abbreviations used:
AGE: advanced glycation end productCyr61: cysteine-rich angiogenic inducer-61ECM: extracellular matrixEGF: epidermal growth factorERK: extracellular regulated kinaseFGF: fibroblast growth factorHGF: hepatocyte growth factorHSP: heat shock proteinIL: interleukinIL-1ra: interleukin-1 receptor antagonistKGF: keratinocyte growth factorLacZ: lactose operon ZLL-37: C-terminal fragment of human
cathelicidin antimicrobial peptide-18MMP: matrix metalloproteinasemTOR: mammalian target of rapamycinNF: nuclear factorNPY: neuropeptide YNramp1: natural resistanceeassociated macro-
phage protein-1Nrf: nuclear factor-E2-related factorPI3K: phosphatidyl inositol-3 kinaseRE: response elementShh: sonic hedgehog
Cutaneous wounds are the result of disruptedskin integrity. The healing process dependson local wound factors, systemic mediators,
the underlying disease, and the type of injury. Thesefactors combine to determine if physiologic or acutewound healing occurs, or if there is an abnormalhealing process, also called chronic wound healing.Chronic wounds are the result of an inadequaterepair process that is unable to restore anatomic andfunctional integrity in an appropriate length of time.Chronic wounds affect about 1% of the Europeanpopulation and are frequently a management chal-lenge, even with an interdisciplinary approach. Inaddition to having an adverse effect on the quality oflife of the affected individuals, chronic wounds alsocreate a significant economic burden: nearly 2% ofhealth budgets are devoted to the care of chronicwounds.1
Our understanding of the mechanisms involved incutaneous wound healing has dramatically increased
SPARC: secreted protein acidic and rich incysteine
TF: tissue factorTGF: transforming growth factorTIMP: tissue inhibitor of matrix
metalloproteinaseTLR: toll-like receptorVEGF: vascular endothelial growth factor
the Departments of Dermatologya and Plastic Surgery,b
gensburg University Hospital.
orted by grants of the German Research Foundation (Deut-
he Forschungsgemeinschaft DFG, BA 3410/3-1) and the
ovartis Foundation (S.S., Novartis Graduate Scholarship).
licts of interest: None declared.
int requests: Stephan Schreml, MD, Department of
ermatology, Regensburg University Hospital, Franz-Josef-
rauss-Allee 11, 93053 Regensburg, Germany. E-mail:
ished online June 24, 2010.
-9622/$36.00
09 by the American Academy of Dermatology, Inc.
0.1016/j.jaad.2009.10.048
in the past few years. Keeping up to date with thecurrent literature is sometimes difficult. Our aim is toprovide researchers and clinicians working in the fieldof wound healing with selected new insights intowound pathogenesis and cutaneous repair
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mechanisms. We consider topics such as genetics,proteases, cytokines, chemokines, and regulatorypeptides, as well as therapeutic strategies. All areviewed in the context of the intertwined phases ofwound healing: (1) inflammatory phase, (2) prolifer-ative phase (neoangiogenesis, granulation, re-epithe-lialization), and (3) remodeling phase (extracellular
CAPSULE SUMMARY
d Since the beginning of the newmillennium a large number of articleshave been published dealing withcutaneous wound healing.
d This article reviews novel findings relatedto the major phases of cutaneous woundhealing: inflammation, proliferation, andtissue remodeling.
d Newly discovered molecular targets andpathways provide the basis for furtherresearch and future clinical studies.
matrix [ECM] remodeling).Detailed figures are providedto facilitate the understandingof the rather complex patho-genetic mechanisms.
Chronic wounds are de-fined as wounds that do notfollow the well-defined step-wise process of physiologichealing. Instead, they aretrapped in an uncoordinatedand self-sustaining phase ofinflammation that impairsthe restoration of anatomicand functional integrity inthe normal period of time.Many of the pathophysio-
logic factors (hypoxia, pH changes, and bacterialcolonization) that contribute to delayed woundhealing are well known. However, the exact patho-genesis of chronic wounds remains unclear.Rather than giving a comprehensive overview onwound healing, the following sections will focus onnewly discovered molecular mechanisms and theirimportance in wound healing.
THE INFLAMMATORY PHASEThe initial phase after cutaneous injury is domi-
nated by inflammatory reactions mediated by cyto-kines, chemokines, growth factors, and their actionson cellular receptors (Fig 1). Intracellular signalingcascades are activated, contributing to cell prolifer-ation, migration, and differentiation. In addition,chemoattractant factors recruit different cell types,such as granulocytes and macrophages, to thewound site, thus initiating wound repair. Thewound milieueconsisting of various proteinases,cytokines, chemokines, pH gradients, and pO2
gradientsehas a major impact on cellular functions.The importance of wound fluid in regulating theresponsiveness of fibroblasts to proliferation signalsmediated by cytokines has been shown by Nedelecet al.2
During the inflammatory phase of wound healing,a variety of membrane-bound receptors play a role inrecruiting leukocytes and other cells. One receptormediating leukocyte-endothelial cell interaction3
is intercellular adhesion molecule-1 (CD54).
Intercellular adhesion molecule-1 interacts with leu-kocytes via CD11a (together with CD18 = lympho-cyte function-associated antigen-1). Nagaoka et al4
found that intercellular adhesion molecule-1edefi-cient mice showed impaired wound healing becauseof a lack of leukocyte and macrophage infiltrationinto the wound site. These cells are required to
establish an inflammatory re-action, which is a major mile-stone on the way toorganized wound repair.
Coordinated inflamma-tory phases require asubtle balance of proin-flammatory cytokines andchemokines and their antag-onists. Although interleukin(IL)-1 is known as a key fac-tor, little is known about thefunctions of the IL-1 receptorantagonist (IL-1ra). Ishidaet al5 found that IL-1ra e/emice showed an interruptionin transforming growth
factor (TGF)-b1 signaling, which resulted in reducedcollagen deposition and vascular endothelial growthfactor (VEGF) expression. IL-1ra is only temporarilyup-regulated until 10 days after skin injury. IL-1radeficiency induces prolonged nuclear factor (NF)-kBp65 nuclear translocation. The prolonged inflamma-tory phase leads to delayed wound healing in thesemice.
Ishida et al6 further studied the role of chemokinereceptors in wound pathogenesis in a full-thicknessexcisional skin mouse model. The chemokine che-mokine C-X3-C motif ligand-1 (CX3CL1) (nomencla-ture according to patterns of conserved cysteines:chemokine C-X3-C motif ligand-1; fractalkine) andits receptor chemokine C-X3-C motif receptor-1(CX3CR1) are up-regulated at wound sites. Theirrole in wound healing was elucidated in an exci-sional wound CX3CR1 e/e mouse model thatshowed reduced macrophage infiltration and thenreduced TGF-b1 and VEGF signaling (because bothare released by macrophages), which in turn led todecreased collagen deposition and neoangiogene-sis, inevitably resulting in delayed wound healing.When IL-1ra is knocked out, different chemokinesare also up-regulated.
Furthermore, the shift in balance between proin-flammatory and anti-inflammatory factors is one ofthe central reasons for persistent inflammation inchronic wound healing. An anti-inflammatory factorinvolved in regulating the balance is secretory leu-kocyte protein inhibitor-1. Its gene expression is
Fig 1. Inflammatory phase. Cells communicate via connexin-43 (Cx43) and proinflammatorycytokines and chemokines, such as CC chemokine ligand (CCL)-2, tumor necrosis factor (TNF ),and interleukin (IL)-1 trigger inflammation. Vascular endothelial growth factor (VEGF ),transforming growth factor (TGF )-b, and keratinocyte growth factor (KGF )-1 are inducedand facilitate the next stage of wound healing (proliferative phase). Reactive oxygen species(ROS ) are being produced, degraded by peroxiredoxin-6, and their effects are reduced byLL-37. asODN, Antisense oligodeoxynucleotides; CX3CL1, chemokine C-X3-C motif ligand-1;CX3CR1, chemokine C-X3-C motif receptor-1; HSP, heat shock protein; ICAM, intercellularadhesion molecule; IL-1ra, IL-1 receptor antagonist; Nrf, nuclear factor-E2-related factor; greenarrows, positive regulation: activation; red arrows, negative regulation: inhibition.
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modulated by natural resistanceeassociated macro-phage protein-1 (Nramp1) (solute carrier family 11member-1), which is also a modulator of inflamma-tory toll-like receptor (TLR)-7 signaling. Secretoryleukocyte protein inhibitor-1 down-regulation leadsto prolonged wound healing as shown in an Nramp1e/e macrophage model. In wild-type Nramp1 1/1mice, secretory leukocyte protein inhibitor-1 levelswere significantly higher and cutaneous woundhealing was significantly faster than in Nramp1knockout mice.7
The regulation of genes encoding growth factorsand cytokines or chemokines is also of great interest.Beyer et al8 summarized the specific role of NF-E2-related factor (Nrf)2 in the regulation of woundhealing. The gene is the target of keratinocyte growthfactor (KGF), and the protein is an important tran-scription factor inducing detoxifying enzymes andantioxidant proteins. Nrf2 messenger RNA was sig-nificantly up-regulated in a murine full-thicknessexcisional wound model, while other members ofthis transcription factor family either remained unaf-fected (Nrf1) or were even down-regulated (Nrf3).Using transgenic mice expressing a dominant-nega-tive Nrf2 variant, Beyer et al8 were able to show thatNrf2 plays a crucial role in inflammatory wound
pathogenesis but not in re-epithelialization. Again,this finding shows that wound closure is a highlyregulated process in terms of time-dependent ex-pression profiles of cytokines/chemokines andgrowth factors. These factors not only account forenhancing inflammation but also set well-definedstop signals to block the inflammatory cascade whenappropriate during the wound-healing process. Thisis important as a prolonged inflammatory reaction isthe main reason for impaired wound healing.Therefore, therapeutic strategies aimed at reducinginflammation at the appropriate time point couldhave a significant impact. Mori et al9 found thatknocking down connexin-43 (gap junction compo-nent that mediates cell migration and proliferation)by antisense oligodeoxynucleotides (asODN) re-sulted in faster wound closure and a reduced inflam-matory reaction, as also reflected by reduced levelsof CC chemokine ligand (CCL)-2 and tumor necrosisfactor. Correspondingly, histologic analysis showedreduced macrophage and leukocyte infiltration. Afew years earlier, Coutinho et al10 had already shownthe dynamic regulation of connexin expression dur-ing wound repair. Therapeutic strategies based onthese observations are being developed; for in-stance, a single topical application of connexin-43
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antisense gel resulted in a transient down-regulationof connexin-43 protein levels and subsequentlyaccelerated wound closure.11
One of the major breakthroughs in our under-standing of wound healing during the past years wasthe discovery that antimicrobial peptides have animpact on both wound healing and on differentcellular and subcellular functions. For example,cathelicidin, more specifically LL-37, a C-terminalfragment of human cathelicidin (human cathelicidinantimicrobial protein-18), is up-regulated by com-mon growth factors, such as insulin-like growthfactor-1 or TGF-a.12 These peptides function asregulatory factors by inhibiting cytokine releaseand triggering respiratory burst (generation of reac-tive oxygen species [ROS]) in the early phase ofwound healing.13 The antagonist role of LL-37 maybe relevant for keeping the level of inflammation atthe wound site under control.14,15 Moreover, LL-37acts as a potent antimicrobial peptide, thereby alsolimiting inflammatory processes.14,15 In contrast,other regulatory peptides such as the human b-de-fensins even trigger the release of proinflammatorycytokines and chemokines.16 Besides, LL-37 hasbeen shown to transactivate the epidermal growthfactor (EGF) receptor and thus to stimulatekeratinocyte proliferation during the subsequentproliferative phase.17,18 The important in vivo activ-ity of LL-37 in wound healing has been studied inob/ob (obese, ob-gene encodes leptin) mice.19
Adenoviral transfer of LL-37 to ob/ob mice withexcisional wounds was shown to result in signifi-cantly improved wound healing. But how can LL-37be therapeutically modified in a clinical setting?Many questions still need to be answered beforeadenoviral transfer is incorporated into routine clin-ical practice. On the other hand, LL-37 expressioncan be modulated by the application of short-chainfatty acids such as butyrate (not in all cell typesinvolved).20 This approach is relatively easy and maybe suitable for daily practice.
A well-known problem in delayed wound healingis oxidative stress induced by reactive oxygen spe-cies (ROS).21,22 Peroxiredoxin-6 has been identifiedas one of the factors protecting endothelial cells andkeratinocytes from ROSeinduced damage to mem-branes, DNA, and proteins.23 The peroxiredoxinfamily comprises 6 members, which reduce hydro-gen peroxide and other organic peroxides by meansof redox-active cysteine. Kumin et al23 have shownin vitro that knocking down peroxiredoxin-6 incultured endothelial cells increases their susceptibil-ity to oxidative stress. In addition, peroxiredoxin-6deficient mice showed massive hemorrhage in gran-ulation tissueedependent on the amount of
oxidative stressewhich counteracted proper woundhealing.
Cell membrane damage leads to a release of a vastvariety of intracellular proteins from the cytoplasmiccompartment into the wound bed. One highly con-served family of intracellular proteins contributing tothe early inflammatory phase after cutaneous injuryis the heat shock protein (HSP) family. Exogenouslyadministered HSP70 and HSP90 (normally located inthe cytoplasm) as well as HSP gp96 (located inendoplasmic reticulum) produced enhanced woundhealing in a full-thickness skin wound mousemodel.24 One mechanism involved is HSP70-medi-ated activation of phagocytosis by macrophages,which are essential for removing cell debris fromthe wound to allow 3-dimensional tissue reconstruc-tion and remodeling.
Chen et al25 reported another factor, which istemporarily overexpressed in the early stage ofwound healing. Tissue factor (TF) (thromboplastinor factor III) plays an important role in the coagula-tion cascade. In a murine diabetic model, TF may beone of the cross-links between the early inflamma-tory phase and the subsequent proliferative phase.Chen et al25 showed that TF was up-regulated asearly as 1 hour after injury and that high TF levels ledto improved wound vascularization. Moreover, arelative deficiency of TF in diabetic mice comparedwith nondiabetic controls resulted in longer wound-healing times. These results underline that a perfectspatial and chronological regulation of proteinasesand their inhibitors is needed for normal woundclosure.
PROLIFERATIVE PHASEThe proliferative phase includes: (1) neoangio-
genesis (Fig 2, A), (2) formation of granulation tissueand ECM (Fig 2, B), and (3) re-epithelialization(Fig 2, C ).
NeoangiogenesisNeoangiogenesis, one of the major processes in
wound healing, is absolutely necessary for properwound healing (Fig 2, A). The switch of macro-phages from producing proinflammatory cytokinesto secreting VEGF is controlled by TLR cross-talkwith adenosine A2A receptors. Myeloid differentia-tion primary response gene (MyD)-88, a cytoplasmicadaptor protein for TLR signaling, is responsible forthis switch. Wounds of myeloid differentiation pri-mary response protein-88 e/e mice heal signifi-cantly slower than those of wild-type mice. Indifferent knockout models, it has been proven thatIL-1 receptoreassociated kinase-4 (IRAK-4) and tu-mor necrosis factor-receptoreassociated factor-6
Fig 2. Continued
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Fig 2. Proliferative phase. A, Neoangiogenesis is induced by activation of mammalian target ofrapamycin (mTOR), sonic hedgehog (Shh), neuropeptide Y (NPY ), and toll-like receptors(TLRs) (cross-talk with adenosine A2A receptors, myeloid differentiation primary responseprotein-88 [MyD88]: important intracellular TLR adaptor in wound healing), leading toproduction of angiogenic factors, such as vascular endothelial growth factor (VEGF ),cysteine-rich angiogenic inducer-61 (Cyr61), and interleukin (IL)-b1. Thus, de novo vesselformation is induced. B, Proliferation and collagen production of fibroblasts is activated byincrease in activin/follistatin ratio, matrilin-2, and activation of sphingosine signaling pathway(sphingosine-1 kinase [Sp1-K]). Secreted protein acidic and rich in cysteine (SPARC ) (part ofextracellular matrix [ECM]) controls excessive fibroblast proliferation and facilitates productionof collagen-1. IL-4-induced wound-healing macrophages contribute to fibroblast proliferationand collagen production. C, Re-epithelialization is triggered via epidermal growth factor (EGF )(its receptor is transactivated by LL-37), activation of mTOR pathway by keratin-17 productionand hepatocyte growth factor (HGF ) (acts via c-Met receptor on keratinocytes). Leptin isimportant for proliferation/migration of keratinocytes, which interact with ECM throughgalectins in their podosomes. AGE, Advanced glycation end product; c-Met, mesenchymalepithelial transition factor = hepatocyte growth factor receptor (HGF-R); EGF-R, epidermalgrowth factor receptor; Fhl2, four and a half LIM domains-2; IGF, insulin-like growth factor;MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF, nuclear factor;PAI, plasminogen activator inhibitor; PI3K, phosphatidyl inositol-3 kinase; RAGE, receptor ofadvanced glycation end products; RELM, resistin-like molecule; Sp1-P, sphingosine-1-phos-phate; TGF, transforming growth factor; TH2-cells, T helper cell type 2; USF, upstreamtranscription factor; YM, chitinase-like protein; Y2-receptor, neuropeptide Y receptor-2; greenarrows, positive regulation: activation; red arrows, negative regulation: inhibition.
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(TRAF-6) participate in this complex process ofmacrophage phenotype change, which is essentialfor vascular proliferation in wounds.26
In addition to VEGF, which has been studied ingreat detail, other barely known angiogenic factorsneed to be investigated. One is cysteine-rich angio-genic inducer-61 (Cyr61), a heparin-binding, ECM-associated protein, which is known to contribute toangiogenesis by modulating cell proliferation, mi-gration, and adhesion of endothelial cells and fibro-blasts. Chen27 was the first to show that Cyr61 issignificantly up-regulated in dermal fibroblasts
during granulation tissue formation and that Cyr61levels return to basal levels thereafter. Remarkably,Cyr61 regulates genes encoding for major proteinsthat are involved in different aspects of cutaneouswound healing, such as angiogenesis and lympho-genesis (VEGF-A and -C), inflammation (IL-1b), ECMremodeling (matrix metalloproteinase [MMP]; tissueinhibitor of MMP [TIMP]), and cell-matrix interac-tions (eg, integrins a3 and a6).
In 2006, Asai28 reported that the sonic hedgehog(Shh) gene is a genetic factor contributing to de novovessel formation in wounds. Shh is known to be
Fig 3. Remodeling phase. Matrix metalloproteinases (MMPs) are positively regulated bymammalian target of rapamycin (mTOR), inhibited by a1-antichymotrypsin (a1-ACT ) and bytissue inhibitors of MMPs (TIMPs). Rather unspecific stimuli, such as bacterial colonization andpH value modifications, alter MMP activity. Syndecan-4 (on fibroblasts) is induced uponcutaneous injury and interacts with integrins and growth factor receptors. These processescontribute to extracellular matrix remodeling (especially MMP1-3), chronic wound pathogen-esis (MMP8/9), and keratinocyte migration (especially MMP28 = epilysin). RE, Responseelement; green arrows, positive regulation: activation; red arrows, negative regulation:inhibition.
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important in epithelial-mesenchymal interactions. Ina patched-1/lactose operon Z (LacZ) mouse model,Asai28 topically applied the Shh gene DNA in form ofa human Shh plasmid. Patched-1 is a receptor forhedgehog signaling that is activated by Shh binding.LacZ encodes for b-galactosidase. The LacZ reportergene is particularly useful for studies of the cis-regulatory element for tissue-specific expression intransgenic mice because of the ease of the enzymeassay and the visualization on sections. In the modelof Asai,28 topical Shh gene therapy increased theangiogenic cytokine production of dermal fibro-blasts, leading to an increase in endothelial cellproliferation, migration, and vessel tube formation.
In 2003, Ekstrand et al29 reported on the role ofneuropeptide Y (NPY), a well-known neurotrans-mitter, in wound healing. NPY acts via interactionwith NPY-receptor-2 and initiates the formation oftreelike vessel structures. In a mouse corneal micro-pocket model and in a chick chorioallantoic mem-brane model using NPY-receptor-2-deficient mice,NPY failed to induce angiogenesis and wound heal-ing was drastically reduced in these mice.29
Furthermore, hypoxia is a major factor contribu-ting to delayed healing of chronic cutaneous
wounds. A key target may be mammalian target ofrapamycin (mTOR) (synonym of FK506 bindingprotein-12/rapamycin-associated protein kinase-1)as it has a significant regulatory impact on hypoxia-induced angiogenesis.30 In normoxic conditions,mTOR does not seem to play a central role inangiogenesis. Hypoxia-induced activation of prolif-eration and angiogenesis by mTOR is a new molec-ular link between exogenous stimuli and cellularresponse. This link may be clinically relevant asmTOR inhibitors (eg, rapamycin, everolimus) areknown to lead to delayed wound healing in the earlypostoperative phase after solid organ transplanta-tion. Another argument for the importance of mTORin wound healing is the observation that blocking oreven knocking down signaling pathways interactingwith mTOR complex-1esuch as extracellular regu-lated kinase (ERK)1/2, p38 mitogen-activated pro-tein kinase, and phosphatidyl inositol-3 kinase(PI3K)—delay, accelerate or even prevent woundclosure.31 mTOR integrates signals from thesepathways,30 and a fragile balance of positivewound-healing signals (eg, p38 mitogen-activatedprotein kinase and ERK1/2) and negative ones (eg,PI3K pathway) is needed for normal healing.31
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Sufficient vascularization is absolutely necessaryfor the delivery of nutrients and oxygen to thewound site during the subsequent energy-consu-ming proliferative phase.
Fibroblast proliferation and collagenproduction
In the proliferative phase (Fig 2, B), nutrients andoxygen are limiting factors because of the tremen-dous metabolic activity. Just as in neoangiogenesis,macrophages are also being rediscovered in theirinteraction with fibroblasts. A special subtype ofwound healing macrophages is induced mainly byIL-4-mediated T helper cell type 2 (TH2) responses.32
This macrophage subtype is characterized by theexpression of membrane-bound molecules that en-able interaction with the ECM, for example chitinase-like protein (YM1) and resistin-like molecule(RELM)-a. In addition, the expression of the anti-inflammatory IL-27 receptor a is induced via IL-4,marking the shift from inflammation to the stimula-tion of fibroblast proliferation and collagen produc-tion, for example via insulin-like growth factor-1.Wound healing macrophages also contribute to col-lagen production as arginase activity in this subtypeis up-regulated, allowing macrophages to convertarginine to ornithine, which is an important precur-sor in collagen production.
A number of other relevant factors important forfibroblast proliferation in cutaneous wound healinghave been investigated in the past years. In 2002,Bradshaw et al33 reported on the involvement of anECM protein, secreted protein acidic and rich incysteine (SPARC) (synonym of osteonectin or BM-40) in cutaneous wound healing. The authors com-pared wound healing in SPARC e/e knockout miceand wild-type mice. SPARC-null mice showedquicker wound healing and higher proliferation ratesof fibroblasts but only half as much collagen Icontent as wild-type mice, as documented by hy-droxyproline levels. The increased contractibility ofcollagen fibers produced by SPARC-null dermalfibroblasts may be partially explained by the de-creased collagen I content.33
Little is known about matrilin-2, a protein involvedin fibroblast proliferation and ECM interaction.Matrilin-2 is a member of the von Willebrand factorA domain-containing protein family and a compo-nent of extracellular filament networks. It interactswith ECM components, fibroblasts, and keratino-cytes. DNp63/bone morphogenic protein-7/Smadsignaling (DNp63, N-terminal isoform of p53; Smad,Sma and mothers against decapentaplegic homolog)has been shown to regulate matrilin-2 transcription inwound healing, elucidating the promising potential
of this pathway.34 As matrilin-2 is known to contrib-ute to wound healing and as skin injury leads totransient DNp63-up-regulation, further study of thiscascade appears promising.35 After completion ofwound healing processes through involvement ofmatrilin-2, DNp63 is down-regulated to basal levels,again showing the fragile complexity of cutaneouswound healing.
Another important, time-dependent mechanisminvolves fibroblast growth factor (FGF) binding pro-tein. FGF itself is a prominent stimulus for fibroblastproliferation and differentiation. This protein is alsoup-regulated during the initial phase after skin injuryand quickly decreases to baseline levels as shown ina severe combined immunodeficiency (SCID) xeno-graft mouse model with human upper eyelid skin.36
In turn, however, excessive proliferation and colla-gen production rates of fibroblasts may lead tohypertrophic scarring or keloid formation.Hypertrophic scars are marked by excessive collagenproduction, leading to scar elevation and hardening.An enzyme needed to form a stable triple helicalcollagen molecule by hydroxylating procollagenproline residues is termed prolyl-4-hydroxylase.Kim et al37 demonstrated that inhibition of thisenzyme by the topically applied inhibitor FG-1648reduced scar hypertrophy in a rabbit ear hypertro-phic scar model. Moreover, wound re-epithelializa-tion and granulation remained unimpaired.
The potential of lysophospholipids (eg, sphingo-myelin, phosphatidylcholine) in modulating fibro-blast functions is far from being fully understood.38
The protein four and a half LIM-domains (Fhl)-239eadownstream effector of sphingosine-1-phosphatesignalingeis translocated to the nucleus in cutaneouswound repair. (LIM domains are named after theirinitial discovery in the proteins Lin-11, Isl-1; andMec-3; LIM is a protein structural domain composedof two contiguous zinc finger domains.) Fhl-defi-cient mice show impaired wound healing as a resultof reduced collagen contractibility and cell migrationto the wound. Furthermore, there is molecular evi-dence for these observations as p130Cas (a proteinimportant for cell migration; Cas, Crk associatedsubstrate) was down-regulated in Fhl e/e mice.39
This new signaling cascade may be an attractivetarget for the relatively new class of sphingosine-1-phosphate receptor inhibitors and activators.
Many other factors also control fibroblast activity,for instance TGF-b and activins (TGF-b-superfamilymembers). Activins are known as important proteinsin wound repair, and the expression of two activin-binding proteinsefollistatin and follistatin-relatedproteinehas been investigated. As a consequenceof wounding, activins are up-regulated, whereas
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follistatin and follistatin-related protein levels remainrelatively constant or even decrease. As more freeactivin is available by increasing the ratio of activinand follistatin-related protein, activin is able to acti-vate TGF-b-mediated signaling, which is importantfor wound repair.40 Of course, receptors not only acton a single cell type but rather mediate cross talkbetween different cell types in wound healing.Ghahary and Ghaffari41 studied the important crosstalk between keratinocytes and fibroblasts with aspecial focus on the regulation of MMPs and TGF-b1 signaling. They showed that wound-edge kerat-inocytes produce significantly higher amounts ofTGF-b1, which is a potent paracrine stimulus forfibroblast and macrophage proliferation as well asmigration. This cross talk between keratinocytes andfibroblasts in different skin layers is very important,particularly for the initiation of the next step:re-epithelialization.
Re-epithelializationRe-epithelialization (Fig 2, C ) aims at covering the
wound surface with a layer of epithelium and isbased on the differentiation, proliferation, andmigration of epidermal keratinocytes. After thewound bed has been properly established withproliferating fibroblasts, a new collagen matrix, andnew vessels, the process of re-epithelialization canstart. Keratinocytes are activated and migrate into thewound site from the edges. Even highly conservedpathways such as the Wnt (combined from winglessWg and Int) pathway are involved in this process.42
New mechanisms in the complex process of re-epithelialization have been discovered during thepast few years. The role of a Ca21-dependente-(g-glutamyl)lysin cross-linking enzyme termedtransglutaminase-1 was investigated in a neonatalmouse skin model.43 This enzyme colocalizes withinvolucrin, which is essential for the composition ofthe cornified envelope of the stratum corneum.Inada et al43 showed that transglutaminase-1 e/emouse skin grafted on athymic mice showed delayedwound healing and uncontrolled keratin 6a mes-senger RNA expression, possibly to compensate fortransglutaminase-1 deficiency.
Another previously unknown mechanism in re-epithelialization was found in a mouse model with atargeted deletion of the signaling domain of a6b4integrin (laminin-332; formerly known as laminin-5).a6b4 Integrin is essential for proper EGF-mediatedERK and Jun-N terminal protein kinase signaling.The crucial finding was that a6b4 integrin is requiredfor the nuclear translocation of mitogen-activatedprotein kinases (MAPK) and NF-kB, which, in turn,
regulates keratinocyte proliferation and migration inwound healing.44
KGF is also known for its potential in woundhealing. Lin et al45 used Sprague-Dawley rats inwhich wound healing was impaired by sepsis as aresult of cecal ligation after punch biopsy. By meansof electroporation transfection techniques usingplasmids containing KGF-1 DNA, they showed sig-nificantly decreased wound healing times. Thismodel even provided evidence that KGF may betherapeutically useful in the future.
Epithelial injury stimulates specific signaling cas-cades, which may be potential therapeutic targets infuture. Providence46 was the first to show a specifickeratinocyte response to epithelial monolayerwounding in a cell culture model. Plasminogen acti-vator inhibitor-1 is a serine protease inhibitor essentialfor barrier proteolysis and cell-to-matrix adhesion.Plasminogen activator inhibitor (PAI)-1 messengerRNA levels are elevated after epithelial injury.Upstream transcription factor-1 (USF-1, a helix-loop-helix transcription factor)—which binds to an E-Boxmotif in the plasminogen activator inhibitor-1 proxi-mal gene promoter—is induced by tissue injury invitro. These data implicate upstream transcriptionfactor-1 as a transcriptional regulator of genes in-volved in wound repair. Even though specific modi-fication of this signal transduction pathway is still farfrombeing routine, it is tempting to speculate onnoveldrugs interacting with upstream transcription factor-1.
mTOR (FK506 binding protein-12/rapamycin-as-sociated protein kinase; RAFT1, rapamycin andFKBP12 target-1) is a highly conserved proteinkinase and a component of two different proteincomplexes: mTOR complex-1 regulates prolifera-tion, DNA synthesis, transcription, and translation,whereas mTOR complex-2 is predominantly in-volved in the control of cell size and actin skeletonultrastructure.30 In 2006, a novel link between ker-atin 17 (an intermediate filament)47 and mTOR wasshown by Kim et al.48 Keratin 17, which is up-regulated in wounded stratified epithelia, stimulatesthe Akt/mTOR (Akt is protein kinase B) pathway bybinding to the adaptor protein 14-3-3s. As a conse-quence, proliferation and protein synthesis of kerat-inocytes increase, which is absolutely necessary forcutaneous wound healing.48 The differential regula-tion of the PI3K/Akt pathway upstream of mTOR30
seems to be involved in wound-healing gene ex-pression patterns.49 In addition, PI3K/Akt signalingis extremely important in preventing keratinocytesfrom going into apoptosis.50
The galectins, ie, b-galactoside binding lectins,are carbohydrate-binding proteins that also play akey role in wound healing. Cao51 reported that
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specific galectins (3 and 7, but not 1) play a role inepithelial wound healing of the cornea of galectin-3e/e mice. Surprisingly, exogenous galectin-3 ad-ministration did not lead to improved wound healingin galectin-3 e/e mice, whereas galectin-7 showedpotential. However, in galectin-3 1/1 mice, galec-tin-3 was of additional benefit when administeredexogenously. The emerging role of galectin-7 inepidermal wound healing was studied in a mousemodel with galectin-7 e/e mice.52 Although the skinstructure remained unaltered in galectin-7 e/e mice,re-epithelialization proved to be significantly slowerthan in wild-type controls. As galectin-7 is located inkeratinocyte podosomes, the authors speculated ona mechanism of impaired cell/ECM interactionthrough reduced galectin-7 expression.52 The galec-tin family may also be a promising wound treatmenttarget, particularly as the topical application ofgalectin-3 or -7 should not be clinically difficult.
Chronic wounds not only occur more frequentlyin patients with diabetes mellitus but these woundsare also more difficult to treat. Diabetes plays apivotal role in chronic wound pathogenesis.53 In thepast years, a molecular link between advancedglycation end products (AGEs) and microvascularas well as macrovascular complications in diabeteshas been established. Glycated proteins (AGEs) actthrough receptors of AGEs (RAGEs).54 The pathwaysactivated by AGEs (RAGEs) include NF-kB-mediatedinflammation, ERK- and PI3K/Akt-signaling. A re-view of these effects has been published recently,54
and the link between carbohydrates and epithelialrepair has been studied in great detail.55 The fact thatAGEs also modify dermal fibroblast proliferation56
underlines the clinical observation of extremelydifficult wound care in patients with diabetes.These pathways are integrated via the highly con-served mTOR30 and represent important signalingcascades in wound healing and pathogenesis.Changes induced by diabetes also comprise alteredmigration, proliferation, and differentiation patternsof keratinocytes in patients with chronic ulcers.57
A deficiency of leptin seems to be co-causative fordiabetes and obesity. Adipocytes secrete leptin,which is the product of the obese (ob) gene and animportant regulatory feedback signal for energyhomeostasis. The impact of leptin on wound healingwas proven by Frank et al,58 who reported improvedwound healing in leptin-deficient mice after topicaland systemic leptin administration. Wild-type micealso showed improved wound healing after leptinadministration. By studying the direct impact ofleptin, Frank et al58 were able to show that theclassic model of ob/ob-mice for impaired re-epithe-lialization can not be explained simply by the mild
diabetic phenotype but rather by the lack of a directmitogenic effect of leptin on hyperproliferativewound-edge keratinocytes via leptin receptor sub-type obese receptor b (ObRb). People with diabetesand obesity may benefit from future leptin treatmentstrategies in wound therapy.
A cytokine with multiple roles in acute andchronic wound healing is hepatocyte growth factor(HGF), which interacts with c-Met (mesenchymalepithelial transition factor is HGF receptor).59
HGF/scatter factor is a paracrine cellular growth,motility and a morphogenic factor. HGF/scatter fac-tor is secreted by mesenchymal cells, targeting andacting primarily on epithelial cells and endothelialcells. The question of whether HGF is differentlyexpressed in acute compared with chronic woundshas been controversially discussed in recent years. In2007, Conway et al60 found that HGF was signifi-cantly higher in chronic ulcers than in acute wounds.This finding confirmed the results by Nayeri et al,61
who studied HGF in chronic wounds. In addition,Conway et al60 showed that HGF expression differsspatially in chronic wounds, being up-regulated atthe wound edge and down-regulated in the sur-rounding normal-appearing skin with levels decreas-ing with increasing distance from the wound. Incontrast, in acute wounds, HGF expression wasdown-regulated at the wound edge and up-regu-lated with increasing distance from the wound.Furthermore, Conway et al60 showed that c-Met(mesenchymal epithelial transition factor, an HGFreceptor) is nearly undetectable in wound edgekeratinocytes of chronic wounds, whereas, in acutewounds, c-Met expression is significantly higher thanin normal-appearing skin. These results are in ac-cordance with animal models using c-Met-deficientkeratinocytes, which are unable to contribute to there-epithelialization process, indicating that c-Met isessential for physiologic wound healing.59
In 2003, Heilborn et al62 found the antimicrobialpeptide LL-37 to be lacking in chronic woundepithelia. The administration of LL-37 antibodiesled to reduced Ki67 levels in the epithelium, reflect-ing a reduced proliferation rate and a subsequentdeceleration of wound healing. LL-37 peaked after48 hours postinjury and returned to baseline levelsuntil wound closure. As bacterial colonization is amajor issue in chronic ulcers, the effects of antimi-crobial peptides, such as LL-37, are interesting. Oneof the recently discovered pathways by which LL-37acts is kallikrein-mediated proteolysis.63 This is ofgreat importance as the balance of proteolytic activ-ity controls innate immune responses at epithelialsurfaces. Against this background, LL-37 may be oneof the most promising targets for future treatment
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regimens in chronic wound healing. It could be ofmajor relevance that antimicrobial peptides affectproliferation and migration of keratinocytes as wellas their cytokine and chemokine production.16
REMODELING PHASEThe tissue remodeling phase (Fig 3) starts as early
as a few days after injury and lasts up to 2 years. Inthis phase, a variety of proteinases contribute tocoordinated wound healing, which are regulated bytime-dependent and spatial modification of expres-sion patterns. Apart from this regulation, almost allproteinases are altered in their activity and confor-mation by the wound milieu itself, for instance by pHchanges caused by wound healing64 as comparedwith physiologic conditions.65
An important group of proteinases are MMPswhich are known to be precisely regulated by thepH value.64,66 They play a central role in woundhealing as they degrade certain constitutes of provi-sional wound tissue, such as collagen I, III, IV, andVII.67 Once the provisional wound tissue has beenremoved, the presence of TIMPs is crucial, as other-wise the continuing degradation would counteracttissue formation and subsequently wound closure.Wound healing is influenced by both the ratio ofcertain MMPs and TIMPs (eg, MMP1/TIMP ratio indiabetic foot ulcers)68,69 and the ratio of MMPs toeach other (eg, high levels of MMP1 are needed forwound healing, whereas excessively high levels ofMMP8 and MMP9 impair wound healing).70 PotentMMP inhibitors and synthetic MMPs are available.
MMP2 (gelatinase 2) has turned out to be impor-tant for angiogenesis, inflammation, and fibrosis inwound healing. Jansen et al71 were able to identifythe essential role of the enhancer element responseelement (RE)-1 in skin injuryeinduced MMP2 ex-pression in a MMP2/LacZ reporter mouse model.RE-1 is known to regulate most of the constitutiveMMP2 promoter activity. Jansen et al71 showed thatRE-1 is an important cis-regulatory element in dermalwound healing. Modulation of MMP2 expressionmay be a way to reorganize cell-cell interactions inskin wounds. Injuries are known to regulate MMPexpression by distinct pathways, for example, MMP2by the enhancer element RE-1.71
A connection between MMPs and mTOR has beeninvestigated.72 Although mTOR up-regulates MMP1and MMP3 expression after ultraviolet BeinitiatedDNA damage, it does not affect IL-1b-mediatedMMP1 and MMP3 production by fibroblasts.Ultraviolet B radiation was suggested as a therapeu-tic option to activate MMPs in wound healing.However, in chronic wounds, excessively high levelsof certain MMPs such as MMP8 and MMP9 exist, and
factors such as a1-antichymotrypsin appear capableof regulating MMP9 function in skin wound heal-ing.70,73 Again, the link between inflammation (IL-1b) and tissue remodeling (MMPs) becomes evidentand shows that wound healing phases are not strictlychronological but rather interwoven. A newly foundmember of the MMP family is MMP28, also known asepilysin. In 2008, Illman et al74 reviewed the role ofepilysin, which is known to be overexpressed inbasal keratinocytes after cutaneous injury. Epilysinnot only plays an important role in wound cellmigration but also in tumor cell invasion.
In recent years, a few other biomarkers forchronic wound pathogenesis have been character-ized. Low serum levels of Factor XIII, for instance,are known to be one factor contributing to delayedchronic venous ulcer healing. FXIIIa can bind to ECMproteins and thereby contribute to wound healing.Gemmati et al75,76 studied a gene polymorphism inthe FXIIIa subunit V34L in 91 patients with chronicvenous ulcer versus 195 healthy control subjects.FXIII V34L carriers are known to exhibit reduceda2-antiplasmin incorporation into fibrin. Gemmatiet al75,76 speculated on a mechanism involving plas-min activation of pro-MMPs by direct plasmin-antiplasmin interaction. The major observation wassignificantly reduced ulcer size in patients withincreasing numbers of polymorphic FXIII L34 alleles,which was independent of total FXIII activity. Ahigher number of FXIII L34 alleles seemed correlatedto a decrease in the higher fibrinolytic activity seen inpatients with chronic venous ulcers.75,76
Cell-ECM interactions are of major relevance forwound healing. Integrins are cell surface receptorsthat interact with the ECM and mediate variousintracellular signals. Factors such as integrins needto interact with cell membraneebound receptors forefficient cell-ECM interactions. One newly studiedco-receptor interacting with b1-integrins and growthfactor tyrosine kinase receptors is syndecan-4. Thisheparan sulfate proteoglycan crosses the cell mem-brane and is up-regulated in response to cutaneousinjury. Echtermeyer et al77 demonstrated its impor-tance by using mice that were heterozygous orhomozygous for mutated syndecan-4 genes. Bothtypes exhibited delayed wound healing as comparedwith wild-type controls but were otherwiseindistinguishable.
NEW AND FUTURE THERAPEUTICAPPROACHES
For the evaluation of different therapeutic ap-proaches, a reliable scoring system is indispensable.The wound bed score78 and the ulcerated leg sever-ity assessment score79 have been established to
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evaluate the stages and progress of wound healing inclinical practice. Not only clinical scoring systems areused for wound evaluation; more sophisticatedmethods are available. Optical wound measurementis in vogue and promising for certain questionsarising in ulcer treatment plans.80,81
One recently introduced, promising technique isthe application of biodegradable polymers thatrelease certain growth factors such as FGF-2 in apH-dependent manner.82 pH dependency is veryimportant as pH values change during wound repair,and enzyme activity may fluctuate as pH changes.1
To make things even more complex in this specialcase, basic FGF is triggered by histamine (a mediatoralso released upon cutaneous injury), leading toaccelerated wound closure.83
In addition, pH changes affect bacterial coloniza-tion84,85 and function, such as causing structuralchanges of the enterotoxin C2 of staphylococci.86
In addition, raised pH values in chronic woundsfacilitate infections by Candida albicans.87 A majorfact highlighting the importance of pH on cells is thateven protein expression patterns and transcriptionalprocesses are altered by extracellular protons, forinstance musculoaponeurotic fibrosarcoma onco-gene homolog G-2 (an important transcription fac-tor) expression may be induced by lower pH values(higher proton concentration).88 Surprisingly, spe-cific pH-dependent signaling pathways exist eventhough pH seems to be a rather unspecific stimulus atthe first glance. Therefore, modifications in woundpH could be promising, simple, and inexpensivestrategies in future chronic wound treatments.89
However, further research is indispensable to eluci-date the impact of wound pH on the healing progressand on available treatment options.
Antimicrobial peptides also play a pivotal role inpreventing bacterial colonization.15 They may there-fore be a promising target for future therapeutics inwound healing.18,20 It could be of major relevancethat antimicrobial peptides also affect proliferationand migration of keratinocytes as well as theircytokine and chemokine production.16 Therefore,influencing the inflammatory and the re-epitheliali-zation phase by stimulating these peptides, forinstance via vitamin D, seems to be feasible.
Another promising target in wound healing ismTOR. For instance, increased levels of mTOR andp70S6 kinase (a downstream target of mTOR)30 arepresent in keloids.90 Therefore, the local inhibitionof mTOR by inhibitors such as rapamycin (sirolimus)or everolimus may be promising in the prevention ofhypertrophic scarring and keloids. On the otherhand, systemic inhibition of mTOR (eg, in posttrans-plantation immunosuppression) leads to impaired
wound healing, which necessitates the assessment ofmTOR activity before treatment. Another problem isthat these inhibitors are not yet available in a topicalformulation.
The endoplasmic chaperone protein calreticulininduces proliferation of keratinocytes, fibroblasts,and endothelial cells in vitro affecting all stages ofwound healing.91 Nanney et al91 showed enhancedmacrophage recruitment to the wound site (inflam-matory phase), improved granulation (proliferativephase), and re-epithelialization in a porcine exci-sional skin wound model after topical administrationof calreticulin. They emphasized the potential ofcalreticulin in wound treatment with the advantagethat it can be applied topically.
A novel therapeutic approach is the use of ex vivogene transfer by allogenic keratinocyte cell suspen-sions. For example, allogenic keratinocytes trans-fected with human EGF by pCEP/h EGF (plasmidchromosomal expression platform/human epider-mal growth factor) plasmids were transplanted in afull-thickness porcine wound model, leading tohigher rates of re-epithelialization.92 This strategymay be applicable for a variety of different genesimportant in wound healing, but there are still toomany unknown factors for using gene transfer in aclinical setting.
In addition, some approaches rely on the use ofintact stem cells instead of modifying the cellspresent in the wound. Of course, this strategy cannot be specifically assigned to any of the phasesdiscussed above. As fetal wound healing differs fromadult wound healing in many aspects, gene expres-sion in foreskin and fetal skin cells was compared.93
The overall TGF-b expression (predominantly TGF-b1) in fetal cells was 6-fold up-regulated. In addition,bone morphogenic protein (BMP)-2 levels wereabout 4-fold higher in fetal cells. However, growthdifferentiation factor (GDF)-10 (synonym of bonemorphogenic protein-3B) was down-regulated 12-fold in fetal cells. The authors concluded that fetalcells offer advantages for cell-based wound therapycompared with foreskin cells.93
A newly discovered field in wound repair is thenetwork between the nervous system and the im-mune system. Numerous important mediators existin this cross talk, which include neuropeptides andcytokines released from nerve fibers, immune cells,and cutaneous cells. A link between wound healingand the nervous system is clinically apparent asperipheral neuropathy is reported in 30% to 50% ofpatients with diabetes, presenting the most commonand sensitive predictor for foot ulceration.94 Oneimportant player in this context is substance P. Inaddition to its role in pain perception, substance P
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acts as an injury-inducible factor early in the woundhealing process and induces CD291 stromal-like cellmobilization.95 Remarkably, mobilization of suchcells also occurs in uninjured mice, rats, and rabbitsif substance P is intravenously injected.95 Both sub-stance P injection and transfusion of autologouslyderived substance Pemobilized CD291 cells fromuninjured rabbits accelerated wound healing in analkali burn model.95 Hong et al95 showed thatepithelial engraftment of transfused cells into injuredtissue occurred during wound healing. Moreover,they showed that substance P can stimulate transmi-gration, cell proliferation, activation of the extracel-lular signalerelated kinases (Erk)-1 and -2, andnuclear translocation of b-catenin in vitro. Thispioneer work highlighted a new function of sub-stance P as a systemically acting messenger of injuryand as a mobilizer of CD291 stromal-like cells inwound healing.
CONCLUSIONSThe variety of molecular and cellular targets and
signaling cascades in wound healing may startspeculations on future treatment strategies basedon these results. However, additional attentionshould be drawn to widely used techniques, eventhough evidence-based data are still missing thatmay underline the promising results in daily clinicalroutine. Prominent examples are negative pressurewound therapy96,97 or hyperbaric oxygen ther-apy.98,99 Regulating the activity of cells involved inwound healing seems to be a hot topic in futurewound therapy as many new and interesting factorsare being discovered and studied in cutaneouswound healing. A promising new strategy forchronic wound treatment is tissue engineering forskin substitutes. However, this procedure involvesthe problem that wound healing gene expressionpatterns are different in fetal and adult cells.93
Nevertheless, fetal cells seem to be promising as afew specific gene expression patterns are known tobe essential for scarless wound healing.93,100 Thesefindings may influence research on bone-marro-wederived stem cells for skin repair101 as little isknown about their specific wound gene expressionpatterns. New studies also focus on the role of hairfollicle-bound epithelial stem cells in cutaneouswound healing.102 These cells may give rise to thedevelopment of new follicles, glands, and epidermalregeneration.
Even though most strategies still have to betransferred from bench to bedside, many of themare promising, and the future will show whichstrategies will be implemented into clinical routine.
The authors would like to thank Walter Burgdorf for theprecise correction of the manuscript and Yvonne Egle forthe thorough proofreading. The editorial assistance ofMonika Schoell is gratefully acknowledged.
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