ORIGINAL ARTICLE The pro-inflammatory environment in recalcitrant diabetic foot wounds Jorge Berlanga Acosta, Diana Garcia del Barco, Danay Cibrian Vera, William Savigne, Pedro Lopez-Saura, Gerardo Guillen Nieto, Gregory S Schultz Acosta JB, del Barco DG, Vera DC, Savigne W, Lopez-Saura P, Nieto GG, Schultz GS. The pro-inflammatory environment in recalcitrant diabetic foot wounds. Int Wound J 2008;5:530–539. ABSTRACT Lower extremity ulceration is one of the serious and long-term diabetic complications rendering a significant social burden in terms of amputation and quality-of-life reduction. Diabetic patients experience a substantial wound-healing deficit. These lesions are featured by an exaggerated and prolonged inflammatory reaction with a significant impairment in local bacterial invasion control. Experimental and clinical evidences document the deleterious consequences of the wound’s pro-inflammatory phenotype for the repair process. From a biochemical standpoint, hyperinflammation favours wound matrix degradation, thus, amplifying a pre-existing granulation tissue productive cells’ invasiveness and recruitment deficit. Tumour necrosis factor perpetuates homing of inflammatory cells, triggers pro-apoptotic genes and impairs reepithelialisation. Advanced glycation end-products act in concert with inflammatory mediators and commit fibroblasts and vascular cells to apoptosis, contributing to granulation tissue demise. Therapeutic approaches aimed to downregulate hyperinflammation and/or attenuate glucolipotoxicity may assist in diabetic wound healing by dismantling downstream effectors. These medical interventions are demanded to reduce amputations in an expanding diabetic population. Key words: Diabetes OVERVIEW OF THE DIABETIC WOUND PROBLEM Key Points • diabetes mellitus (DM) is the • • Granulation tissue • Hyperglycaemia • Inflammation • Wounds only endocrine metabolic disorder with an expanding proportion that approaches to a worldwide pandemic disease lower extremity ulceration is one of the various and serious long-term complications associated with DM, which is sustained and/or amplified by the underlying failure in the repair process of peripheral soft tissues 530 Diabetes mellitus (DM) is the only endocrine metabolic disorder with an expanding proportion that is approaching worldwide pandemic levels (1). The connection between diabetes and foot ulceration was stated as early as 1887 by the surgeon T.D. Pryce. For the first time, Pryce claimed in an article published in Lancet that ‘diabetes itself may play an active part in the causation of perforating ulcers’ (2). Lower extremity ulceration is one of the various and serious long-term complications associated with DM, which is sustained and/or amplified by the underlying failure in the repair Authors: JB Acosta, PhD, Tissue Repair and Cytoprotection Research Project, Biomedical Research Direction, Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, Avenida 31 e/158 y 190, Playa, PO Box 6162, Havana 10600, Cuba; DG del Barco, MD, Tissue Repair and Cytoprotection Research Project, Biomedical Research Direction, Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, Avenida 31 e/158 y 190, Playa, PO Box 6162, Havana 10600, Cuba; DC Vera, BSc, Tissue Repair and Cytoprotection Research Project, Pharmaceutical Division, Biomedical Research Direction, Center for Genetic Engineering and Biotechnology, Avenida 31 e/158 y 190, Playa, PO Box 6162, Havana 10600, Cuba; W Savigne, MD, Instituto Nacional de Angiologı́a y Cirugı́a Vascular, Hospital Salvador Allende, Calzada del Cerro S/N, Cerro, Havana, Cuba; P Lopez-Saura, MD, Center for Biological Research, 134 St. e/23 and 25, Cubanacan, Playa, Havana, Cuba; G Guillen Nieto, PhD, Biomedical Research Direction, Center for Genetic Engineering and Biotechnology, Avenida 31 e/158 y 190, Playa, PO Box 6162, Havana 10600, Cuba; GS Schultz, PhD, Department of Obstetrics and Gynecology, Institute for Wound Research, University of Florida, Gainesville, FL 32610-0294, USA Address for correspondence: Dr JB Acosta, PhD, Tissue Repair and Cytoprotection Research Project, Biomedical Research Direction, Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, Avenue 31 e/158 & 190, Cubanacan, Playa, PO Box 6162, Havana 10600, Cuba E-mail: jorge.berlanga@cigb.edu.cu ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc • International Wound Journal • Vol 5 No 4 Recalcitrant diabetic foot wounds process of peripheral soft tissues. The impaired healing in diabetes affects the resolution of both acute and chronic wounds and represents a significant health care expenditure even for the most developed economies. Poor-healing, lower extremity ulcerations are a significant cause of hospitalisation and the first step towards limb amputation. Diabetes increases the risk of lower extremity amputation by at least 15-fold and contributes to 85% of all non traumatic amputations (3,4). The term diabetic foot was coined to distinguish the specific qualities about the feet of diabetic patients that set this disease apart from other conditions that affect the lower extremity. Infection, ulceration and/or destruction of deep tissues associated with neurological abnormalities and various degrees of peripheral vascular disease in the lower limb define the ‘diabetic foot’ (5). Although the molecular pathophysiology of the diabetic foot disease itself is beyond the scope of this review, it is pertinent to highlight that from the pathophysiological point of view; diabetic foot ulcers are classified into two main clinical arms: neuropathic or ischaemic (6). Although both neuropathy and vasculopathy as individual entities may coexist and interact in a diabetic foot, substantial clinical and histological differences can be distinguished between neuropathic and ischaemic wound beds. Thus, neuropathy and tissue hypoperfusion are hyperglycaemic long-term complications that, along with wound dimension and the host’s incapability to control local infection, contribute to the prognosis of an unfavourable outcome. Wound healing is a highly dynamic, finely orchestrated and complex mega-process of multiple overlapping phases that can be divided into three main stages: (i) inflammatory, (ii) proliferative and (iii) remodelling. Large-scale clinical trials have identified hyperglycaemia as a key determinant for the development of biochemical disturbances that steers the onset and progression of diabetic chronic complications that interact and interamplify each other (7). Among these complications, the poorness in triggering and/or sustaining a physiological wound repair mechanism is dismal. The diabetic wound is mostly distinguished by its chronicity and by the asynchrony in the healing phases within a specific wound niche (8). No diabetic ulcer deserves to be clinically underestimated or considered as a ‘naı̈ve’ wound. Broadly speaking, diabetes impairs most. if not all, the events encompassed within the healing process, including haemostasis, inflammation, matrix deposition, angiogenesis, contraction and remodelling. While healing diabetic wounds in a timely manner is instrumental in any plan for amputation prevention, they pose an overwhelming molecular puzzle demanding research and elucidation. Although the diabetic foot wound is multidisciplinarily approached within comprehensive therapeutic systems (9), success in the treatment of recalcitrant lesions remains discouraging. The clinical challenge of the diabetic healing failure is the gross expression of an outstanding array of biochemical and cellular disturbances. Herein, we examine the role of the wound’s inflammatory burden as a pivotal ingredient for the stubbornness of diabetic repair process. Key Points • diabetes increases the risk of • • • DISSECTING THE DIABETIC WOUND: THE INFLAMMATORY COMPONENT The inflammatory response is required for optimal wound healing. Polymorphonuclear neutrophils (PMN) preside the first wave of immune cells that invade the wound bed. Although these cells have long been considered to be confined to host defence against a variety of infectious agents, recent studies have shown that PMN also contribute to the production of inflammatory cytokines (10). The wound macrophage is temporally second to PMN. Monocytes begin infiltrating the wound site
24 hours after injury, attracted by chemotactic factors including complement factor 5a, fibrin by-products and transforming growth factor beta (TGF-b). In response to cytokines in the wound, monocytes differentiate into wound macrophages that are necessary for wound repair. They are an important source of growth factors and their depletion provokes a delayed healing process (11). The diabetic foot ulcer is generally a chronic wound that does not exhibit the orderly cascade of events that characterise normal wound healing. In contrast to the physiological process, the inflammatory reaction in diabetic wounds appears prolonged. Our experience on serial biopsies from both neuropathic and ischaemic ulcer-derived granulation tissue have indicated that even in the absence of infection, PMN infiltration is intense, prolonged and topographically not polarised, particularly ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc • • • • • lower extremity amputation by at least 15-fold and contributes to 85% of all non traumatic amputations infection, ulceration and/or destruction of deep tissues associated with neurological abnormalities and various degrees of peripheral vascular disease in the lower limb define the diabetic foot neuropathy and tissue hypoperfusion are hyperglycaemic longterm complications that, along with wound dimension and the host’s incapability to control local infection, contribute to the prognosis of an unfavourable outcome wound healing is a highly dynamic, finely orchestrated and complex mega-process of multiple overlapping phases that can be divided into three main stages: (i) inflammatory, (ii) proliferative and (iii) remodelling while healing diabetic wounds in a timely manner is instrumental in any plan for amputation prevention, they pose an overwhelming molecular puzzle demanding research and elucidation the clinical challenge of the diabetic healing failure is the gross expression of an outstanding array of biochemical and cellular disturbances herein, we examine the role of the wound’s inflammatory burden as a pivotal ingredient for the stubbornness of diabetic repair process the inflammatory response is required for optimal wound healing in contrast to the physiological process, the inflammatory reaction in diabetic wounds appears prolonged 531 Recalcitrant diabetic foot wounds Key Points • although influx of inflammatory • • • • • • • cells into the wound is initially slow and inadequate, once it is turned on, potent inflammatory forces exclusive for the diabetic environment arise to sustain the generation of pro-inflammatory cytokines and a deregulated production of tissue matrix metalloproteinases (MMPs), which sharply limit the process of granulation tissue formation and maturation the search for explanations on this exaggerated inflammatory reaction has identified critical elements aiming at both soluble and cellular factors compelling evidences indicate that PMN are critical towards the acquisition of a prodegradative phenotype resulting from the imbalance between matrix synthesis and degradation as a matter of fact, high circulating PMN elastase levels are associated to a poor glycaemic control and are currently considered as a risk marker for the development of diabetic angiopathy another link between wound MMPs and pro- 10 inflammatory cytokines is that some MMPs are regulated via NFkB pathway, involving the participation of IL-1 and TNF-a as triggering signals an alternative explanation for the abnormal diabetic proinflammatory portrait suggests that inflammatory cells are able to evade apoptosis contrary to normal counterparts, inflammatory cells’ apoptosis, matrix deposition and epithelial restitution appear blunted in diabetic wounds TNF-a has been largely implicated in this contraventional event and has proved to negatively impact the repair process 532 in neuropathic wounds. It is not uncommon to observe a chronic infiltration presided by an ‘acute’ inflammatory effector cell, coexisting with a scarce extracellular matrix accumulation in which collagen deposit is impoverished. On the contrary, a broadly spread infiltration of round cells predominate in those patients suffering from wound bed hypoperfusion because of macro- or microangiopathy. Although influx of inflammatory cells into the wound is initially slow and inadequate, once it is turned on, potent inflammatory forces exclusive for the diabetic environment arise to sustain the generation of pro-inflammatory cytokines and a deregulated production of tissue matrix metalloproteinases (MMPs), which sharply limit the process of granulation tissue formation and maturation. Although it is generally accepted that the chronic diabetic ulcer is stalled in the inflammatory phase of the normal healing process, this inflammatory reaction does not necessarily imply a physiological bacterial local control. On the contrary, clinical practice teaches that diabetic individuals are more susceptible to both wound infection and hyperinflammation, which is not pathogenically detachable from the elevated levels of tumour necrosis factor-alpha (TNF-a) and interleukin (IL)-6 (12). Experimental studies have shown that macrophages from diabetic mice break down cell detritus much slower and far less efficiently than their non diabetic counterparts (13). The search for explanations on this exaggerated inflammatory reaction has identified critical elements aiming at both soluble and cellular factors. Data derived from diabetic rodent models have documented a prolonged expression of macrophage inflammatory protein-2 and macrophage chemoattractant protein-1. Neutralising antibodies against these chemokines provided evidence that their dysregulation and sustained expression appears to be directly associated with the increased and protracted infiltration of both PMN and macrophages into the diabetic wound (14). Compelling evidence indicates that PMN are critical towards the acquisition of a prodegradative phenotype resulting from the imbalance between matrix synthesis and degradation. The granulocytes secrete pro-inflammatory cytokines, particularly TNF-a and IL-1b. Both cytokines are capable of directly stimulating the synthesis of MMPs. PMN secrete, among others, MMP-8 that has been identified as the predominant collagenase in non healing wounds (15). Prolonged PMN infiltration is also linked to elastase, overproduction of reactive oxygen species (ROS) and reactive nitrogen species within the wound site; all with a remarkable cytotoxic and prodegradative potential (16,17). As a matter of fact, high circulating PMN elastase levels are associated to a poor glycaemic control and are currently considered as a risk marker for the development of diabetic angiopathy (18). Another link between wound MMPs and proinflammatory cytokines is that some MMPs are regulated via nuclear factor-kappa B (NFkB) pathway, involving the participation of IL-1 and TNF-a as triggering signals. In this context, the molecular targets of MMPs are myriad and include not only elements of the extracellular matrix but also locally secreted growth factors and their receptors. The observation that diabetic wounds are enriched in MMPs provides support for the premise that impaired growth factor availability may act as a healing and limiting factor (14,19). An alternative explanation for the abnormal diabetic pro-inflammatory portrait suggests that inflammatory cells are able to evade apoptosis. Contrary to normal counterparts, inflammatory cells’ apoptosis, matrix deposition and epithelial restitution appear blunted in diabetic wounds. Although some forces seem to prevent the apoptotic commitment of the inflammatory cells, other cells that are essential for granulation tissue outgrowth stand as extremely prone to suicide (20). TNF-a has been largely implicated in this contraventional event. In addition to MMPs, high levels of TNF-a in the wound have been identified as a molecular predictive factor for closure failure (14). Type 2 diabetes is associated with high serum levels of inflammatory cytokines such as TNF-a (21). Within the wound, TNF-a stimulates its own secretion and that of IL-1b that contributes to a persistent inflammatory status (22). TNF-a has proved to negatively impact the repair process. Its deregulation is not only associated with persistent inflammation but also to connective tissue destruction, thus pushing towards a catabolic slope (23). In line with this, TNF-a application causes a decrease in wound tensile strength, which is likely because of decreased collagen types I and III expression (24,25). On the contrary, its inhibition or deletion generally enhances the repair processes. Genetic ablation of ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc Recalcitrant diabetic foot wounds TNF-a receptor-1 improves the general wound healing profile by enhancing angiogenesis, collagen production and reepithelialisation (26). Systemic administration of neutralising antibodies against TNF-a into wounded ob/ob mice attenuated local inflammation by reducing the number of viable macrophages, while triggering a rapid and complete neoepidermal coverage (27). This indicates that TNF-a inhibition is apparently sufficient to neutralise the misbehaving inflammatory machinery in non healing wounds, so as to assist in reprogramming the whole local microenvironment. The effects associated with this intervention include both the restitution of the epithelial sector, as the restoration of granulation tissue outgrowth in terms of cellularity, and extracellular matrix synthesis. Graves’ studies have illustrated the proapoptogenic effect of wound-secreted TNF-a on fibroblasts populations in experimental diabetic wounds. The intervention with a TNF-a-specific inhibitor during wound healing significantly reduced caspase-3 activity and fibroblast apoptosis by almost 50%. The effect of the inhibitor assisted in repopulating the wound bed by increasing fibroblast number by 31% as well as activating these cells for making up new matrix by 72% (26). Another contribution of this group is the recent identification of 71 overexpressed genes in diabetic animals that directly or indirectly regulate apoptosis and that significantly enhance cas- pases activity. The functional significance of diabetes-induced apoptosis was studied by treating diabetic mice with a pancaspase inhibitor. The study confirmed that apoptosis inhibition significantly improved several parameters within the healing process, including fibroblast density, enhanced collagens I and III mRNA levels and increased matrix formation. Converging evidences indicate that there is a remarkable increase in the apoptotic process of granulation tissue producing cells in those wounds from individuals with poorly controlled blood sugar and concomitant microangiopathy (28). Thus, the high level of fibroblastic cells apoptosis is a meaningful contributing factor for a deficient healing response in diabetic individuals. Summarising the above ideas, TNF-a is a major driving force towards the onset and perpetuation of the wound pro-inflammatory and pro-catabolic phenotype. By mean of an active crosstalk between inflammatory cells and TNF-a, a larger recruitment of these cells lineages is ensued, which renders more local TNF. These woundhomed inflammatory cells become refractory or somehow spared from the apoptotic commitment. As a concomitant side effect, TNF-a intoxicates fibroblasts and vascular precursor cells. Fibroblasts proliferation and migration are arrested, matrix ingredients secretion is impaired and at the end, these fibroblasts are bound to suicide (Figure 1). Key Points • systemic administration of neu- • tralising antibodies against TNF-a into wounded ob/ob mice attenuated local inflammation by reducing the number of viable macrophages, while triggering a rapid and complete neo-epidermal coverage the effects associated with this intervention include both the restitution of the epithelial sector, as the restoration of granulation tissue outgrowth in terms of cellularity, and extracellular matrix synthesis Figure 1. The cytotoxic effect of pro-inflammatory cytokines and glycation products in diabetic wound failure. AGE, advanced glycation end products; IL, interleukin; EN-RAGE, extracellular newly identified receptor for advanced glycation end products; TNF-a, tumour necrosis factor-alpha. ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc 533 Recalcitrant diabetic foot wounds Key Points • a brief anti-inflammatory inter- • • • • • vention towards TNF-a neutralisation is able to restore insulin sensitivity by reestablishing the expression and functionality of the hormone s receptor as a bridge between clinical and molecular evidences and to further validate the relevance of the inflammatory ingredient against the normal healing rhythm in diabetic ulcers, a clinical study showed that pressure relief of neuropathic ulcers by total contact casts significantly enhanced wound healing after 20 days compared with a concurrent control group with similar lesions and appropriate glycaemic but lacking casts this result confirms that relieving the irritating pressure reduces the hyperinflammatory ingredient and accelerates the repair processes many recent evidences indicate that the biology of the advanced glycation end-products/receptor (AGE/RAGE) system in diabetic individuals is a molecular trigger and/or an amplifier of most, if not all, the accumulative disease complications, particularly within the vascular system it has been established that AGE accumulate and interfere with tissue matrix proteins through inappropriate crosslinking once AGE(s) are formed, their interaction with RAGE triggers the generation of proinflammatory cytokines, adhesion molecules and chemokines, thus enhancing the attraction of inflammatory cells into the tissue for a sustained inflammatory response 534 An alternative pathogenic cascade establishes a connection between TNF-a levels and tissues insulin resistance (29) from where the skin is not excluded. This notion has been recently enriched with the confirmation by Goren et al. that systemic TNF-a levels are linked to an impaired healing response under diabetic and obese conditions in ob/ob mice (2). Chronic exposure of keratinocytes to TNF-a inhibits insulin-stimulated glucose uptake. Most importantly, it is the merit of this work to show that a brief anti-inflammatory intervention towards TNF-a neutralisation is able to restore insulin sensitivity by reestablishing the expression and functionality of the hormone’s receptor. As stated by the authors, the extension of this concept to other soluble mediators might partially explain why topical application of growth factors into chronically wounded tissue has failed to improve the healing process (2). According to in silico simulation methods, any therapeutic approach aimed to neutralise TNF-a or to increase the local availability of active TGF-b1 would be similarly effective regardless of the initial assumption of the derangement that underlies diabetic foot ulcers (DFU) (elevated TNF versus reduced TGF-b1) (30). Generally speaking, diabetes complications have the tremendous aetiopathogenic imprint of an increased expression of TNF-a and fas/fas-ligand (31). As a bridge between clinical and molecular evidences and to further validate the relevance of the inflammatory ingredient against the normal healing rhythm in diabetic ulcers, a clinical study (32) showed that pressure relief of neuropathic ulcers by total contact casts significantly enhanced wound healing after 20 days compared with a concurrent control group with similar lesions and appropriate glycaemic but lacking casts. The histopathological characterisation of the two study groups indicated that inflammation along with matrix and angiogenesis alterations persisted in the control group. On the contrary, total contact casts steered the wound towards a repair profile with no major inflammatory evidences. This result confirms that relieving the irritating pressure reduces the hyperinflammatory ingredient and accelerates the repair processes. Several years ago, Mast and Schultz (33) had anticipated the concept that repeated tissue injury leads to prolonged inflammation that causes elevated levels of proteases, thus re- tarding the healing process. This turns envisionable that the introduction of a wound hyperinflammation counter-regulating therapy paralleled with a timely balanced protease/antiprotease intervention could be of major benefit. ADVANCED GLYCATION ENDPRODUCTS, THE RECEPTOR AND THE WOUND PROINFLAMMATORY PROCESS Many recent evidences indicate that the biology of the advanced glycation end-products/receptor (AGE/RAGE) system in diabetic individuals is a molecular trigger and/or an amplifier of most, if not all, the accumulative disease complications, particularly within the vascular system. In both types 1 and 2 diabetes, the persistent hyperglycaemia seems to be a major requisite in the non enzymatic glycation process that originates the AGE as a heterogeneous group of cytotoxic compounds. As a corollary, ROS are also formed along the AGE generation process and correspondingly ROS hasten AGE formation, thus paving the way for a selfperpetuating pathogenic cycle of ROS-AGE, which somehow characterises the biochemistry of diabetes (34). It has been established that AGE accumulate and interfere with tissue matrix proteins through inappropriate crosslinking. Similarly, AGE accumulate intracellularly and disturb a variety of cell physiological activities. AGE act via cell surface receptormediated interactions, such as RAGE, which coincidently appears overrepresented in tissues from most diabetic individuals (35). Generally speaking, once AGE(s) are formed, their interaction with RAGE triggers the generation of pro-inflammatory cytokines, adhesion molecules and chemokines, thus enhancing the attraction of inflammatory cells into the tissue for a sustained inflammatory response (Figure 1). Glycoxidation products accumulate in the non labile dermal cutaneous collagen, which means that a diabetic’s skin is physicochemically altered, further complicating biological processes that mechanistically depend on the cells’ physiological integrity such as anchorage, chemotaxis, migration, proliferation, terminal differentiation and scaffolding and branching so as to create new blood vessels. Unpublished ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc Recalcitrant diabetic foot wounds data from our group, based on routine staining and immunohistochemistry on granulation tissue biopsies, have suggested that an intense accumulation of AGE and a concomitant RAGE overexpression in productive cells are associated with a frustrated angiogenesis characterised by the abundance of degenerated endothelial precursor cells as by scattered angioblastoma cells incapable of lining up to establish an appropriate co-opting. We recently confirmed the immediate toxic consequences of AGE accumulation in rats systemically challenged with methylglyoxal. As expected, the animals floridly reproduced most of the diabetic systemic complications but within a normal glycaemic environment throughout their experimental life. It was shown that the uncontrolled preponderance of the AGE/RAGE binomium impairs the appropriate balance required between antagonistic forces in operational processes like pro-inflammation/counterinflammation, regeneration/degeneration and vasodilation/vasoconstriction. With this notion, it is intuitive to speculate that the AGE/RAGE axis perturbs tissue repair as it has been consistently proved in experimental systems (36). Experimental data teach that diet-derived AGE significantly influences the rate of wound healing, which appears associated with AGE skin deposits. Mice fed with lower AGE levels exhibited increased reepithelialisation, angiogenesis and granulation tissue deposition than those fed with high AGE diets (37). RAGE is expressed on the most important cells involved in the normal wound healing, including endothelial cells, monocytes, fibroblasts and smooth muscle cells. RAGE also binds non AGEs, members of the S100/calgranulin family of polypeptides, termed extracellular newly (EN) identified RAGE-binding proteins (EN-RAGEs) (38). EN-RAGEs are synthesised by leucocytes and trigger inflammatory cells’ activation leading to the synthesis of pro-inflammatory cytokines. This autocrine and paracrine loop propagates and sustains the inflammatory response, further contributing to the wound chronic inflammatory state that contains both types of RAGE ligands. In mononuclear phagocytes, ligation of RAGE by AGEs or EN-RAGEs increases generation of cytokines, such as TNF-a, IL-6 and IL-1, as to an enhanced production of O2 , which is typical of diabetics compared with non diabetic counterparts (39). Endothelial cells express RAGE and are sensitive to the ligand interaction, resulting in the induction of the procoagulant initiator tissue factor, enhanced permeability and expression of adhesion molecules such as VCAM-1 (vascular cell adhesion molecule-1) and cytokines such as IL-6 (40). Accumulation of AGE on the vessel wall has been implicated in the pathogenesis of diabetes complications (34). Accordingly, RAGE has been recently shown to be involved in both microvascular (41) and macrovascular (42) complications that establishes an alternative path for the diabetic healing failure, with the additional burden of an ischaemic component. In addition to what has been achieved on fibroblasts’ and myofibroblasts’ biology, the involvement of RAGE within the vascular sector has also gained delineation. Shoji et al. (43) have published data, which define RAGE’s participation in the diabetic undermined angiogenic response. They show that RAGE stimulation leads to a decreased proliferation and to an increased rate of vascular precursor cells death, whereas both events are prevented under a RAGE-deficient context. In line with the proapoptogenic consequences of the AGE–RAGE interaction for endothelial cells, other evidences extend this concept to fibroblastic cells. Graves’ group has established a signalling pathway leading to fibroblast apoptosis when these cells are exposed to a particularly relevant form of AGE (CML collagen). In this model, RAGE agonistic stimulation proved to activate p38 and JNK pathways via different intermediate triggers (ROS, NOS and ceramides) leading eventually to an enhanced caspase-3 activity and in which pro-apoptotic gene-enhanced expression was likely mediated by FOXO1 transcription factor (44). This meaningful evidence along with other demonstrations derived from Ann Marie Schmidt’s group, currently place the AGE/ RAGE at the centre of the toxic and proinflammatory cascade of events that disturbs wound healing in diabetes (45). Few years ago, Schmidt’s laboratory examined the concept that quenching cellular RAGE stimulation in diabetic wounded mice would positively impact in the healing response. The functional neutralisation of autologous RAGE primarily resulted to equilibrate the inflammatory forces in a timely manner, followed by an increase in wound closure according to histological evidences of thick granulation tissue and reepithelialisation. These findings highlight the premise that in ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc Key Points • experimental data teach that • • diet-derived AGE significantly influences the rate of wound healing, which appears associated with AGE skin deposits RAGE is expressed on the most important cells involved in the normal wound healing, including endothelial cells, monocytes, fibroblasts and smooth muscle cells meaningful evidence along with other demonstrations derived from Ann Marie Schmidt’s group, currently place the AGE/RAGE at the centre of the toxic and proinflammatory cascade of events that disturbs wound healing in diabetes 535 Recalcitrant diabetic foot wounds Key Points • diabetes is contemporarily expanding worldwide; its victims will welcome novel drugs and smart prophylactic interventions, which would assist in preventing/reducing the systemic complications such as lower extremity amputations contrast to therapeutic strategies interfering with distal effector pathways, such as growth factors administration, the RAGE blockade may exert a more proximal and global effect on the biology of the diabetic wound by modulating the expression of instrumental local ingredients such as growth factors, cytokines and MMPs. Taken together, silencing of RAGE resets the balance between effective and deleterious inflammation, thereby promoting wound closure (36). CONCLUSIONS High-grade diabetic wounds either ischaemic or neuropathic remain as an empty clinical niche, largely contributing to lower limb amputations within the diabetic population. The molecular bases of the diabetic wound healing impairment are complex and multifactorial. Nevertheless, for these wounds to gain the classic clinical expression of arrest, non progression, phases’ asynchrony, slow and/or poor collagenisation and refractoriness to reepithelialise, the convergence of both systemic and local pathogenic factors seems to play a critical role. Several studies have underscored the role of sustained hyperglycaemia as a systemic factor heavily hindering wound healing. Converging data from a myriad of classic experiments indicate that even in culture medium conditions, certain glucose levels appear toxic enough to characteristically perturb fibroblast, endothelial cells and keratinocyte homeostasis, thus recreating some of the in vivo impairments that distinguish the diabetic granulation tissue histopathology. Inflammation acts in a paradoxical manner within the diabetic wound by acting as a potent limiting factor in many respects without contributing to an effective control of local infection. A perpetuating vicious circle is established between the prolonged cellular recruitment and the spillover of pro-inflammatory cytokines such as TNF-a and IL-1b. In addition to enhance matrix degradation via proteases activation, TNF-a has a more upstream deleterious effect as it reduces granulation tissue cellularity by promoting apoptosis in fibroblast and endothelial cells. Another amplifying vicious circle involves the trilogy of AGE/RAGE þ TNF-a in perpetuating a harmful inflammatory reaction within the wound. In addition to the direct cytotoxic effect of both extracellularly and intracellularly accu536 mulated AGE, the ligation of the receptor is considered a key factor for a lengthened inflammatory reaction as a pro-apoptogenic threat for fibroblasts and angioblasts. The jugulation of TNF-a/inflammation and/or that of the AGE/RAGE activation would be a theoretically perfect therapeutic target. The deactivation of these two interdigitated arms must act in a concerted fashion to prevent the strike of downstream negative effectors. Diabetes is contemporarily expanding worldwide. Its victims will welcome novel drugs and smart prophylactic interventions, which would assist in preventing/reducing the systemic complications such as lower extremity amputations. Besides, it is tempting to study why the most universal cells’ fuel appears as toxic for such an indispensable and ancestral mechanism as tissue repair. Diabetes is characterised by pro-inflammatory cytokines spillover (TNF-a, IL-1 and IL-6) particularly secreted by inflammatory cells. These cytokines act as potent chemoattractants within the wound bed recruiting more inflammatory cells and protracting their in-wound homing. The local oversecretion of pro-inflammatory cytokines exerts a negative effect in wound cells such as fibroblasts, vascular precursors, pericytes and smooth muscle cells. The excessive AGE accumulation achieved in a diabetic’s tissues as a result of prolonged hyperglycaemic conditions severely intoxicates the above-mentioned cells. AGE toxicity can be attributed to the direct glycation of cellular proteins or by the receptor’s ligation (RAGE). RAGE also binds a non AGE family member: the EN-RAGE. This leucocytes’ product triggers inflammatory cells’ activation further enhancing the synthesis of pro-inflammatory cytokines enriching the toxic microenvironment. These three interdependent factors contribute to the diabetic wound chronification. Their cytotoxic actions on granulation tissue productive cells include proliferative arrest associated with senescence and unscheduled apoptosis. Granulation tissue productive cells’ population is reduced, while inflammatory cells appear to be increased. This cellular deficit is ensued by a substantial reduction of extracellular matrix synthesis and accumulation, while the wound slips into a catabolic balance characterised by a hostile milieu of oxygen- and nitrogen-derived species, matrix proteases and soluble mediators of inflammation. ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc Recalcitrant diabetic foot wounds REFERENCES 1 Engelgau MM, Geiss LS, Saaddine JB, Boyle JP, Benjamin SM, Gregg EW, Tierney EF, RiosBurrows N, Mokdad AH, Ford ES, Imperatore G, Narayan KMV. The evolving diabetes burden in the United States. Ann Intern Med 2004;140: 945–50. 2 Goren I, Muller E, Pfeilschifter J, Frank S. Severely impaired insulin signaling in chronic wounds of diabetic ob/ob mice: a potential role of tumor necrosis factor-alpha. Am J Pathol 2006;168: 765–77. 3 Most RS, Sinnock P. The epidemiology of lower extremity amputations in diabetic individuals. Diabetes Care 1983;6:87–91. 4 Bild DE, Selby JV, Sinnock P, Browner WS, Braveman P, Showstack JA. Lower-extremity amputation in people with diabetes. Epidemiology and prevention. Diabetes Care 1989;12:24–31. 5 Singh N, Armstrong DG, Lipsky BA. Preventing foot ulcers in patients with diabetes. JAMA 2005;293: 217–28. 6 Edmonds ME. The diabetic foot: pathophysiology and treatment. Clin Endocrinol Metab 1986;15: 889–16. 7 Stratton IM, Adler AI, Neil HA, Matthews DR, Manley SE, Cull CA, Hadden D, Turner RC, Holman RR. Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ 2000;321:405–12. 8 LeRoith D, Fonseca V, Vinik A. Metabolic memory in diabetes–focus on insulin. Diabetes Metab Res Rev 2005;21:85–90. 9 Boulton AJ, Meneses P, Ennis WJ. Diabetic foot ulcers: a framework for prevention and care. Wound Repair Regen 1999;7:7–16. 10 Hubner G, Brauchle M, Smola H, Madlener M, Fassler R, Werner S. Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice. Cytokine 1996;8:548–56. 11 DiPietro LA. Wound healing: the role of the macrophage and other immune cells. Shock 1995; 4:233–40. 12 Borst SE. The role of TNF-alpha in insulin resistance. Endocrine 2004;23:177–82. 13 O’Brien BA, Geng X, Orteu CH, Huang Y, Ghoreishi M, Zhang Y, Bush JA, Li G, Finegood DT, Dutz JP. A deficiency in the in vivo clearance of apoptotic cells is a feature of the NOD mouse. J Autoimmun 2006;26:104–15. 14 Wetzler C, Kampfer H, Stallmeyer B, Pfeilschifter J, Frank S. Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair. J Invest Dermatol 2000;115:245–53. 15 Nwomeh BC, Liang HX, Cohen IK, Yager DR. MMP-8 is the predominant collagenase in healing wounds and nonhealing ulcers. J Surg Res 1999;81: 189–95. 16 Schonfelder U, Abel M, Wiegand C, Klemm D, Elsner P, Hipler UC. Influence of selected wound 538 17 18 19 20 21 22 23 24 25 26 27 28 29 30 dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro. Biomaterials 2005;26:6664–673. Dinh T, Pham H, Veves A. Emerging treatments in diabetic wound care. Wounds 2002;14:2–10. Piwowar A, Knapik-Kordecka M, Warwas M. Concentration of leukocyte elastase in plasma and polymorphonuclear neutrophil extracts in type 2 diabetes. Clin Chem Lab Med 2000;38:1257–261. Inoue N, Nishikata S, Furuya E, Takita H, Kawamura M, Nishikaze O. Streptozotocin diabetes: prolonged inflammatory response with delay in granuloma formation. Int J Tissue React 1985;7:27–33. Goova MT, Li J, Kislinger T, Qu W, Lu Y, Bucciarelli LG, Nowygrod S, Wolf BM, Caliste X, Yan SF, Stern DM, Schmidt DM. Blockade of receptor for advanced glycation end-products restores effective wound healing in diabetic mice. Am J Pathol 2001; 159:513–25. Mishima Y, Kuyama A, Tada A, Takahashi K, Ishioka T, Kibata M. Relationship between serum tumor necrosis factor-alpha and insulin resistance in obese men with Type 2 diabetes mellitus. Diabetes Res Clin Pract 2001;52:119–23. Nwomeh BC, Yager DR, Cohen IK. Physiology of the chronic wound. Clin Plast Surg 1998;25: 341–56. Naguib G, Al Mashat H, Desta T, Graves DT. Diabetes prolongs the inflammatory response to a bacterial stimulus through cytokine dysregulation. J Invest Dermatol 2004;123:87–92. Salomon GD, Kasid A, Cromack DT, Director E, Talbot TL, Sank A, Norton JA. The local effects of cachectin/tumor necrosis factor on wound healing. Ann Surg 1991;214:175–80. Rapala K, Laato M, Niinikoski J, Kujari H, Soder O, Mauviel A, Pujol JP. Tumor necrosis factor alpha inhibits wound healing in the rat. Eur Surg Res 1991;23:261–68. Liu R, Bal HS, Desta T, Behl Y, Graves DT. Tumor necrosis factor-alpha mediates diabetesenhanced apoptosis of matrix-producing cells and impairs diabetic healing. Am J Pathol 2006; 168:757–64. Goren I, Muller E, Schiefelbein D, Christen U, Pfeilschifter J, Mühl H, Frank S. Systemic antiTNF-alpha treatment restores diabetes-impaired skin repair in ob/ob mice by inactivation of macrophages. J Invest Dermatol 2007;127: 2259–267. Rai NK, Suryabhan, Ansari M, Kumar M, Shukla VK, Tripathi K. Effect of glycaemic control on apoptosis in diabetic wounds. J Wound Care 2005; 14:277–81. Mantzoros CS, Moschos S, Avramopoulos I, Kaklamani V, Liolios A, Doulgerakis DE, et al. Leptin concentrations in relation to body mass index and the tumor necrosis factor-alpha system in humans. J Clin Endocrinol Metab 1997;82:3408–413. Mi Q, Riviere B, Clermont G, Steed DL, Vodovotz Y. Agent-based model of inflammation and wound healing: insights into diabetic foot ulcer pathology and the role of transforming growth factor-beta1. Wound Repair Regen 2007;15:671–82. ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc Recalcitrant diabetic foot wounds 31 Joussen AM, Poulaki V, Mitsiades N, Cai WY, Suzuma I, Pak J, Ju ST, Rook SL, Esser P, Mitsiades CS, Kirchhof B, Adamis AP, Aiello LP. Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes. FASEB J 2003;17:76–78. 32 Piaggesi A, Viacava P, Rizzo L, Naccarato G, Baccetti F, Romanelli M, Zampa V, Del Prato S. Semiquantitative analysis of the histopathological features of the neuropathic foot ulcer: effects of pressure relief. Diabetes Care 2003;26:3123–128. 33 Mast BA, Schultz GS. Interactions of cytokines, growth factors, and proteases in acute and chronic wounds. Wound Repair Regen 1996;4:411–20. 34 Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:813–20. 35 Schmidt AM, Hofmann M, Taguchi A, Yan SD, Stern DM. RAGE: a multiligand receptor contributing to the cellular response in diabetic vasculopathy and inflammation. Semin Thromb Hemost 2000;26: 485–93. 36 Berlanga J, Cibrian D, Guillen I, Freyre F, Alba JS, Lopez-Saura P, Merino N, Aldama A, Quintela AM, Triana ME, Montequin JF, Ajamieh H, Urquiza D, Ahmed N, Thornalley PJ. Methylglyoxal administration induces diabetes-like microvascular changes and perturbs the healing process of cutaneous wounds. Clin Sci (Lond) 2005;109:83–95. 37 Cai W, He JC, Zhu L, Chen X, Wallenstein S, Striker GE, Vlassara H. Reduced oxidant stress and extended lifespan in mice exposed to a low glycotoxin diet: association with increased AGER1 expression. Am J Pathol 2007;170:1893–902. 38 Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, Avila C, Kambham N, Bierhaus A, Nawroth P, Neurath MF, Slattery T, Beach D, McClary J, Nagashima M, Morser J, Stern D, Schmidt AM. RAGE mediates a novel proinflammatory axis: 39 40 41 42 43 44 45 a central cell surface receptor for S100/calgranulin polypeptides. Cell 1999;97:889–901. Ding Y, Kantarci A, Hasturk H, Trackman PC, Malabanan A, Van Dyke TE. Activation of RAGE induces elevated O2- generation by mononuclear phagocytes in diabetes. J Leukoc Biol 2007;81: 520–27. Wautier JL, Zoukourian C, Chappey O, Wautier MP, Guillausseau PJ, Cao R, Hori O, Stern D, Schmidt AM. Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats. J Clin Invest 1996;97: 238–43. Yamamoto Y, Kato I, Doi T, Yonekura H, Ohashi S, Takeuchi M, Watanabe T, Yamagishi S, Sakurai S, Takasawa S, Okamoto H, Yamamoto H. Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice. J Clin Invest 2001;108:261–68. Nawroth P, Bierhaus A, Marrero M, Yamamoto H, Stern DM. Atherosclerosis and restenosis: is there a role for RAGE? Curr Diab Rep 2005;5:11–16. Shoji T, Koyama H, Morioka T, Tanaka S, Kizu A, Motoyama K, Mori K, Fukumoto S, Shioi A, Shimogaito N, Takeuchi M, Yamamoto Y, Yonekura H, Yamamoto H, Nishizawa Y. Receptor for advanced glycation end products is involved in impaired angiogenic response in diabetes. Diabetes 2006;55:2245–255. Alikhani M, MacLellan CM, Raptis M, Vora S, Trackman PC, Graves DT. Advanced glycation end products induce apoptosis in fibroblasts through activation of ROS, MAP kinases, and the FOXO1 transcription factor. Am J Physiol Cell Physiol 2007;292:C850–56. Pierce GF. Inflammation in nonhealing diabetic wounds: the space-time continuum does matter. Am J Pathol 2001;159:399–403. ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd and Medicalhelplines.com Inc 539