BioMed Research International

BioMed Research International / 2013 / Article
Special Issue

Chronic Wounds with Emphasis in Diabetic Foot Ulcers

View this Special Issue

Review Article | Open Access

Volume 2013 |Article ID 256043 |

Jorge Berlanga-Acosta, Gregory S. Schultz, Ernesto López-Mola, Gerardo Guillen-Nieto, Marianela García-Siverio, Luis Herrera-Martínez, "Glucose Toxic Effects on Granulation Tissue Productive Cells: The Diabetics’ Impaired Healing", BioMed Research International, vol. 2013, Article ID 256043, 15 pages, 2013.

Glucose Toxic Effects on Granulation Tissue Productive Cells: The Diabetics’ Impaired Healing

Academic Editor: David G. Armstrong
Received18 Aug 2012
Accepted24 Nov 2012
Published26 Dec 2012


Type 2 diabetes mellitus is a metabolic noncommunicable disease with an expanding pandemic magnitude. Diabetes predisposes to lower extremities ulceration and impairs the healing process leading to wound chronification. Diabetes also dismantles innate immunity favoring wound infection. Amputation is therefore acknowledged as one of the disease’s complications. Hyperglycemia is the proximal detonator of systemic and local toxic effectors including proinflammation, acute-phase proteins elevation, and spillover of reactive oxygen and nitrogen species. Insulin axis deficiency weakens wounds’ anabolism and predisposes to inflammation. The systemic accumulation of advanced glycation end-products irreversibly impairs the entire physiology from cells-to-organs. These factors in concert hamper fibroblasts and endothelial cells proliferation, migration, homing, secretion, and organization of a productive granulation tissue. Diabetic wound bed may turn chronically inflammed, procatabolic, and an additional source of circulating pro-inflammatory cytokines, establishing a self-perpetuating loop. Diabetic fibroblasts and endothelial cells may bear mitochondrial damages becoming prone to apoptosis, which impairs granulation tissue cellularity and perfusion. Endothelial progenitor cells recruitment and tubulogenesis are also impaired. Failure of wound reepithelialization remains a clinical challenge while it appears to be biologically multifactorial. Ulcer prevention by primary care surveillance, education, and attention programs is of outmost importance to reduce worldwide amputation figures.

1. Introduction

What represents today a worldwide pandemic of a noncommunicable disease, diabetes mellitus, has two principal clinical forms identified as types 1 and 2. The former is a condition in which by autoimmune mechanisms pancreatic -cells are eventually destroyed with an absolute insulin deficiency [1]. Type 2 diabetes mellitus (T2DM) is the most prevalent form of the disease and recently acknowledged not as a single clinical condition, but importantly, as a group of metabolic disorders. Diabetes, in general, causes chronic hyperglycemia and a wide range of downstream metabolic disturbances and multiorgan complications [2]. It is notorious, however, that although insulin secretion collapse, peripheral insulin resistance, and/or receptors’ activity failure play a definitive role for the onset of sustained hyperglycemia in T2DM, a large portion of body glucose is cleared by insulin-independent mechanisms, derived from the ability of plasma glucose to influence its own clearance by a mass action effect [3]. T2DM usually most common in adult subjects exhibits a slow, silent, and insidious evolution. Hyperglycemia and its adjoined biochemical consequences undermine the whole tissues being sufficient to orchestrate irreversible systemic complications, from which the cells comprised in soft peripheral tissues and vascular structures do not escape. Lower extremities ulcerations and the potential for amputation are currently acknowledged as members of the list of diabetes complications [4].

Surgeon Davies Pryce put forward as early as 1887 the link between diabetes and foot ulceration by writing in The Lancet that “diabetes itself may play an active part in the causation of perforating ulcers” [5]. However, despite the years of efforts and research, the pathogenesis of impaired wound healing in diabetes remains incompletely elucidated [6]. This poor-healing condition appears to be a multifactorial process, which includes the amalgamation of systemic and local factors that ensure a perpetual forward loop up to chronification. Along this path, the cells seem to progressively wipe out their ability to trigger evolutionarily imprinted mechanisms as migration, proliferation, and transdifferentiation becoming increasingly statics. Thus, diabetic wounds do not only become chronic by a concept of aberrant healing trajectory within a physiological time frame, but also by the asynchrony on the sequence of overlapping events that make up the tissue repair megaprocess. Broadly speaking, diabetes impairs most if not all these events. Thus, the challenge that represents the diabetic wound healing failure is the clinical gross expression of an outstanding array of biochemical and cellular disorders [7]. These ideas are supported in the clinical arena by the alarming statistics of amputations around the world every year [8].

The healing process in diabetes is also jeopardized by the patient’s susceptibility to infection due to deficiencies on the innate immunity. Although the diabetic wound bed may be adversely overwhelmed by inflammatory cells, it does not represent an overt antibacterial protection. By the contrary, the diversion of glucose to the polyol pathway affects bacterial killing by reducing neutrophil opsonophagocytosis. Furthermore, hyperglycemia-induced reactive oxygen species (ROS) deregulated the innate immunity via an overactivation of NF-kappaB (NF- B), thus amplifying the absurd inflammation and intoxicating the wound milieu [9, 10]. Peripheral arterial disease, leading to ischemia or lower limb hypoperfusion is associated with the most severe outcomes, including lower probability of healing, longer healing times, higher probability of ulcer recurrence, greater risk of amputations, and potentially higher mortality [4]. Cells harvested and cultured from hypoperfused granulation tissues orchestrate a molecular program of arrest and senescence (Jorge Berlanga-Acosta. Manuscript accepted. Int Wound Journal). The outcome of the combination “healing failure” and “infection susceptibility” untowardly contributes to amputation. Here we review the current lines of evidences on the toxic resonance of acute and long-term exposure to high glucose on the two main cells for the granulation tissue organization: fibroblasts and endothelial cells. We have included a characterization of the diabetic granulation tissue organizational disorders and the challenge that represents its ultimate process, wound reepithelialization. The literature search was based on English language articles downloaded from Pubmed and Bioline International ( data sources.

2. Consequences of Glucose Overload Toxicity on Fibroblasts and Endothelial Cells

2.1. Fibroblasts

The fibroblast is central to the wound healing process by secreting, contracting, and remodeling the extracellular matrix (ECM). They also secrete growth factors as important messengers for mesenchymal-to-mesenchymal and epithelial-mesenchymal communication, especially for establishing the emerging basement membrane and subsequent reepithelialization. Therefore, any impediment to fibroblast function is deterrent for normal wound healing and may result in chronic, nonhealing wounds. The fibroblast, when engaged in fibrogenesis, displays the highly activated phenotype characteristic of myofibroblasts. Although their origin has not yet been definitely elucidated; proliferation of preexisting adjacent dermal fibroblasts and, probably, recruited from the bone marrow has been documented [11]. Under the high glucose burden imposed by diabetes, cutaneous and extra cutaneous fibroblasts appear perturbed; and for many years, in vitro models recreating “clinical hyperglycemia” have proved to disrupt normal fibroblasts physiology and derange the secretion of extracellular matrix ingredients. These experiments have suggested that high glucose concentration is the proximal detonator of a downstream cascade of molecular disturbances for the skin fibroblast [12].

Rowe and coworkers pioneered the in vitro models that demonstrated that in diabetics’ cutaneous fibroblasts; synthetic, proliferative, and secreting capabilities are reduced [13]. Other parallel studies, in which high glucose concentrations were introduced, proved to inhibit fibroblast proliferation, while the cells turned resistant to proliferate to growth factors such as insulin-like growth factor type-I (IGF-I) and epidermal growth factor (EGF) [14]. Following this attractive targets, Goldstein’s findings allowed for establishing the hypothesis that diabetics fibroblasts replicative life span did proportionally decline with diabetics’ predisposition under normal glucose concentrations, concluding that a persistent, heritable abnormality is present in mesenchymal tissues of overt diabetics and genetically predisposed subjects [15]. Years later, Goldstein also announced that cells obtained from insulin-dependent or insulin-independent diabetics not only exhibit abnormal replicative capacity in vitro, but that the aging process appeared more precociously than in nondiabetic counterparts [16]. Other studies showed that the addition of conditioned media from non-insulin-dependent diabetes mellitus wound fibroblasts induced a dose-dependent inhibition in normal fibroblast proliferation which appeared related to elevated L-lactate levels [17]. This replicative refractoriness of diabetic fibroblasts has been reproduced by different groups in subsequent years [18], thus confirming the need for additional external supplements to ensure cell cycle progression [19]. Accordingly, Loots and coworkers demonstrated the need of the simultaneous rather than the sequential addition of different growth factors combinations for diabetic ulcer fibroblasts in order to induce a proliferative response [20]. In addition to the onset of a quiescent and senescent phenotype of diabetic wound fibroblasts, their ability for horizontal and vertical migration is also dramatically impaired when compared to normal donor cells in different migration assay as in the modified Boyden chamber haptotaxis assay [21]. Most of these attributes are reproduced under acute exposures to high glucose concentrations so that migration speed is reduced by ~40% associated to a decrease in cell directionality and to nonproductive protrusive events, as loss of cell polarization, consistent with the increased activity of Rac1 and the projection of multiple lamellipodia. This experiment concluded that the generation of reactive oxygen species (ROS) may lie behind these abnormalities as they were partially or completely rescued by treatment with N-Acetyl-Cysteine (NAC) [2]. In contrast to the cellular reactions when exposed to high glucose in vitro, full-thickness wounds induced in nondiabetic pigs exposed to a local hyperglycemic environment exhibited no difference in wound closure when compared with normoglycemic controls, suggesting that delayed wound healing by diabetes is a far more complex phenomenon than circumscribed high-glucose concentration itself [22]. As a consequence of the cutaneous accumulation of advanced glycation-end products (AGEs), the skin increases its chronological age. One of the AGEs precursors is 3-deoxyglucosone (3DG). Fibroblasts cultured on 3DG-treated collagen reduce the ability to migrate efficiently since 3DG increases its adherence to the matrix. Additionally, the authors describe a higher level of misfolded proteins [23]. Using the same experimental system, this group demonstrated two years later that the inhibition in fibroblast migration, proliferation, and collagen expression by exposure to 3DG-collagen was mediated via extracellular regulated kinase 1/2 (ERK1/2) and Akt downregulation through activation of p38 MAPK (Mitogen-Activated Protein Kinase), indicating that p38 is a key signaling molecule that plays an opposite role during times of cellular growth and cellular stress [24]. Enriching the above findings, this group also demonstrated that 3DG-modified collagen induces oxidative stress, endoplasmic reticulum stress, and apoptosis via caspase-3 activation.

Oxidative stress appeared dependent on the upregulation of NAD(P)H oxidase 4 (Nox4), a reactive oxygen species Nox homologue, which appeared activated by p38 MAPK. Proximal to this cascade is the effect caused by the interaction of the modified collagen with 3DG, which signals to the fibroblast by interacting with integrins alpha-1/beta-1 (α1β1) and not through the canonical receptor for advanced glycation end-products (RAGE) [25]. Other groups have also demonstrated the induction of cutaneous fibroblasts apoptosis through cytoplasmic and mitochondrial pathways by plating the cells in an AGE-enriched environment made up by Ne-(carboxymethyl)lysine (CML)-collagen, which primarily activated the classic AGE receptor (RAGE) [26]. A subsequent study elegantly demonstrated that after AGEs-RAGE interaction, oxygen species generation is increased, activating both NOS and ceramides, which in turn activates p38 and c-Jun N-terminal protein kinase (JNK). Activated p38 and JNK triggers a cascade that leads to amplify caspase-3 activity, whereas activation of Forkhead box O class 1 (FOXO1) increases the likelihood of apoptosis through enhanced expression of proapoptotic genes [27]. Under a number of circumstances, FOXO transcription factors induce BIM and other proapoptotic genes expression.

In addition to the deleterious effects of glucose and its derivatives, diabetic fibroblasts exhibit particular features. Literature documents that diabetic mice fibroblasts show a severe impairment in VEGF production under normoxic and hypoxic conditions in addition to an increased pro-degradative activity due to the high expression of matrix metalloprotease type 9 (MMP-9) [28]. Similarly, diabetic pigs exhibit an impaired healing that is accompanied by a reduction of IGF-1 in the wound milieu [22]. Studies with human fibroblasts have confirmed the prodegradative phenotype by the increased MMP-2 and MMP-3 production and reduced collagens gene expression [29]. Human diabetic fibroblasts also exhibit a failure in nitric oxide (NO) production, which is concomitant to elevations in MMP-8 and -9 [30]. The fact that these fibroblasts fail in secreting NO is particularly negative given its role for wound healing. Conversely, NO donors’ administration has shown to stimulate cell proliferation and restore the balance of MMPs [31].

It seems that amplification of oxidative stress acts as a primary culprit in harming fibroblasts biology in diabetes, involving electron transport in mitochondria. High intracellular glucose levels increase the electron transport chain in mitochondria during oxidative respiration, leading to formation of O2- and the generation of various reactive oxygen species derivatives in the mitochondria. Other sources of oxidative stress in diabetes include glucose autooxidation, the polyol pathway with ensued depletion of antioxidant reserves and the formation of AGEs [32]. Chronic hyperglycemia-induced mitochondrial ROS stimulate various signaling pathways that amplify inflammation and cell death. They include protein kinase C (PKC), JNK, and p38/MAPK [31]. According to an excellent review by Ponugoti et al. [33]; ROS leads to the activation of members of the FOXO family. This is a family of transcription factors with apparently opposing roles that may defend cells against oxidative stress but also promote cell-cycle arrest in G1 by inducing p27kip1 [34]. FOXO1 activation appears elevated in diabetic connective tissue cells and mediates AGEs and tumor necrosis factor-alpha- (TNF-α-) induced apoptosis both of which are abundant in diabetic connective tissue [35]. FOXO1 limits wound healing by inhibiting fibroblasts proliferation and enhancing their apoptosis [35, 36]. Interestingly, insulin inactivates FOXO1 via Akt leading to its nuclear export and degradation. Defective insulin action in the skin has been proposed as an important mechanism contributing to wound healing defects in diabetes. Perhaps the assorted constellation of the hormone’s pharmacological bounties (increased expression of endothelial nitric oxide synthase, vascular endothelial growth factor, and stromal-derived factor-1α/SDF-1α) observed in experimental and clinical wounds when insulin is topically administered may be attributable to FOXO1 neutralization. Curiously, the acceleration of wound healing occurs in parallel to a local recovery in the expression of proteins involved in insulin signaling pathways [37]. Aside from the above arguments, these preclinical and clinical findings are not surprising in light of the potent anti-inflammatory, proanabolic, and cytoprotective actions of insulin [38], which extend beyond the exclusive regulation of glucose homeostasis [3]. Figure 1 depicts the main pathogenic players and their interconnection as an attempt to summarize the high glucose-triggered fibroblasts damage process.

Despite the prolific investigation conducted during all these years, still questions remain to be answered in relation to ex vivo diabetics’ fibroblasts behavior.(1)Why diabetics’ fibroblasts evoke behavioral traits in culture mirroring the donor’s tissue, even when grown under optimized oxygenation, nutrient, growth factors, and glucose supply?(2)Is there any sort of “behavioral imprinting” so that they are reminiscent from a diabetic donor?(3)Why cultured fibroblasts from both ischemic and neuropathic ulcers exhibit different ultrastructural morphology and organize the monolayer in a privative manner? (4)Is there any epiphenomenon beyond the irreversible glycation sustaining the “impersonation” of the in vivo traits?

2.2. Endothelial Cells

Angiogenesis is a comprehensive term that indicates the physiological process involving the growth of new blood vessels or neovascularization. This is a vital process for embryological growth, tissue development, and wound healing. Different growth factors families as vascular endothelial growth factors (VEGF), fibroblast growth factor (FGF), angiopoietins, platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), in collaboration with other proteins as integrins, cadherins, and ephrins, regulate angiogenesis by promoting endothelial cells recruitment, proliferation, migration, coopting, and collar stabilization. There is an enormous and ever-growing body of evidence indicating the close correlation between hyperglycemia and the abnormalities in endothelial function and morphology [39]. The UK Prospective Diabetes Study (UKPDS) and Diabetes Control of Complications Trial (DCCT) found microvascular disease and hyperglycaemia to be intrinsically related. Thus, anomalous angiogenesis is a hallmark of both type forms of diabetes, which is clearly and early observable during the process of granulation tissue growth, a condition that has been successfully reproduced in animal models [40]. For subjects with macrovascular disease, the defective angiogenesis prolongs and disturbs the healing process. The concept of abnormal angiogenesis extends beyond the wound, given the inability of these patients to create collateral circuits due to VEGF-dependent monocytes dysfunction [41]. Furthermore, insulin has a dramatic impact on the endothelial homeostasis by its ability to stimulate NO release via a cascade that involves activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling and endothelial nitric oxide synthase (e-NOS) phosphorylation. The later being of paramount importance in angiogenesis and wound healing as described below [42].

As depicted for fibroblasts, high glucose and the glycated by-products exert a toxic effect on endothelial cells and the vascular wall in general. In parallel, the endothelial cells per se seem to be a very sensitive target to high glucose. Endothelial dysfunction is intricately related to insulin resistance through the stimulatory effects of insulin on glucose disposal and NO production in the endothelium. Today, vascular dysfunction remains as a major cause of morbidity, amputation/disability, and mortality in diabetic patients. Even after achieving the successful reperfusion of an ulcerated lower extremity, the healing process is slow and torpid. Therapeutic angiogenesis has been pursued for years, but the clinical results have shown relatively limited outcomes [4345]. High glucose concentrations have been associated with endothelial metabolic dysfunction in vitro and in vivo and as for multiple physiological processes; insulin and its downstream signaling regulate most of the endothelial cell functions [46]. High glucose ambient has been shown to disturb endothelial cells cycle, increase DNA damage, and delay endothelial cells replication, as inducing excessive cell death [47]. In addition, high glucose also prevents NO-induced inhibition of vascular smooth muscle cells (VSMC) migration [27] thus contributing to Monckeberg’s media thickening. In vitro models simulating “normoglycemia” and “hyperglycemia” have demonstrated that under high glucose ambient, proliferation and tube formation of dermal microvascular endothelial cells appear impaired [48]. Furthermore, high glucose levels selectively trigger apoptosis in cultured endothelial cells as has been demonstrated by different laboratories [49]. High glucose induces the upregulation of TNF-α level concomitant to the death receptors TNF-R1 and Fas in a variety of cultured endothelial cells [50]. Under this ambient, Bax protein expression increases, cytochrome c is released, subsequently conjugating to Apaf-1 and triggering a caspase cascade-induced death [51].

Hyperglycemia-induced oxidative stress promotes inflammation through increased endothelial cells damage, microvascular permeability, and uncontrolled release of proinflammatory cytokines, including TNF-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6), ultimately leading to decreased insulin sensitivity and diabetic vascular complications. Moreover, hyperglycemia-induced FOXO also plays an important role in the induction and amplification of proinflammatory cytokines production. FOXO1 directly binds to IL-1β promoter and increases its expression in macrophages [52].

Hyperglycemia and the accumulation of AGEs disturb the role of angiogenic growth factors as VEGF, and its receptor, its signaling pathway, thus disrupting endothelial proliferation, migration, and endothelial progenitor cells (EPCs) recruitment and release from bone marrow [53]. Insulin resistance disrupts the NO-mediated angiogenic positive regulation over angiogenic growth factors such as VEGF, FGF, and TGF-β [53]. Studies using streptozotocin-induced diabetic mice with simultaneous hind-limb ischemia have suggested that the angiogenic responses remain preserved even under the diabetic state, and that 40 to 50% reduction of platelet-derived growth factor-BB (PDGF-BB) expression is responsible for the induction of functional and morphological vascular abnormalities and pericytes apoptosis. Conversely, PDGF-BB external supplementation was sufficient to prevent limb autoamputation, an event also reproduced with a PKC inhibitor that restored the expression of endogenous PDGF-BB [54].

The glycation of collagen and other proteins within the wound extracellular matrix and AGEs accumulation bring catastrophic consequences for the angiogenic reaction with inhibition of angiogenesis in vivo. The fact that angiogenesis is restored by aminoguanidine treatment reinforces the antiangiogenic role of AGEs [55]. Angiogenesis is a multifaceted process demanding an appropriate, nonglycated extracellular substrate. This is clearly illustrated by the fact that PDGF-BB anchors to different components of the ECM under physiological conditions acting as a natural depot and slow release system for the growth factor. Local PDGF unavailability has proved to impair the coverage of newly formed vessels with mural cells and local pericytes [56]. This evidence reinforces the pathophysiological impact of high glucose toxicity, the release of proinflammatory cytokines, and the activation of the intrinsic mitochondrial-mediated apoptotic signaling pathway on endothelial cells. In summary, endothelial cells exposed to excess glucose trigger the onset of a pro-inflammatory profile turning these cells into a cytokines and ROS manufacturing plant. The agonistic stimulation of the AGEs receptor is able to mount the same response leading to apoptosis and vascular ruin. The pathogenic effects of hyperglucose associated to insulin axis failure on endothelial cells are summarized in Figure 2.

Compelling evidence indicates that at least a portion of the hyperglycemia-mediated endothelial damages and dysfunctions are associated with an impaired mitochondrial activity resulting in mutations of mitochondrial DNA, due to a disproportionate reactive oxygen radicals production, leading to an inflammatory reaction and apoptosis [57]. As a matter of fact mitochondrial DNA has a much higher mutation rate than nuclear DNA because it lacks histones and is exposed to the direct action of oxygen radicals while its repair system is limited. Therefore, ROS appear to play a pivotal role in systemic endothelial deterioration and biological aging [58]. As described, ROS generation enhances FOXO1 activation and induction of several classes of genes that regulate endothelial cell behavior, including pro-inflammatory factors and eventually the execution of apoptosis of endothelial and adjacent cells [33]. ROS-mediated lipid peroxidation appears to impair most healing events, contributing to growth factors reduction, keratinocytes migration failure, slow or torpid fibroplasia, delayed contraction, and matrix remodeling, not to mention abnormal angiogenesis [59]. Under experimental conditions, the pharmacological intervention with a chemical inhibitor of lipid peroxidation proved to reduce the local edema and to stimulate reepithelialization, neovascularization, proliferation of fibroblasts, and synthesis and maturation of the extracellular matrix. A parallel finding was the normalization of VEGF mRNA expression and secretion in those diabetic mice. This further supports the view that lipid peroxidation perturbs VEGF production [60]. An extraordinary background has accumulated about the role of NO in vascular biology in diverse horizons as ischemia, inflammation, and neovascularization. Impaired endothelium-dependent NO-mediated relaxation occurs in both cellular and in vivo models [61]. Many of the metabolic conditions associated with diabetes are conditioned by failure in NO synthesis or its degradation. In this respect, the integrity of the Akt/e-NOS coupling pathway for a normal endothelial function appears compulsory [62]. Hyperglycemia is also associated to a deficit in tetrahydrobiopterin (BH4) and to an increase in arginase expression, which attempt against NO synthesis and normal endothelial functions such as vascular remodeling responses [63]. The increased generation of peroxynitrite levels under high glucose conditions contributed to deplete cellular antioxidant reserves as to activate NF- B and consequently the expression of the inducible form of nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), and other inflammatory mediators [64].

Endothelial Progenitor Cells (EPCs) are active players for the maintenance and repair of endothelial cells. They participate in angiogenesis as they proliferate, migrate, and differentiate and are a source for proangiogenic factors and cytokines [65]. Multiple lines of evidence indicate that the number of circulating EPCs is decreased under both clinical forms of diabetes, which is likely to be involved in the pathogenesis of vascular complications [66]. Under experimental diabetic conditions, the EPCs number appears significantly, decreased in the bone marrow as in the peripheral blood, which was reverted by treating the mice with insulin [67]. In general, the bone-marrow-derived EPCs in the diabetic patients are considered as dysfunctional, producing fewer endothelial cells with reduced replicative and migratory potential [68]. Tamarat and coworkers have described a limited capacity of diabetic animals-derived bone marrow mononuclear cell to differentiate into endothelial progenitor cells in vitro as to organize tubulogenic structures when subcutaneously implanted in a matrigel plug, thus hindering the revascularization of damaged areas [66]. Over again, the activation of p38 MAPK mediated by an excessive ROS generation has been aimed as responsible for the EPCs impaired proangiogenic potential in vivo by limiting cells proliferation and differentiation [69]. As to fully divert the physiological role of EPCs in tissue repair and angiogenesis, the duet hyperglycemia-ROS stimulate the EPCs to produce pro-inflammatory cytokines and to shift NO production by elevating i-NOS and decreasing e-NOS [70]. As described for other cells, AGE treatment disrupts EPCs physiology thus leading to a downregulation of e-NOS and Bcl-2 expression, as well as an elevation in cyclooxygenase-2, Bax, NF- B, and caspase-3 in a MAPK- (ERK/P38/JNK-) dependent manner [69].

The diabetes-mediated vascular damage is perhaps the most outspoken and ancestrally identified emblem of diabetes. It is varied and broad as it is the concept of systemic endothelial dysfunction. Diabetes distorts the angiogenic program to ironically culminate with a maldistribution of soluble angiogenic factors: shortage where and when required (lower extremities skin) but overproduced where and when not needed (retina). It is also challenging, to understand how and why microvascular morphological changes that recreate chronic, life-time processes are readily identified in a 7-day-old granulation tissue fragment, even in compensated patients. This incites to investigate which are the diabetes’ operational local and/or systemic forces that can disrupt vascular morphogenesis.

3. Failure of Granulation Tissue Onset and Progression

Once we have described the main consequences of high glucose/hyperglycemia on the two principal architects of the granulation tissue: fibroblasts and endothelial cells, we are intended to recapitulate the most distinguishing features on the onset of the granulation process in diabetic cutaneous wound healing.

Tissues’ regenerative capabilities have been neglected along the species evolution; thus, scarring process has emerged as an urgent alternative to favor the structural and functional restoration of a wounded zone. Within these events, the process of granulation tissue formation is pivotal as it constitutes a sort of living, temporary aggregate of cells and proteins, acting as a welding material until the tissue’s continuity is restored. However, the reluctance to trigger and sustain the out-growth of a productive granulation tissue with an appropriate extracellular matrix is typical in diabetic patients, and particularly if ischemia concurs. As mentioned, these wounds are characterized by a proliferative arrest, proinflammed, prooxidant, and prodegradative phenotype [71].

This stubbornness and slowness to heal in diabetes is conditioned by systemic and local factors that in complicity counteract intrinsic reparative mechanisms. In a broad systemic context, inflammation and the anabolic deficit can be conceptually mentioned. Diabetic patients with foot ulceration bear a specific and nonrandom alteration of the immune status with an active upregulation of circulating levels of acute-phase proteins, cytokines, and chemokines that impose a chronic systemic inflammatory profile and amplify local wound inflammatory networks [72]. The systemically elevated levels of pro-inflammatory response markers and the wound’s expression of cytokines and chemokines are among the culprits of the abnormal repair mechanism [73]. Another factor to be considered is that diabetes per se is a metabolic disease in which fuels metabolism is perturbed given the rupture of one of the most important anabolic axis of the organism: insulin/insulin-like growth factor type-I. The role of insulin in wound healing is well known by its anabolic effect on wound protein balance favoring synthesis and preventing degradation [74, 75]. IGF-1 has a similar effect on stimulating wound tissue anabolism. Both insulin and IGF-1 appear to act in part by the induction of ATF4 (CREB2), essential for the activation of mammalian target of rapamycin complex 1 (m-TORC1), which in turn is required for protein synthesis via FOXO-dependent genes repression [76]. We do not rule out that the diabetes-concomitant deficit of incretins could participate in the negative anabolic balance observed in such wounds. Glucagon-like peptide-1 (GLP-1) in addition to its antihyperglycemic actions is endowed with a vast number of multi-organ cytoprotective, trophic and antiinflammatory effects [77]. In support to the GLP-1 action is the study by Ta and coworkers with alogliptin, a specific inhibitor of dipeptidyl peptidase-4 (DPP-4) which showed to inhibit macrophage-mediated inflammation response and to speculatively promote tissue remodeling by inhibiting the expression of different matrix metalloproteases [78].

Rapid formation and deposition of an appropriate extracellular matrix, in particular by fibroblasts, is required for an efficient cellular anchoring and homing at the wound bed. As mentioned above, the cutaneous fibroblast is a sensitive cell to high glucose, AGE-precursors, AGEs, ROS, and TNF-α, rapidly undergoing premature senesce, arrest or apoptosis. Fibroblasts are the main source of collagen, and the number of fibroblasts can be taken for a measure of repair by their collagen synthesis ability. It is very likely that the deficit of growth factors such as TGF-β1, IGF-I and PDGF could translate into a DFU with scarce extracellular matrix acumulation and impoverished cellularity. Numerous growth factors (TGF-β1, IGF-I, PDGF) are able to regulate the balanced expression of matrix metalloproteases and tissue inhibitors of metalloproteases (MMPs/TIMPs), while most of them exhibit an altered expression in DFU [79]. Moreover, the imbalance in the DFU milieu between TGF-β1 and TGF-β3, in which the former appears downregulated, may explain fibroblasts quiescence in terms of proliferation and secretion [80]. This phenomenon represents the deficit of one of the most potent profibrogenic and fibroblasts-mitogenic growth factors, which at the same time is able to downregulate macrophage activation [81]. The extracellular matrix represents the granulation tissue dynamic stroma that provides support for inflammatory cells, fibroblasts, and endothelial cells and allows for the chemotaxis of epithelial cells, thus hosting the reepithelialization process [82]. One of the main challenges for the diabetic wound healing is the structuring of a normal matrix in quantity and quality. In general, a poor extracellular matrix formation distinguishes DFUs, which can result from (a) diminished synthesis, (b) increased rate of degradation by proteolytic enzymes, (c) toxicity due to glycated by-products accumulation, and (d) toxicity by biofilm bacterial contaminants diffusion [83]. We deem that an important cause of the clinical dilemma of the high rate of reulcerations and ipsilateral amputations in DFU patients’ shortly after reepithelialization [84] may be inherent to the qualitative composition of the scar matrix to tolerate tensile forces and mechanical stress.

The diabetic granulation process does not generally exhibit the orderly cascade of events that characterize normal wound healing. This has been confirmed through the histopathological analysis of granulation tissue biopsies by Loots and coworkers who described the lesions as “frozen” in a chronic low-grade inflammatory state associated to a scarce provisional extracellular matrix [85]. Our group’s serial biopsies from both neuropathic and ischemic ulcers-derived granulation tissue have identified histological differences for both types of wounds in the absence of clinical infection. Polymorphonuclear cells (PMN) infiltration is intense and prolonged particularly in neuropathic wounds, co-existing with a scarce extracellular matrix accumulation in which collagen deposit is impoverished (Figure 3).

Under more mature stages, the neuropathics may also show an abnormal sprout of new small vessels and capillaries that may derive not from a normal angiogenic response but due to arteriovenous shunts. Our observations remind us with those of Black and coworkers who demonstrated that in neuropathic patients there exists a decrease in fibroblast proliferation and a scarce amount of collagen accumulation within the wound bed [86]. On the contrary, a broadly spread infiltration of round cells predominate in those patients suffering from wound bed ischemia, associated to a fibrohyaline matrix of “hardened” aspect and abnormal angiogenesis in which vascular wall cellular mosaicism, precocious media thickening, endothelial nuclei hypertrophy and many other defects can be identified (Figure 4). It is likely that the combination of arterial hypoperfusion and glucose toxic derivatives imprints a particular pattern of damage to the morphogenesis of vessels in the wound [87]. These observations incite to speculate that the biochemical microenvironment in ischemic and neuropathic diabetic wounds is different and that the inflammatory “badge” is in correspondence with the wound’s most prevalent pathogenic component [88]. In contrast to acute wounds in nondiabetic subjects, the inflammatory reaction in diabetics appears prolonged [89] which sharply delays granulation tissue formation and maturation [90]. Data derived from murine diabetic models indicate that the exaggerated inflammatory reaction is related to the prolonged expression of macrophage inflammatory protein-2 (MIP-2) and macrophage chemoattractant protein-1 (MCP-1) [91]. Furthermore, the downregulation of the anti-inflammatory cytokine IL-10 in DFUs environment represents the collapse of an important inflammatory restrainer [73]. Another evidence indicates that PMNs are critical cells toward the acquisition and perpetuation of inflammation and a degradative phenotype. The granulocytes secrete TNF-α and IL-1β, which act as a triggering signal for MMPs expression via the common NF- B signaling pathway. Within the wound context, TNF-α stimulates its own secretion and that of IL-1β, which contributes to a persistent inflammatory status [92]. TNF-α has proved to negatively impact the repair process as it is early secreted since the inflammatory phase. Its deregulation is not only associated with persistent inflammation but also to connective tissue degradation [93]. Concomitantly, TNF-α mediates its antagonistic effects on TGF-β1 through the JNK pathway via inhibition of Smad phosphorylation, consequently reducing the expression of TGF-β1, and that of several downstream matrix proteins [94]. In this highly proteolytic milieu, fibronectin, collagens, growth factors, and their receptors are degraded while the wound is way down to a catabolic state [95].

Importantly, the perpetuated homing of PMN within the wound bed is associated to high local levels of elastase secretion, ROS, and reactive nitrogen species [96]. High circulating and PMNs-associated elastase levels are attributable to a poor glycemia control and are currently considered as a risk marker for the development of diabetic angiopathy [97]. Fibronectin degradation, for instance, is referred as one amongst the several causes of diabetic reepithelialization failure. Epidermal keratinocytes require of the interaction between fibronectin and its surface receptor integrin α5β1 to effectively migrate [98]. Curiously, insulin-degrading activity has also been demonstrated in the fluid of diabetic experimental and human wounds, which have been shown to correlate with the glycated hemoglobin levels [99]. The connection between NO metabolism and foot ulcer proteases profile has been described. In contrast to elevated MMP-8 and 9 displayed by the nonhealing diabetic foot wound, the concentration of NO appears significantly reduced. Diabetic skin fibroblasts treated with NO donor compounds, selectively raised NO production, increased cell proliferation, and decreased the expression of MMP-8 and -9 in a dose-dependent manner. Thus, that NO resumes the cell proliferation program and promotes the reestablishment of an antiproteases effect has emerged as argument in favor of the NO salutary effect in wound healing [30].

The link between wound cells and apoptosis was described above; we just wish to comment that in sharp contrast to wound-infiltrated inflammatory cells, which become refractory to apoptosis, granulation tissue-producing cells are sensitive to commit suicide where TNF-α stands as a major driving force. The negative impact of TNF-α level on the sensitivity of tissues to insulin has been consistently documented. Skin cells are not excluded from this effect [100]. Conclusively, any therapeutic approach aimed to neutralize TNF-α or to increase the wound local availability of active TGF-β1 would be similarly effective for stimulating granulation tissue and wound closure [101].

Chronic wounds and especially diabetic foot ulcers exhibit a highly pro-oxidant microenvironment that amplifies the cytotoxic cascade. Endothelial cells and fibroblasts, in particular senescent fibroblasts, are a prominent source for oxygen radicals, but at the same time they turn into these radicals targets which by converging mechanisms arrest cell proliferation and induce apoptosis [102]. Thus, the disturbed oxidant/antioxidant balance as the AGEs accumulation within the chronic wound microenvironment is considered a major factor, which amplifies the unrestrained and persistent inflammatory, toxic, and catabolic state of nonhealing wounds [96].

The failure of wound contraction is a clinical hallmark of diabetic granulation tissue. Fibroblast-to-myofibroblast transdifferentiation represents a key event during wound healing and tissue repair. The contractile force, generated by myofibroblasts as a highly specialized cell, speeds the healing process of dermal wounds in healthy humans, accounting for an 80–90% of scar tissue reduction [103]. In addition, the contraction process reduces the area to be resurfaced by reepithelialization, which represents a sort of ergonomic response. In diabetic subjects, however, contraction is impaired and deep ulcers heal by the combination of granulation and reepithelialization. The classical view on dermal wound healing implies recruitment of local fibroblasts [104] followed by a subsequent process of transdifferentiation in which the fibroblasts gain a definitive phenotype of differentiated myofibroblasts by neo-expressing a-smooth muscle actin (α-SMA). Nevertheless, α-SMA expression is precisely controlled by the joint action of growth factors like TGF-β1 and extracellular matrix proteins like the fibronectin (FN) splice variant ED-A, as by the local mechanical microenvironment [104]. It should be noted, however, that indwelling fibroblasts in diabetic wounds are refractory to proliferate and adopt a senescent phenotype, and that TGF-β1, fibronectin, and other matrix proteins may appear in deficit. Hence, all these factors may contribute to the poor contractile activity. Furthermore, Goldberg and coworkers have shown that among the deleterious activities of TNF-α within the wound is to suppress α-SMA expression in human dermal fibroblasts [94]. Figure 5 integrates the cascade of deleterious factors that impact on diabetic granulation tissue onset.

If the animals-derived evidence that a high fraction of the wound myofibroblasts potentially derives from bone marrow fibrocytes is valid for humans [105]; we have already learned that diabetes negatively impacts on the general bone marrow physiology [106] and that beyond this, stromal-derived factor-1alpha (SDF-1α), which acts as a recruiting factor and its CXCR4 chemokine receptor are also impaired by diabetes [107]. Finally, it has been documented that the circulating acute inflammatory reactants involved in insulin resistance inhibit fibrocytes differentiation [108].

There are numerous cellular and molecular aspects unknown and that remain to be answered on the granulation tissue biology.(1)What are the molecular and cellular driving forces supporting the microscopic structural differences between neuropathic and ischemic ulcers beds?(2)What is the explanation for the “inheritance” of vascular changes as a dramatic Monckeberg media thickening in nascent arteries within an early hatching granulation tissue?(3)Why granulation tissue is histomorphologically abnormal even in metabolically compensated patients?Reepithelialization at the clinical level, it is not a lesser important problem as most of the diabetic wounds may granulate in time, while reepithelialization is even far slower, arrhythmic, and torpid. Reepithelialization is accomplished through the combined actions of keratinocytes’ dedifferentiation, proliferation, and migration requiring a complex basement membrane, emerged of the mutual interaction between mesenchymal and epithelial cells. Reepithelialization failure is therefore one of the landmarks of diabetic and other chronic wounds. The epidermal edge of a chronic wound is thick and hyperproliferative with mitotically active keratinocytes unable to migrate along the surface, and by the contrary, moving down deep into the neodermis. Therefore, it has been speculated that the nonhealing edge keratinocytes do not successfully complete either of two possible pathways: activation or differentiation. In consonance with this, one of the major issues in chronic wounds treatments is how to revert the chronic wound keratinocytes’ phenotype to a proper differentiating and migratory program [109].

Glucose has shown to exhibit a direct toxic effect on keratinocytes. As for other cells grown in the presence of high glucose concentrations, human epidermal keratinocytes significantly reduce their proliferation rate and replicative life span and were rendered more susceptible to commit apoptosis [110]. Other studies also confirmed that hyperglycemic conditions abort keratinocytes’ proliferative ability and their migratory response [111]. Aside from the glucose-mediated direct cytotoxic effect on the keratinocytes, AGE modification of type-I collagen and other ECM proteins impairs the integrin-mediated adhesion of keratinocytes to the basement matrix and could thus contribute to the pathogenesis of diabetic reepithelialization failure [112]. In this context, epithelial-mesenchymal interaction plays a role in establishing the profile and order of released factors regulating keratinocytes proliferation and differentiation [113].

The fact that insulin is biologically relevant for skin cells derives from the observation that insulin is an essential component for cultured human keratinocytes, demonstrating its involvement in the regulation of proliferation, survival, and metabolism [114]. Recent studies in this field document that among other roles, insulin contributes to VEGF release in skin wound cells through an Akt1-mediated posttranscriptional mechanism [115]. Glucose is known to affect insulin action by regulating the expression of several genes including insulin receptor at both the transcriptional and translational levels [116]. Lack of insulin receptor expression derives in reduced skin proliferation and abnormal differentiation in vivo [117]. Furthermore, TNF-α has also been implicated in epithelial cells arrest by deeply perturbing critical elements of keratinocytes’ physiology, including insulin sensitivity [118].

A notorious study has provided evidence aiming at the roles of c-myc and β-catenin in impairing epithelial edges migration. Nuclear beta-catenin stabilization inhibits keratinocytes migration by blocking epidermal growth factor response via c-myc induction, and repressing keratins 6 and 16 expression, depleting at the end the pool of epidermal stem cells at the nonhealing edge [119]. It is therefore evident that keratinocytes migration incapability plays an important role in reepithelialization failure since cytoskeletal keratins K2, K6, and K10 have been observed diminished in DFUs [120]. Moreover, the observation that EGF response appeared blocked may have further deleterious impact. Many peptide growth factors, including members of the EGF family, accelerate wound reepithelialization in vitro and in vivo [121]. Among them, the activation of the EGF family of ligands and the receptor is of physiological significance. Furthermore, EGF receptor (EGFR) expression is transiently increased at wound margins, suggesting an active role for this receptor in wound repair. EGF stimulates both cell proliferation and motility [122], with the later being dependent on EGFR autophosphorylation and the subsequent activation of phospholipase C -1 (PLC- 1). On the other hand, EGFR activation also leads to membrane ruffling and focal adhesions through activation of members of the Rho subfamily of GTP-binding proteins [123]. Recent experiments document the negative effect of the Slug null mutation as a downstream EGFR catalytic mediator for wound reepithelialization. Thus, any interference with the EGFR cascade will hamper epithelial resurfacing [124]. Classic experiments provide illustrative examples on the relevance of the epithelial-mesenchymal cross-talk and on the irreplaceable role of growth factor as a networking bridge [125] for reepithelialization. Skin-reconstitution studies have shown that bone marrow stromal cells (BMSCs), in addition to dermis-localized preadipocytes and fibroblasts distinctively promote epidermal regeneration [126]. As diabetes courses with a deficient secretion of growth factors and other chemotactic mediators in areas of tissue repair, recruitment of circulating stromal cells appears reduced; which may turn into an additional hit to that of high glucose-associated toxicity [127]. At the end, there are so many factors which may interact to obstruct chronic wounds reepithelialization that it may turn into a puzzle.

Above all, questions from the clinical practice remain and pose as a challenge for basic researchers: (1) why after wound contours surgical debridement keratinocytes migration resumes, for soon after become stunted and arrested again? (2) Why the biopsies invariably show a hypertrophic lip of epithelial cells in vertical, downward growth in spite of a horizontal polarization?

4. Concluding Remarks

Although diabetes per se is a complex disease, our contemporary understanding on the molecular mechanisms impairing wound healing in diabetes has indefectibly expanded over the last 20 years. The last few years have witnessed the birth of the notion that type 2 diabetes is not a single, unique process, whereas the concept of group of diseases has flourishing. Type 2 diabetes seems to be ethiopathogenically multifactorial and behaves as individual as the affected subject is. So is the pattern of the clinical complications, including the wound itself.

To date, all the evidence aims to high glucose burden as the proximal trigger to unleash acute and chronic self-perpetuating loops, which include but are not limited to ROS-lipid peroxidation, hyperinflammation/disimmunity, AGE-RAGE toxicity, mitochondrial dysfunction, and nytrosilation end-products accumulation. The concerted action of these factors enforces fibroblasts, pericytes, and endothelial cells to a precocious senescence, arrest, and apoptosis. Indeed, the failure of the agonistic stimulation of the insulin axis deeply impacts on the biology of diabetics’ cells. This is a seminal axis that connects the anabolic role of insulin via aminoacids transporters translocation and protein synthesis with cell survival and proliferation mechanisms, thus preventing apoptosis, autophagy (cells-self catabolism), and arrest. Fatal for the cells is silencing the agonistic stimulation of tyrosine kinase-growth factors receptors which would ensure cell cycle progression. At both experimental and clinical levels, the diabetic wound phenotype is the expression of countless molecular factors that operate through a complex biochemistry and lead to an aberrant cellular behavior. The pathway to chronification has not been fully elucidated but by all means it represents a form of cells’ biological disobedience and entails the need of continuous surgical “cuttings” in order to transiently restore an acute behavior by “refreshing” the cellular environment. It is likely that the scarcity of insulin and growth factors-induced tyrosine kinase receptors downstream signaling may lie, at least in part, behind chronification. Diabetes takes away the resources that ensure wound cells perpetuation and turn-over.

Although type 2 diabetes worldwide expansion is undeniable, primary care ulcer prevention plans together with the emergence of first-line pharmaceuticals and smart devices like engineered skin equivalents will certainly prevent and reduce contemporary amputation figures.


GFs/TK-R’s:Growth factors/tyrosine kinase receptors
MMPs: Matrix metalloproteases
ECM: Extracellular matrix
ROS: reactive oxygen species
OXPHOS: oxidative phosphorylation
TNF-α: Tumor necrosis factor
FOXO: Forkhead box O family of transcription factors
E.R. Stress: Endoplasmic reticulum stress
AGE: Advanced glycation end-products
RAGE: receptor for the AGE.


  1. D. Daneman, “Type 1 diabetes,” Lancet, vol. 367, no. 9513, pp. 847–858, 2006. View at: Publisher Site | Google Scholar
  2. M. L. Lamers, M. E. S. Almeida, M. Vicente-Manzanares, A. F. Horwitz, and M. F. Santos, “High glucose-mediated oxidative stress impairs cell migration,” PLoS ONE, vol. 6, no. 8, Article ID e22865, 2011. View at: Publisher Site | Google Scholar
  3. C. Bouché, S. Serdy, C. R. Kahn, and A. B. Goldfine, “The cellular fate of glucose and its relevance in type 2 diabetes,” Endocrine Reviews, vol. 25, no. 5, pp. 807–830, 2004. View at: Publisher Site | Google Scholar
  4. D. G. Armstrong, K. Cohen, S. Courric et al., “Diabetic foot ulcers and vascular insufficiency: our population has changed, but our methods have not,” Journal of Diabetes Science and Technology, vol. 5, no. 6, pp. 1591–1595, 2011. View at: Google Scholar
  5. T. Davies Pryce, “A case of perforating ulcers of both feet associated with diabetes and ataxic symptoms,” The Lancet, vol. 130, no. 3331, pp. 11–12, 1887. View at: Google Scholar
  6. P. D. Nguyen, J. P. Tutela, V. D. Thanik et al., “Improved diabetic wound healing through topical silencing of p53 is associated with augmented vasculogenic mediators,” Wound Repair and Regeneration, vol. 18, no. 6, pp. 553–559, 2010. View at: Publisher Site | Google Scholar
  7. J. Berlanga-Acosta, C. Valdéz-Pérez, W. Savigne-Gutiérrez et al., “Cellular and molecular insights into the wound healing mechanism in diabetes,” Biotecnologia Aplicada, vol. 27, pp. 255–261, 2010. View at: Google Scholar
  8. A. J. Boulton, L. Vileikyte, G. Ragnarson-Tennvall, and J. Apelqvist, “The global burden of diabetic foot disease,” Lancet, vol. 366, no. 9498, pp. 1719–1724, 2005. View at: Publisher Site | Google Scholar
  9. G. Gloire, S. Legrand-Poels, and J. Piette, “NF-κB activation by reactive oxygen species: fifteen years later,” Biochemical Pharmacology, vol. 72, no. 11, pp. 1493–1505, 2006. View at: Publisher Site | Google Scholar
  10. S. Mitra and E. Abraham, “Participation of superoxide in neutrophil activation and cytokine production,” Biochimica et Biophysica Acta, vol. 1762, no. 8, pp. 732–741, 2006. View at: Publisher Site | Google Scholar
  11. M. Guarino, A. Tosoni, and M. Nebuloni, “Direct contribution of epithelium to organ fibrosis: epithelial-mesenchymal transition,” Human Pathology, vol. 40, no. 10, pp. 1365–1376, 2009. View at: Publisher Site | Google Scholar
  12. N. Y. Yevdokimova, “High glucose-induced alterations of extracellular matrix of human skin fibroblasts are not dependent on TSP-1-TGFβ1 pathway,” Journal of Diabetes and its Complications, vol. 17, no. 6, pp. 355–364, 2003. View at: Publisher Site | Google Scholar
  13. D. W. Rowe, B. J. Starman, W. Y. Fujimoto, and R. H. Williams, “Abnormalities in proliferation and protein synthesis in skin fibroblast cultures from patients with diabetes mellitus,” Diabetes, vol. 26, no. 4, pp. 284–290, 1977. View at: Google Scholar
  14. K. Hehenberger and A. Hansson, “High glucose-induced growth factor resistance in human fibroblasts can be reversed by antioxidants and protein kinase C-inhibitors,” Cell Biochemistry and Function, vol. 15, no. 3, pp. 197–201, 1997. View at: Google Scholar
  15. S. Goldstein, E. J. Moerman, and J. S. Soeldner, “Diabetes mellitus and genetic prediabetes. Decreased replicative capacity of cultured skin fibroblasts,” Journal of Clinical Investigation, vol. 63, no. 3, pp. 358–370, 1979. View at: Google Scholar
  16. S. Goldstein, “Cellular and molecular biological studies on diabetes mellitus,” Pathologie Biologie, vol. 32, no. 2, pp. 99–106, 1984. View at: Google Scholar
  17. K. Hehenberger, J. D. Heilborn, K. Brismar, and A. Hansson, “Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of L-lactate,” Wound Repair and Regeneration, vol. 6, no. 2, pp. 135–141, 1998. View at: Google Scholar
  18. M. A. M. Loots, E. N. Lamme, J. R. Mekkes, J. D. Bos, and E. Middelkoop, “Cultured fibroblasts from chronic diabetic wounds on the lower extremity (non-insulin-dependent diabetes mellitus) show disturbed proliferation,” Archives of Dermatological Research, vol. 291, no. 2-3, pp. 93–99, 1999. View at: Publisher Site | Google Scholar
  19. A. T. Grazul-Bilska, G. Luthra, L. P. Reynolds et al., “Effects of basic fibroblast growth factor (FGF-2) on proliferation of human skin fibroblasts in type II diabetes mellitus,” Experimental and Clinical Endocrinology and Diabetes, vol. 110, no. 4, pp. 176–181, 2002. View at: Publisher Site | Google Scholar
  20. M. A. M. Loots, S. B. Kenter, F. L. Au et al., “Fibroblasts derived from chronic diabetic ulcers differ in their response to stimulation with EGF, IGF-I, bFGF and PDGF-AB compared to controls,” European Journal of Cell Biology, vol. 81, no. 3, pp. 153–160, 2002. View at: Google Scholar
  21. S. N. Xue, J. Lei, C. Yang et al., “The biological behaviors of rat dermal fibroblasts can be inhibited by high levels of MMP9,” Experimental Diabetes Research, vol. 2012, Article ID 494579, 7 pages, 2012. View at: Publisher Site | Google Scholar
  22. P. Velander, C. Theopold, T. Hirsch et al., “Impaired wound healing in an acute diabetic pig model and the effects of local hyperglycemia,” Wound Repair and Regeneration, vol. 16, no. 2, pp. 288–293, 2008. View at: Publisher Site | Google Scholar
  23. D. T. Loughlin and C. M. Artlett, “3-Deoxyglucosone-collagen alters human dermal fibroblast migration and adhesion: implications for impaired wound healing in patients with diabetes,” Wound Repair and Regeneration, vol. 17, no. 5, pp. 739–749, 2009. View at: Publisher Site | Google Scholar
  24. D. T. Loughlin and C. M. Artlett, “Modification of collagen by 3-deoxyglucosone alters wound healing through differential regulation of p38 MAP kinase,” PLoS ONE, vol. 6, no. 5, Article ID e18676, 2011. View at: Publisher Site | Google Scholar
  25. D. T. Loughlin and C. M. Artlett, “Precursor of advanced glycation end products mediates ER-stress-induced caspase-3 activation of human dermal fibroblasts through NAD(P)H oxidase 4,” PloS ONE, vol. 5, no. 6, Article ID e11093, 2010. View at: Publisher Site | Google Scholar
  26. M. Alikhani, C. M. MacLellan, M. Raptis, S. Vora, P. C. Trackman, and D. T. Graves, “Advanced glycation end products induce apoptosis in fibroblasts through activation of ROS, MAP kinases, and the FOXO1 transcription factor,” American Journal of Physiology, vol. 292, no. 2, pp. C850–C856, 2007. View at: Publisher Site | Google Scholar
  27. X. Tong, J. Ying, D. R. Pimentel, M. Trucillo, T. Adachi, and R. A. Cohen, “High glucose oxidizes SERCA cysteine-674 and prevents inhibition by nitric oxide of smooth muscle cell migration,” Journal of Molecular and Cellular Cardiology, vol. 44, no. 2, pp. 361–369, 2008. View at: Publisher Site | Google Scholar
  28. O. Z. Lerman, R. D. Galiano, M. Armour, J. P. Levine, and G. C. Gurtner, “Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia,” American Journal of Pathology, vol. 162, no. 1, pp. 303–312, 2003. View at: Google Scholar
  29. S. J. Wall, M. J. Sampson, N. Levell, and G. Murphy, “Elevated matrix metalloproteinase-2 and -3 production from human diabetic dermal fibroblasts,” British Journal of Dermatology, vol. 149, no. 1, pp. 13–16, 2003. View at: Publisher Site | Google Scholar
  30. J. W. Burrow, J. A. Koch, H. H. Chuang, W. Zhong, D. D. Dean, and V. L. Sylvia, “Nitric oxide donors selectively reduce the expression of matrix metalloproteinases-8 and -9 by human diabetic skin fibroblasts,” Journal of Surgical Research, vol. 140, no. 1, pp. 90–98, 2007. View at: Publisher Site | Google Scholar
  31. W. I. Sivitz and M. A. Yorek, “Mitochondrial dysfunction in diabetes: from molecular mechanisms to functional significance and therapeutic opportunities,” Antioxidants and Redox Signaling, vol. 12, no. 4, pp. 537–577, 2010. View at: Publisher Site | Google Scholar
  32. F. Giacco and M. Brownlee, “Oxidative stress and diabetic complications,” Circulation Research, vol. 107, no. 9, pp. 1058–1070, 2010. View at: Google Scholar
  33. B. Ponugoti, G. Dong, D. T. Graves et al., “Role of forkhead transcription factors in diabetes-induced oxidative stress,” Experimental Diabetes Research, vol. 2012, Article ID 939751, 7 pages, 2012. View at: Publisher Site | Google Scholar
  34. P. Storz, “Forkhead homeobox type O transcription factors in the responses to oxidative stress,” Antioxidants and Redox Signaling, vol. 14, no. 4, pp. 593–605, 2011. View at: Publisher Site | Google Scholar
  35. M. F. Siqueira, J. Li, L. Chehab et al., “Impaired wound healing in mouse models of diabetes is mediated by TNF-α dysregulation and associated with enhanced activation of forkhead box O1 (FOXO1),” Diabetologia, vol. 53, no. 2, pp. 378–388, 2010. View at: Publisher Site | Google Scholar
  36. M. E. Obrenovich and V. M. Monnier, “Apoptotic killing of fibroblasts by matrix-bound advanced glycation endproducts,” Science of Aging Knowledge Environment, vol. 2005, no. 4, p. e3, 2005. View at: Google Scholar
  37. M. H. Lima, A. M. Caricilli, L. L. de Abreu et al., “Topical insulin accelerates wound healing in diabetes by enhancing the AKT and ERK pathways: a double-blind placebo-controlled clinical trial,” PLoS ONE, vol. 7, no. 5, Article ID e36974, 2012. View at: Google Scholar
  38. P. Dandona, A. Chaudhuri, H. Ghanim, and P. Mohanty, “Insulin as an Anti-Inflammatory and Antiatherogenic Modulator,” Journal of the American College of Cardiology, vol. 53, no. 5, pp. S14–S20, 2009. View at: Publisher Site | Google Scholar
  39. L. Pirola, A. Balcerczyk, J. Okabe, and A. El-Osta, “Epigenetic phenomena linked to diabetic complications,” Nature Reviews Endocrinology, vol. 6, no. 12, pp. 665–675, 2010. View at: Publisher Site | Google Scholar
  40. T. Sonta, T. Inoguchi, H. Tsubouchi et al., “Evidence for contribution of vascular NAD(P)H oxidase to increased oxidative stress in animal models of diabetes and obesity,” Free Radical Biology and Medicine, vol. 37, no. 1, pp. 115–123, 2004. View at: Publisher Site | Google Scholar
  41. J. Waltenberger, J. Lange, and A. Kranz, “Vascular endothelial growth factor-A-induced chemotaxis of monocytes is attenuated in patients with diabetes mellitus: a potential predictor for the individual capacity to develop collaterals,” Circulation, vol. 102, no. 2, pp. 185–190, 2000. View at: Google Scholar
  42. D. T. Efron, D. Most, and A. Barbul, “Role of nitric oxide in wound healing,” Current Opinion in Clinical Nutrition and Metabolic Care, vol. 3, no. 3, pp. 197–204, 2000. View at: Publisher Site | Google Scholar
  43. S. E. Epstein, R. Kornowski, S. Fuchs, and H. F. Dvorak, “Angiogenesis therapy: amidst the hype, the neglected potential for serious side effects,” Circulation, vol. 104, no. 1, pp. 115–119, 2001. View at: Google Scholar
  44. S. E. Epstein, S. Fuchs, Y. F. Zhou, R. Baffour, and R. Kornowski, “Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards,” Cardiovascular Research, vol. 49, no. 3, pp. 532–542, 2001. View at: Publisher Site | Google Scholar
  45. T. A. Khan, F. W. Sellke, and R. J. Laham, “Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia,” Gene Therapy, vol. 10, no. 4, pp. 285–291, 2003. View at: Publisher Site | Google Scholar
  46. E. Duncan, V. Ezzat, and M. Kearney, “Insulin and endothelial function: physiological environment defines effect on atherosclerotic risk,” Current Diabetes Reviews, vol. 2, no. 1, pp. 51–60, 2006. View at: Publisher Site | Google Scholar
  47. U. Hink, H. Li, H. Mollnau et al., “Mechanisms underlying endothelial dysfunction in diabetes mellitus,” Circulation Research, vol. 88, no. 2, pp. E14–E22, 2001. View at: Google Scholar
  48. M. Jain, F. W. Logerfo, P. Guthrie, and L. Pradhan, “Effect of hyperglycemia and neuropeptides on interleukin-8 expression and angiogenesis in dermal microvascular endothelial cells,” Journal of Vascular Surgery, vol. 53, no. 6, pp. 1654–1660, 2011. View at: Publisher Site | Google Scholar
  49. J. V. Busik, S. Mohr, and M. B. Grant, “Hyperglycemia-Induced reactive oxygen species toxicity to endothelial cells is dependent on paracrine mediators,” Diabetes, vol. 57, no. 7, pp. 1952–1965, 2008. View at: Publisher Site | Google Scholar
  50. N. K. Rai, Suryabhan, M. Ansari, M. Kumar, V. K. Shukla, and K. Tripathi, “Effect of glycaemic control on apoptosis in diabetic wounds,” Journal of Wound Care, vol. 14, no. 6, pp. 277–281, 2005. View at: Google Scholar
  51. S. I. Kageyama, H. Yokoo, K. Tomita et al., “High glucose-induced apoptosis in human coronary artery endothelial cells involves up-regulation of death receptors,” Cardiovascular Diabetology, vol. 10, p. 73, 2011. View at: Publisher Site | Google Scholar
  52. D. Su, G. M. Coudriet, H. K. Dae et al., “FoxO1 links insulin resistance to proinflammatory cytokine IL-1β production in macrophages,” Diabetes, vol. 58, no. 11, pp. 2624–2633, 2009. View at: Publisher Site | Google Scholar
  53. M. Simons, “Angiogenesis, arteriogenesis, and diabetes: paradigm reassessed?” Journal of the American College of Cardiology, vol. 46, no. 5, pp. 835–837, 2005. View at: Publisher Site | Google Scholar
  54. M. Tanii, Y. Yonemitsu, T. Fujii et al., “Diabetic microangiopathy in ischemic limb is a disease of disturbance of the platelet-derived growth factor-BB/protein kinase C axis but not of impaired expression of angiogenic factors,” Circulation Research, vol. 98, no. 1, pp. 55–62, 2006. View at: Publisher Site | Google Scholar
  55. R. Tamarat, J. S. Silvestre, M. Huijberts et al., “Blockade of advanced glycation end-product formation restores ischemia-induced angiogenesis in diabetic mice,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 14, pp. 8555–8560, 2003. View at: Publisher Site | Google Scholar
  56. P. U. Magnusson, C. Looman, A. Åhgren, Y. Wu, L. Claesson-Welsh, and R. L. Heuchel, “Platelet-derived growth factor receptor-β constitutive activity promotes angiogenesis in vivo and in vitro,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 27, no. 10, pp. 2142–2149, 2007. View at: Publisher Site | Google Scholar
  57. S. M. Davidson and M. R. Duchen, “Endothelial mitochondria: contributing to vascular function and disease,” Circulation Research, vol. 100, no. 8, pp. 1128–1141, 2007. View at: Publisher Site | Google Scholar
  58. E. Fosslien, “Mitochondrial medicine—molecular pathology of defective oxidative phosphorylation,” Annals of Clinical and Laboratory Science, vol. 31, no. 1, pp. 25–67, 2001. View at: Google Scholar
  59. A. Martin, M. R. Komada, and D. C. Sane, “Abnormal angiogenesis in diabetes mellitus,” Medicinal Research Reviews, vol. 23, no. 2, pp. 117–145, 2003. View at: Publisher Site | Google Scholar
  60. D. Altavilla, A. Saitta, D. Cucinotta et al., “Inhibition of lipid peroxidation restores impaired vascular endothelial growth factor expression and stimulates wound healing and angiogenesis in the genetically diabetic mouse,” Diabetes, vol. 50, no. 3, pp. 667–674, 2001. View at: Google Scholar
  61. J. P. Cooke and D. W. Losordo, “Nitric oxide and angiogenesis,” Circulation, vol. 105, no. 18, pp. 2133–2135, 2002. View at: Publisher Site | Google Scholar
  62. C. H. Leo, J. L. Hart, and O. L. Woodman, “Impairment of both nitric oxide-mediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes,” British Journal of Pharmacology, vol. 162, no. 2, pp. 365–377, 2011. View at: Publisher Site | Google Scholar
  63. H. Kämpfer, J. Pfeilschifter, and S. Frank, “Expression and activity of arginase isoenzymes during normal and diabetes-impaired skin repair,” Journal of Investigative Dermatology, vol. 121, no. 6, pp. 1544–1551, 2003. View at: Publisher Site | Google Scholar
  64. C. L. M. Cooke and S. T. Davidge, “Peroxynitrite increases iNOS through NF-κB and decreases prostacyclin synthase in endothelial cells,” American Journal of Physiology, vol. 282, no. 2, pp. C395–C402, 2002. View at: Google Scholar
  65. Y. Higashi, K. Noma, M. Yoshizumi, and Y. Kihara, “Endothelial function and oxidative stress in cardiovascular diseases,” Circulation Journal, vol. 73, no. 3, pp. 411–418, 2009. View at: Publisher Site | Google Scholar
  66. R. Tamarat, J. S. Silvestre, S. Le Ricousse-Roussanne et al., “Impairment in ischemia-induced neovascularization in diabetes: bone marrow mononuclear cell dysfunction and therapeutic potential of placenta growth factor treatment,” American Journal of Pathology, vol. 164, no. 2, pp. 457–466, 2004. View at: Google Scholar
  67. H. Saito, Y. Yamamoto, and H. Yamamoto, “Diabetes alters subsets of endothelial progenitor cells that reside in blood, bone marrow, and spleen,” American Journal of Physiology, vol. 302, no. 6, pp. C892–C901, 2012. View at: Google Scholar
  68. C. J. M. Loomans, E. J. P. De Koning, F. J. T. Staal et al., “Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes,” Diabetes, vol. 53, no. 1, pp. 195–199, 2004. View at: Publisher Site | Google Scholar
  69. C. Shen, Q. Li, Y. C. Zhang et al., “Advanced glycation endproducts increase EPC apoptosis and decrease nitric oxide release via MAPK pathways,” Biomedicine and Pharmacotherapy, vol. 64, no. 1, pp. 35–43, 2010. View at: Publisher Site | Google Scholar
  70. H. Reinhard, P. Karl Jacobsen, M. Lajer et al., “Multifactorial treatment increases endothelial progenitor cells in patients with type 2 diabetes,” Diabetologia, vol. 53, no. 10, pp. 2129–2133, 2010. View at: Publisher Site | Google Scholar
  71. J. B. Acosta, D. Garcia Del Barco, D. Cibrian Vera et al., “The pro-inflammatory environment in recalcitrant diabetic foot wounds,” International Wound Journal, vol. 5, no. 4, pp. 530–539, 2008. View at: Publisher Site | Google Scholar
  72. M. Jagannathan-Bogdan, M. E. McDonnell, H. Shin et al., “Elevated proinflammatory cytokine production by a skewed T cell compartment requires monocytes and promotes inflammation in type 2 diabetes,” Journal of Immunology, vol. 186, no. 2, pp. 1162–1172, 2011. View at: Publisher Site | Google Scholar
  73. C. Weigelt, B. Rose, U. Poschen et al., “Immune mediators in patients with acute diabetic foot syndrome,” Diabetes Care, vol. 32, no. 8, pp. 1491–1496, 2009. View at: Publisher Site | Google Scholar
  74. D. Tuvdendorj, X. J. Zhang, D. L. Chinkes et al., “Intensive insulin treatment increases donor site wound protein synthesis in burn patients,” Surgery, vol. 149, no. 4, pp. 512–518, 2011. View at: Publisher Site | Google Scholar
  75. X. J. Zhang, D. L. Chinkes, Ø. Irtun, and R. R. Wolfe, “Anabolic action of insulin on skin wound protein is augmented by exogenous amino acids,” American Journal of Physiology, vol. 282, no. 6, pp. E1308–E1315, 2002. View at: Google Scholar
  76. C. M. Adams, “Role of the transcription factor ATF4 in the anabolic actions of insulin and the anti-anabolic actions of glucocorticoids,” Journal of Biological Chemistry, vol. 282, no. 23, pp. 16744–16753, 2007. View at: Publisher Site | Google Scholar
  77. V. Gupta, “Pleiotropic effects of incretins,” Indian Journal of Endocrinology and Metabolism, vol. 16, supplement 1, pp. S47–S56, 2012. View at: Google Scholar
  78. N. N. Ta, Y. Li, C. A. Schuyler, M. F. Lopes-Virella, and Y. Huang, “DPP-4 (CD26) inhibitor alogliptin inhibits TLR4-mediated ERK activation and ERK-dependent MMP-1 expression by U937 histiocytes,” Atherosclerosis, vol. 213, no. 2, pp. 429–435, 2010. View at: Publisher Site | Google Scholar
  79. H. Galkowska, U. Wojewodzka, and W. L. Olszewski, “Chemokines, cytokines, and growth factors in keratinocytes and dermal endothelial cells in the margin of chronic diabetic foot ulcers,” Wound Repair and Regeneration, vol. 14, no. 5, pp. 558–565, 2006. View at: Publisher Site | Google Scholar
  80. R. Blakytny and E. B. Jude, “Altered molecular mechanisms of diabetic foot ulcers,” International Journal of Lower Extremity Wounds, vol. 8, no. 2, pp. 95–104, 2009. View at: Publisher Site | Google Scholar
  81. C. D. Surh and J. Sprent, “TGF-beta puts the brakes on homeostatic proliferation,” Nature Immunology, vol. 13, no. 7, pp. 628–630, 2012. View at: Google Scholar
  82. W. P. Daley, S. B. Peters, and M. Larsen, “Extracellular matrix dynamics in development and regenerative medicine,” Journal of Cell Science, vol. 121, no. 3, pp. 255–264, 2008. View at: Publisher Site | Google Scholar
  83. G. Schultz, J. Berlanga-Acosta, L. Cowan, and J. Stechmiller, “Linking the advanced glycation endproducts/receptor for advanced glycation endproducts pathway in diabetics with inflammation and topical antiinflammatory treatments of chronic wounds,” in Advances in Wound Care, C. K. Sen, Ed., vol. 1, The Ohio State University Medical Center, Ohio, USA, 2010. View at: Google Scholar
  84. D. Skoutas, N. Papanas, G. S. Georgiadis et al., “Risk factors for ipsilateral reamputation in patients with diabetic foot lesions,” International Journal of Lower Extremity Wounds, vol. 8, no. 2, pp. 69–74, 2009. View at: Publisher Site | Google Scholar
  85. M. A. M. Loots, E. N. Lamme, J. Zeegelaar, J. R. Mekkes, J. D. Bos, and E. Middelkoop, “Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds,” Journal of Investigative Dermatology, vol. 111, no. 5, pp. 850–857, 1998. View at: Publisher Site | Google Scholar
  86. E. Black, J. Vibe-Petersen, L. N. Jorgensen et al., “Decrease of collagen deposition in wound repair in type 1 diabetes independent of glycemic control,” Archives of Surgery, vol. 138, no. 1, pp. 34–40, 2003. View at: Google Scholar
  87. S. M. Sliman, T. D. Eubank, S. R. Kotha et al., “Hyperglycemic oxoaldehyde, glyoxal, causes barrier dysfunction, cytoskeletal alterations, and inhibition of angiogenesis in vascular endothelial cells: aminoguanidine protection,” Molecular and Cellular Biochemistry, vol. 333, no. 1-2, pp. 9–26, 2010. View at: Publisher Site | Google Scholar
  88. J. Berlanga-Acosta, G. S. Schultz, and P. Lopez-Saura, “Biology of the diabetic wound,” in Foot Ulcers: Causes, Diagnosis and Treatments, P. E. Overhaussen, Ed., Nova Science Publishers, Hauppauge, NY, USA, 2009. View at: Google Scholar
  89. G. D. Mulder, D. K. Lee, and N. S. Jeppesen, “Comprehensive review of the clinical application of autologous mesenchymal stem cells in the treatment of chronic wounds and diabetic bone healing,” International Wound Journal, vol. 9, no. 6, pp. 595–600, 2012. View at: Google Scholar
  90. M. L. Usui, J. N. Mansbridge, W. G. Carter, M. Fujita, and J. E. Olerud, “Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds,” Journal of Histochemistry and Cytochemistry, vol. 56, no. 7, pp. 687–696, 2008. View at: Publisher Site | Google Scholar
  91. C. Wetzler, H. Kampfer, B. Stallmeyer, J. Pfeilschifter, and S. Frank, “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,” Journal of Investigative Dermatology, vol. 115, no. 2, pp. 245–253, 2000. View at: Publisher Site | Google Scholar
  92. B. C. Nwomeh, D. R. Yager, and I. K. Cohen, “Physiology of the chronic wound,” Clinics in Plastic Surgery, vol. 25, no. 3, pp. 341–356, 1998. View at: Google Scholar
  93. G. Naguib, H. Al-Mashat, T. Desta, and D. T. Graves, “Diabetes prolongs the inflammatory response to a bacterial stimulus through cytokine dysregulation,” Journal of Investigative Dermatology, vol. 123, no. 1, pp. 87–92, 2004. View at: Publisher Site | Google Scholar
  94. M. T. Goldberg, Y. P. Han, C. Yan, M. C. Shaw, and W. L. Garner, “TNF-α suppresses α-smooth muscle actin expression in human dermal fibroblasts: an implication for abnormal wound healing,” Journal of Investigative Dermatology, vol. 127, no. 11, pp. 2645–2655, 2007. View at: Publisher Site | Google Scholar
  95. B. A. Mast and G. S. Schultz, “Interactions of cytokines, growth factors, and proteases in acute and chronic wounds,” Wound Repair and Regeneration, vol. 4, no. 4, pp. 411–420, 1996. View at: Google Scholar
  96. S. J. Weiss, “Tissue destruction by neutrophils,” New England Journal of Medicine, vol. 320, no. 6, pp. 365–376, 1989. View at: Google Scholar
  97. A. Piwowar, M. Knapik-Kordecka, and M. Warwas, “Concentration of leukocyte elastase in plasma and polymorphonuclear neutrophil extracts in type 2 diabetes,” Clinical Chemistry and Laboratory Medicine, vol. 38, no. 12, pp. 1257–1261, 2000. View at: Publisher Site | Google Scholar
  98. C. M. Stanley, Y. Wang, S. Pal et al., “Fibronectin fragmentation is a feature of periodontal disease sites and diabetic foot and leg wounds and modifies cell behavior,” Journal of Periodontology, vol. 79, no. 5, pp. 861–875, 2008. View at: Publisher Site | Google Scholar
  99. W. C. Duckworth, J. Fawcett, S. Reddy, and J. C. Page, “Insulin-degrading activity in wound fluid,” Journal of Clinical Endocrinology and Metabolism, vol. 89, no. 2, pp. 847–851, 2004. View at: Publisher Site | Google Scholar
  100. C. S. Mantzoros, S. Moschos, I. Avramopoulos et al., “Leptin concentrations in relation to body mass index and the tumor necrosis factor-α system in humans,” Journal of Clinical Endocrinology and Metabolism, vol. 82, no. 10, pp. 3408–3413, 1997. View at: Publisher Site | Google Scholar
  101. Q. Mi, B. Rivière, G. Clermont, D. L. Steed, and Y. Vodovotz, “Agent-based model of inflammation and wound healing: insights into diabetic foot ulcer pathology and the role of transforming growth factor-β1,” Wound Repair and Regeneration, vol. 15, no. 5, pp. 671–682, 2007. View at: Publisher Site | Google Scholar
  102. M. H. Zou, C. Shi, and R. A. Cohen, “High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H2 receptor-mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells,” Diabetes, vol. 51, no. 1, pp. 198–203, 2002. View at: Google Scholar
  103. N. Petrova and M. Edmonds, “Emerging drugs for diabetic foot ulcers,” Expert Opinion on Emerging Drugs, vol. 11, no. 4, pp. 709–724, 2006. View at: Publisher Site | Google Scholar
  104. A. Desmoulière, C. Chaponnier, and G. Gabbiani, “Tissue repair, contraction, and the myofibroblast,” Wound Repair and Regeneration, vol. 13, no. 1, pp. 7–12, 2005. View at: Publisher Site | Google Scholar
  105. L. Mori, A. Bellini, M. A. Stacey, M. Schmidt, and S. Mattoli, “Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow,” Experimental Cell Research, vol. 304, no. 1, pp. 81–90, 2005. View at: Publisher Site | Google Scholar
  106. G. P. Fadini and A. Avogaro, “It is all in the blood: the multifaceted contribution of circulating progenitor cells in diabetic complications,” Experimental Diabetes Research, vol. 2012, Article ID 742976, 8 pages, 2012. View at: Publisher Site | Google Scholar
  107. D. M. Bermudez, J. Xu, B. J. Herdrich, A. Radu, M. E. Mitchell, and K. W. Liechty, “Inhibition of stromal cell-derived factor-1α further impairs diabetic wound healing,” Journal of Vascular Surgery, vol. 53, no. 3, pp. 774–784, 2011. View at: Publisher Site | Google Scholar
  108. D. Pilling, C. D. Buckley, M. Salmon, and R. H. Gomer, “Inhibition of fibrocyte differentiation by serum amyloid P,” Journal of Immunology, vol. 171, no. 10, pp. 5537–5546, 2003. View at: Google Scholar
  109. M. I. Morasso and M. Tomic-Canic, “Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing,” Biology of the Cell, vol. 97, no. 3, pp. 173–183, 2005. View at: Publisher Site | Google Scholar
  110. M. Deveci, R. R. Gilmont, W. R. Dunham, B. P. Mudge, D. J. Smith, and C. L. Marcelo, “Glutathione enhances fibroblast collagen contraction and protects keratinocytes from apoptosis in hyperglycaemic culture,” British Journal of Dermatology, vol. 152, no. 2, pp. 217–224, 2005. View at: Publisher Site | Google Scholar
  111. C. C. E. Lan, I. H. Liu, A. H. Fang, C. H. Wen, and C. S. Wu, “Hyperglycaemic conditions decrease cultured keratinocyte mobility: implications for impaired wound healing in patients with diabetes,” British Journal of Dermatology, vol. 159, no. 5, pp. 1103–1115, 2008. View at: Publisher Site | Google Scholar
  112. P. Zhu, C. Yang, L. H. Chen, M. Ren, G. J. Lao, and L. Yan, “Impairment of human keratinocyte mobility and proliferation by advanced glycation end products-modified BSA,” Archives of Dermatological Research, vol. 303, no. 5, pp. 339–350, 2011. View at: Publisher Site | Google Scholar
  113. A. El Ghalbzouri, P. Hensbergen, S. Gibbs, J. Kempenaar, R. Van Der Schors, and M. Ponec, “Fibroblasts facilitate re-epithelialization in wounded human skin equivalents,” Laboratory Investigation, vol. 84, no. 1, pp. 102–112, 2004. View at: Publisher Site | Google Scholar
  114. Y. Liu, M. Petreaca, M. Yao, and M. Martins-Green, “Cell and molecular mechanisms of keratinocyte function stimulated by insulin during wound healing,” BMC Cell Biology, vol. 10, article 1, 2009. View at: Publisher Site | Google Scholar
  115. I. Goren, E. Müller, D. Schiefelbein et al., “Akt1 controls insulin-driven VEGF biosynthesis from keratinocytes: implications for normal and diabetes-impaired skin repair in mice,” Journal of Investigative Dermatology, vol. 129, no. 3, pp. 752–764, 2009. View at: Publisher Site | Google Scholar
  116. H. Terashi, K. Izumi, M. Deveci, L. M. Rhodes, and C. L. Marcelo, “High glucose inhibits human epidermal keratinocyte proliferation for cellular studies on diabetes mellitus,” International Wound Journal, vol. 2, no. 4, pp. 298–326, 2005. View at: Publisher Site | Google Scholar
  117. E. Wertheimer, N. Spravchikov, M. Trebicz et al., “The regulation of skin proliferation and differentiation in the IR null mouse: implications for skin complications of diabetes,” Endocrinology, vol. 142, no. 3, pp. 1234–1241, 2001. View at: Publisher Site | Google Scholar
  118. T. Banno, A. Gazel, and M. Blumenberg, “Effects of tumor necrosis factor-α (TNFα) in epidermal keratinocytes revealed using global transcriptional profiling,” Journal of Biological Chemistry, vol. 279, no. 31, pp. 32633–32642, 2004. View at: Publisher Site | Google Scholar
  119. O. Stojadinovic, H. Brem, C. Vouthounis et al., “Molecular pathogenesis of chronic wounds: the role of β-catenin and c-myc in the inhibition of epithelialization and wound healing,” American Journal of Pathology, vol. 167, no. 1, pp. 59–69, 2005. View at: Google Scholar
  120. R. K. Sivamani, M. S. Garcia, and R. Rivkah Isseroff, “Wound re-epithelialization: modulating keratinocyte migration in wound healing,” Frontiers in Bioscience, vol. 12, no. 8, pp. 2849–2868, 2007. View at: Publisher Site | Google Scholar
  121. P. A. Coulombe, “Wound epithelialization: accelerating the pace of discovery,” The Journal of Investigative Dermatology, vol. 121, no. 2, pp. 219–230, 2003. View at: Google Scholar
  122. L. B. Nanney, S. Paulsen, M. K. Davidson, N. L. Cardwell, J. S. Whitsitt, and J. M. Davidson, “Boosting epidermal growth factor receptor expression by gene gun transfection stimulates epidermal growth in vivo,” Wound Repair and Regeneration, vol. 8, no. 2, pp. 117–127, 2000. View at: Publisher Site | Google Scholar
  123. S. Li, Q. Wang, Y. Wang, X. Chen, and Z. Wang, “PLC-γ1 and Rac1 coregulate EGF-induced cytoskeleton remodeling and cell migration,” Molecular Endocrinology, vol. 23, no. 6, pp. 901–913, 2009. View at: Publisher Site | Google Scholar
  124. D. F. Kusewitt, C. Choi, K. M. Newkirk et al., “Slug/Snai2 is a downstream mediator of epidermal growth factor receptor-stimulated reepithelialization,” Journal of Investigative Dermatology, vol. 129, no. 2, pp. 491–495, 2009. View at: Publisher Site | Google Scholar
  125. S. Frank, G. Hubner, G. Breier, M. T. Longaker, D. G. Greenhalgh, and S. Werner, “Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing,” Journal of Biological Chemistry, vol. 270, no. 21, pp. 12607–12613, 1995. View at: Publisher Site | Google Scholar
  126. S. Aoki, S. Toda, T. Ando, and H. Sugihara, “Bone marrow stromal cells, preadipocytes, and dermal fibroblasts promote epidermal regeneration in their distinctive fashions,” Molecular Biology of the Cell, vol. 15, no. 10, pp. 4647–4657, 2004. View at: Publisher Site | Google Scholar
  127. V. Gopalakrishnan, R. C. Vignesh, J. Arunakaran, M. M. Aruldhas, and N. Srinivasan, “Effects of glucose and its modulation by insulin and estradiol on BMSC differentiation into osteoblastic lineages,” Biochemistry and Cell Biology, vol. 84, no. 1, pp. 93–101, 2006. View at: Publisher Site | Google Scholar

Copyright © 2013 Jorge Berlanga-Acosta et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

More related articles

 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly as possible. We will be providing unlimited waivers of publication charges for accepted research articles as well as case reports and case series related to COVID-19. Review articles are excluded from this waiver policy. Sign up here as a reviewer to help fast-track new submissions.