Hindawi Publishing Corporation
BioMed Research International
Volume 2013, Article ID 256043, 15 pages
http://dx.doi.org/10.1155/2013/256043
Review Article
Glucose Toxic Effects on Granulation Tissue Productive Cells: The
Diabetics’ Impaired Healing
Jorge Berlanga-Acosta,1 Gregory S. Schultz,2 Ernesto López-Mola,1 Gerardo Guillen-Nieto,3
Marianela García-Siverio,1 and Luis Herrera-Martínez4
1
Tissue Repair and Cytoprotection Research Group, Center for Genetic Engineering and Biotechnology, Playa, CP 10600 Havana, Cuba
Institute for Wound Research, University of Florida, Gainesville, FL, USA
3
Biomedical Research Direction, Center for Genetic Engineering and Biotechnology, Playa, CP 10600 Havana, Cuba
4
General Direction, Center for Genetic Engineering and Biotechnology, Playa, CP 10600 Havana, Cuba
2
Correspondence should be addressed to Jorge Berlanga-Acosta; jorge.berlanga@cigb.edu.cu
Received 18 August 2012; Accepted 24 November 2012
Academic Editor: David G. Armstrong
Copyright © 2013 Jorge Berlanga-Acosta et al. is 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.
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 chroni�cation. 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 e�ectors including proin�ammation, acute-phase proteins elevation,
and spillover of reactive oxygen and nitrogen species. Insulin axis de�ciency weakens wounds’ anabolism and predisposes to
in�ammation. e systemic accumulation of advanced glycation end-products irreversibly impairs the entire physiology from
cells-to-organs. ese factors in concert hamper �broblasts and endothelial cells proliferation, migration, homing, secretion,
and organization of a productive granulation tissue. Diabetic wound bed may turn chronically in�ammed, procatabolic, and
an additional source of circulating pro-in�ammatory cytokines, establishing a self-perpetuating loop. Diabetic �broblasts 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 �gures.
1. Introduction
What represents today a worldwide pandemic of a noncommunicable disease, diabetes mellitus, has two principal clinical forms identi�ed as types 1 and 2. e former is a condition
in which by autoimmune mechanisms pancreatic �-cells are
eventually destroyed with an absolute insulin de�ciency [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 de�nitive 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 in�uence its own clearance by a mass action e�ect [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 so 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 e Lancet
2
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].
is 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
chroni�cation. 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. us, 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. us, the challenge that
represents the diabetic wound healing failure is the clinical
gross expression of an outstanding array of biochemical and
cellular disorders [7]. ese ideas are supported in the clinical
arena by the alarming statistics of amputations around the
world every year [8].
e healing process in diabetes is also jeopardized by the
patient’s susceptibility to infection due to de�ciencies on the
innate immunity. Although the diabetic wound bed may be
adversely overwhelmed by in�ammatory 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 NFkappaB (NF-�B), thus amplifying the absurd in�ammation
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 BerlangaAcosta. Manuscript accepted. Int Wound Journal). e 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: �broblasts
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. e literature search was based on English
language articles downloaded from Pubmed and Bioline
International (http://www.bioline.org.br/) data sources.
2. Consequences of Glucose Overload Toxicity
on Fibroblasts and Endothelial Cells
2.1. Fibroblasts. e �broblast is central to the wound healing process by secreting, contracting, and remodeling the
extracellular matrix (ECM). ey also secrete growth factors
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as important messengers for mesenchymal-to-mesenchymal
and epithelial-mesenchymal communication, especially for
establishing the emerging basement membrane and subsequent reepithelialization. erefore, any impediment to
�broblast function is deterrent for normal wound healing and
may result in chronic, nonhealing wounds. e �broblast,
when engaged in �brogenesis, displays the highly activated
phenotype characteristic of myo�broblasts. Although their
origin has not yet been de�nitely elucidated� proliferation
of preexisting adjacent dermal �broblasts and, probably,
recruited from the bone marrow has been documented
[11]. Under the high glucose burden imposed by diabetes,
cutaneous and extra cutaneous �broblasts appear perturbed�
and for many years, in vitro models recreating “clinical
hyperglycemia” have proved to disrupt normal �broblasts
physiology and derange the secretion of extracellular matrix
ingredients. ese experiments have suggested that high
glucose concentration is the proximal detonator of a downstream cascade of molecular disturbances for the skin �broblast [12].
Rowe and coworkers pioneered the in vitro models
that demonstrated that in diabetics’ cutaneous �broblasts�
synthetic, proliferative, and secreting capabilities are reduced
[13]. Other parallel studies, in which high glucose concentrations were introduced, proved to inhibit �broblast 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 �ndings allowed for establishing the
hypothesis that diabetics �broblasts 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-insulindependent diabetes mellitus wound �broblasts induced a
dose-dependent inhibition in normal �broblast proliferation
which appeared related to elevated L-lactate levels [17]. is
replicative refractoriness of diabetic �broblasts has been
reproduced by different groups in subsequent years [18],
thus con�rming 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 �broblasts in order to induce
a proliferative response [20]. In addition to the onset of a
quiescent and senescent phenotype of diabetic wound �broblasts, their ability for horizontal and vertical migration is
also dramatically impaired when compared to normal donor
cells in different migration assay as in the modi�ed 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
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protrusive events, as loss of cell polarization, consistent
with the increased activity of Rac1 and the projection of
multiple lamellipodia. is 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 �broblast 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 �ndings, this group also demonstrated that 3DG-modi�ed 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 modi�ed collagen with 3DG, which signals to
the �broblast by interacting with integrins alpha-1/beta-1
(�1�1) and not through the canonical receptor for advanced
glycation end-products (RAGE) [25]. Other groups have
also demonstrated the induction of cutaneous �broblasts
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 aer AGEsRAGE 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 caspase3 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 �broblasts exhibit particular features.
Literature documents that diabetic mice �broblasts show a
severe impairment in VEGF production under normoxic
3
and hypoxic conditions in addition to an increased prodegradative 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 �broblasts have con�rmed the prodegradative
phenotype by the increased MMP-2 and MMP-3 production
and reduced collagens gene expression [29]. Human diabetic �broblasts also exhibit a failure in nitric oxide (NO)
production, which is concomitant to elevations in MMP-8
and -9 [30]. e fact that these �broblasts 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 ampli�cation of oxidative stress acts as
a primary culprit in harming �broblasts 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 in�ammation
and cell death. ey 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. is 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-�-) induced apoptosis
both of which are abundant in diabetic connective tissue
[35]. FOXO1 limits wound healing by inhibiting �broblasts
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 stromalderived factor-1�/SDF-1�) 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 �ndings are not surprising
in light of the potent anti-in�ammatory, 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
�broblasts damage process.
4
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High glucose burden
Inflammatory cytokines
GFs/TK-R’s
MMPs
ROS
AGEs
Mitochondrial damage
TNF-α
ECM deposition
OXPHOS
FOXO
catabolism
Fibroblasts recruitment,
anchorage, proliferation
Antioxidant
reserves
E.R. stress
Granulation
refractoriness
Arrest, senescence,
apoptosis
F 1: Negative impact of high glucose levels on cutaneous �broblasts biology. Short- or long-term exposure to high glucose
concentrations is toxic for cutaneous �broblasts suppressing the cells’ biological activities. e �broblasts become reactive but not active. e
high glucose burden engenders and uncontrolled production of ROS within the mitochondria with three major consequences: detriment on
the OXP�OS reactions, depletion of the cells antioxidant reserves, and ampli�cation of the mitochondrial dysfunction due to ROS-mediated
attack to its DNA. Under this scenario apoptosis may prevail. ROS also may lead to cell cycle arrest due to p53 and p21 upregulation and
nuclear compartmentalization. Alternatively, high glucose concentrations may impose a proin�ammatory program within the wound by
perpetuating a special population of macrophages (M1) so that �broblasts become intoxicated and suppress the secretion of ECM ingredients.
Conversely, the in�ammation mediators fuel the secretion of MMPs. e negative balance of ECM inhibits �broblasts chemotaxis, homing,
anchoring, and proliferation. e proin�ammatory environment inhibits the secretion of numerous growth factors with �bro angiogenic
potential as TGF-�, PDF, EGF, and so forth and interferes with the signaling pathways of the TK-R’s. Inhibition of the TK-R’s downstream
networking entails the suppression of positive forces for a balanced control of granulation tissue repopulation with productive cells. e
accumulation of AGEs activates the AGE�RAGE axis, which further ampli�es local in�ammation and reactivity by increasing the secretion of
TNF-alpha and adhesion molecules. is cytokine interferes with insulin and growth factors signaling, TGF-�1 for instance, which further
ampli�es the obstruction of the PI3K�Akt�mTOR�Cyclin D axis. e balance against this vital axis promotes the nuclear compartmentalization
of representative of the FOXO family members, which contributes to catabolism, senescence, arrest, and apoptosis. In this prooxidative
environment, it is common to activate cells autophagy in response to the accumulation of missfolded proteins. us, all these factors converge
to slow down granulation tissue outgrowth.
Despite the proli�c investigation conducted during all
these years, still questions remain to be answered in relation
to ex vivo diabetics’ �broblasts behavior.
(1) �hy diabetics’ �broblasts 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) �hy cultured �broblasts 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. is is a vital
process for embryological growth, tissue development, and
wound healing. Different growth factors families as vascular
endothelial growth factors (�EGF), �broblast growth factor
(FGF), angiopoietins, platelet-derived growth factor (PDGF),
transforming growth factor-� (TGF-�), in collaboration with
other proteins as integrins, cadherins, and ephrins, regulate
angiogenesis by promoting endothelial cells recruitment,
proliferation, migration, coopting, and collar stabilization.
ere is an enormous and ever-growing body of evidence
indicating the close correlation between hyperglycemia and
the abnormalities in endothelial function and morphology
[39]. e UK Prospective Diabetes Study (UKPDS) and
Diabetes Control of Complications Trial (DCCT) found
microvascular disease and hyperglycaemia to be intrinsically
related. us, anomalous angiogenesis is a hallmark of both
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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.
e 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. e later being of
paramount importance in angiogenesis and wound healing as
described below [42].
As depicted for �broblasts, 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 aer achieving the
successful reperfusion of an ulcerated lower extremity, the
healing process is slow and torpid. erapeutic angiogenesis
has been pursued for years, but the clinical results have shown
relatively limited outcomes [43–45]. 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-� 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 cascadeinduced death [51].
Hyperglycemia-induced oxidative stress promotes in�ammation through increased endothelial cells damage,
microvascular permeability, and uncontrolled release of
proin�ammatory cytokines, including TNF-�, interleukin1� (IL-1�), 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 ampli�cation of proin�ammatory cytokines production. FOXO1 directly binds to
5
IL-1� 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-� [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].
e 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. e 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. is 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]. is
evidence reinforces the pathophysiological impact of high
glucose toxicity, the release of proin�ammatory 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-in�ammatory pro�le turning these cells into
a cytokines and ROS manufacturing plant. e agonistic
stimulation of the AGEs receptor is able to mount the
same response leading to apoptosis and vascular ruin. e
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 in�ammatory 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. erefore, 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-in�ammatory factors
and eventually the execution of apoptosis of endothelial and
6
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High glucose burden
Insulin axis failure
Endothelial cells and pericytes
ROS
NEP
AGEs
TNF-α
Reduction of angiogenic growth factors:
TGF-β, VEGF, PDGF, FGFs.
Reduction of SDF-1α expression
Reduction of HIF-1α expression
Reduction of angiopoietin-1 expression
e-NOS
• Reduction of endothelial cells
detachment and migration
• Reduction or abnormal cellular
coopting and tubulogenesis
• Onset of an endothelial reactive
inflammatory program
• Onset of a proapoptogenic
program
F 2: Negative impact of high glucose levels and failure of the insulin system on vascular cells. Endothelial cells are a sensitive target for
high glucose concentration and especially for the insulin receptor downstream signaling attenuation. Similarly, pericytes, which are key cells
for the angiogenic process to succeed, are targeted by these factors. e endothelial cells metabolic response to the above proximal triggers
engenders the accumulation of superoxide and hydroxyl reactive groups. ese prooxidative elements disrupt the physiological pathways for
NO metabolism, accumulating toxic nitrosylation end-products. In this context, AGEs are precipitately formed and accumulated within the
vascular wall. Different pathways converge to induce TNF-� overproduction. e proximal triggers and all these factors also contribute to
disrupt e-NOS activity having as net result a de�cit in endothelial NO, the inability for vasodilation, and the suppression of endothelial cells
proliferative reserves. is circle is further ampli�ed due to the concomitant reduction in the pool of critical angiogenic factors involved in
the regulation of vascular regeneration. Eventually endothelial cells and pericytes may onset a pro-apoptogenic program, which will hinder
granulation tissue perfusion and wound healing.
adjacent cells [33]. ROS-mediated lipid peroxidation appears
to impair most healing events, contributing to growth factors
reduction, keratinocytes migration failure, slow or torpid
�broplasia, 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 �broblasts, and synthesis and maturation of the extracellular matrix. A parallel �nding was the normalization of VEGF mRNA expression and secretion in those
diabetic mice. is 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, in�ammation, and
neovascularization. Impaired endothelium-dependent NOmediated 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/eNOS coupling pathway for a normal endothelial function
appears compulsory [62]. Hyperglycemia is also associated to
a de�cit 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]. e 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 (ICAM1), and other in�ammatory mediators [64].
Endothelial Progenitor Cells (EPCs) are active players
for the maintenance and repair of endothelial cells. ey
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
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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-in�ammatory cytokines and to shi� 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].
e diabetes-mediated vascular damage is perhaps the
most outspoken and ancestrally identi�ed 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 identi�ed in a 7day-old granulation tissue fragment, even in compensated
patients. is 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: �broblasts 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,
proin�ammed, prooxidant, and prodegradative phenotype
[71].
is stubbornness and slowness to heal in diabetes is
conditioned by systemic and local factors that in complicity counteract intrinsic reparative mechanisms. In a broad
7
systemic context, in�ammation and the anabolic de�cit
can be conceptually mentioned. Diabetic patients with foot
ulceration bear a speci�c 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 in�ammatory pro�le and amplify
local wound in�ammatory networks [72]. e systemically
elevated levels of pro-in�ammatory 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.
e 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 de�cit
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
antiin�ammatory effects [77]. In support to the GLP-1 action
is the study by Ta and coworkers with alogliptin, a speci�c
inhibitor of dipeptidyl peptidase-4 (DPP-4) which showed
to inhibit macrophage-mediated in�ammation 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 �broblasts, is required for
an efficient cellular anchoring and homing at the wound
bed. As mentioned above, the cutaneous �broblast is a
sensitive cell to high glucose, AGE-precursors, AGEs, ROS,
and TNF-�, rapidly undergoing premature senesce, arrest
or apoptosis. Fibroblasts are the main source of collagen,
and the number of �broblasts can be taken for a measure
of repair by their collagen synthesis ability. It is very likely
that the de�cit of growth factors such as TGF-�1, IGF-I and
PDGF could translate into a DFU with scarce extracellular
matrix acumulation and impoverished cellularity. Numerous
growth factors (TGF-�1, 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�1 and TGF-�3, in which the former appears downregulated,
may explain �broblasts quiescence in terms of proliferation
and secretion [80]. is phenomenon represents the de�cit
of one of the most potent pro�brogenic and �broblastsmitogenic growth factors, which at the same time is able to
downregulate macrophage activation [81]. e extracellular
matrix represents the granulation tissue dynamic stroma
that provides support for in�ammatory cells, �broblasts, and
endothelial cells and allows for the chemotaxis of epithelial
8
F 3: Common histological aspect of a neuropathic granulation
tissue. Neuropathic granulation tissue exhibiting a scarce deposition
of extracellular matrix. Note a central blood vessel with abundant
surrounding heterogeneous cellularity and abundant �brin accumulation, suggesting hyperpermeability. Hematoxylin/eosin staining,
×10 magni�cation.
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 bio�lm 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 aer reepithelialization [84] may be inherent
to the qualitative composition of the scar matrix to tolerate
tensile forces and mechanical stress.
e diabetic granulation process does not generally
exhibit the orderly cascade of events that characterize normal wound healing. is has been con�rmed through the
histopathological analysis of granulation tissue biopsies by
Loots and coworkers who described the lesions as “frozen” in
a chronic low-grade in�ammatory state associated to a scarce
provisional extracellular matrix [85]. Our group’s serial
biopsies from both neuropathic and ischemic ulcers-derived
granulation tissue have identi�ed histological differences for
both types of wounds in the absence of clinical infection.
Polymorphonuclear cells (PMN) in�ltration 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 �broblast
proliferation and a scarce amount of collagen accumulation
within the wound bed [86]. On the contrary, a broadly spread
in�ltration of round cells predominate in those patients suffering from wound bed ischemia, associated to a �brohyaline
matrix of “hardened” aspect and abnormal angiogenesis in
which vascular wall cellular mosaicism, precocious media
BioMed Research International
F 4: Common histological aspects of ischemic granulation
tissue. Note the presence of an intense in�ammatory in�ltrate of
round cells spread all over the tissue area. e emerging vessels
appear unfunctional with thickened walls of �brohyaline material,
“hardened aspect”, and endothelial nuclei hypertrophy. Hematoxylin/eosin staining, ×10 magni�cation.
thickening, endothelial nuclei hypertrophy and many other
defects can be identi�ed (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]. ese observations
incite to speculate that the biochemical microenvironment
in ischemic and neuropathic diabetic wounds is different
and that the in�ammatory “badge” is in correspondence
with the wound’s most prevalent pathogenic component
[88]. In contrast to acute wounds in nondiabetic subjects,
the in�ammatory 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 in�ammatory reaction is related
to the prolonged expression of macrophage in�ammatory
protein-2 (MIP-2) and macrophage chemoattractant protein1 (MCP-1) [91]. Furthermore, the downregulation of the
anti-in�ammatory cytokine IL-10 in DFUs environment represents the collapse of an important in�ammatory restrainer
[73]. Another evidence indicates that PMNs are critical cells
toward the acquisition and perpetuation of in�ammation and
a degradative phenotype. e granulocytes secrete TNF-�
and IL-1�, which act as a triggering signal for MMPs
expression via the common NF-�B signaling pathway. Within
the wound context, TNF-� stimulates its own secretion and
that of IL-1�, which contributes to a persistent in�ammatory
status [92]. TNF-� has proved to negatively impact the
repair process as it is early secreted since the in�ammatory
phase. Its deregulation is not only associated with persistent
in�ammation but also to connective tissue degradation [93].
Concomitantly, TNF-� mediates its antagonistic effects on
TGF-�1 through the JNK pathway via inhibition of Smad
phosphorylation, consequently reducing the expression of
TGF-�1, and that of several downstream matrix proteins
[94]. In this highly proteolytic milieu, �bronectin, collagens,
growth factors, and their receptors are degraded while the
wound is way down to a catabolic state [95].
BioMed Research International
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 �bronectin and its surface receptor integrin �5�1 to effectively migrate [98]. Curiously, insulindegrading activity has also been demonstrated in the �uid
of diabetic experimental and human wounds, which have
been shown to correlate with the glycated hemoglobin levels
[99]. e connection between NO metabolism and foot
ulcer proteases pro�le has been described. In contrast to
elevated MMP-8 and 9 displayed by the nonhealing diabetic
foot wound, the concentration of NO appears signi�cantly
reduced. Diabetic skin �broblasts treated with NO donor
compounds, selectively raised NO production, increased
cell proliferation, and decreased the expression of MMP8 and -9 in a dose-dependent manner. us, 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].
e link between wound cells and apoptosis was
described above; we just wish to comment that in sharp contrast to wound-in�ltrated in�ammatory cells, which become
refractory to apoptosis, granulation tissue-producing cells
are sensitive to commit suicide where TNF-� stands as a
major driving force. e negative impact of TNF-� 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-� or to increase the wound local availability
of active TGF-�1 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 ampli�es the cytotoxic cascade. Endothelial cells and �broblasts,
in particular senescent �broblasts, 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]. us, the
disturbed oxidant/antioxidant balance as the AGEs accumulation within the chronic wound microenvironment is
considered a major factor, which ampli�es the unrestrained
and persistent in�ammatory, toxic, and catabolic state of
nonhealing wounds [96].
e failure of wound contraction is a clinical hallmark
of diabetic granulation tissue. Fibroblast-to-myo�broblast
transdifferentiation represents a key event during wound
healing and tissue repair. e contractile force, generated
by myo�broblasts 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
9
ergonomic response. In diabetic subjects, however, contraction is impaired and deep ulcers heal by the combination
of granulation and reepithelialization. e classical view on
dermal wound healing implies recruitment of local �broblasts
[104] followed by a subsequent process of transdifferentiation
in which the �broblasts gain a de�nitive phenotype of
differentiated myo�broblasts by neo-expressing a-smooth
muscle actin (�-SMA). Nevertheless, �-SMA expression is
precisely controlled by the joint action of growth factors like
TGF-�1 and extracellular matrix proteins like the �bronectin
(FN) splice variant ED-A, as by the local mechanical
microenvironment [104]. It should be noted, however, that
indwelling �broblasts in diabetic wounds are refractory to
proliferate and adopt a senescent phenotype, and that TGF�1, �bronectin, and other matrix proteins may appear in
de�cit. Hence, all these factors may contribute to the poor
contractile activity. Furthermore, Goldberg and coworkers
have shown that among the deleterious activities of TNF-�
within the wound is to suppress �-SMA expression in human
dermal �broblasts [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 myo�broblasts potentially derives from bone
marrow �brocytes is valid for humans [105]; we have already
learned that diabetes negatively impacts on the general bone
marrow physiology [106] and that beyond this, stromalderived factor-1alpha (SDF-1�), 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 in�ammatory reactants involved in insulin
resistance inhibit �brocytes differentiation [108].
ere 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. e 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. erefore,
10
BioMed Research International
Systemic cytotoxicity
Hyperglycemia
Insulin resistance
Systemic proinflammatory program
AGE-RAGE
TNF-α
ROS
Enhanced and prolonged inflammatory cells
recruitment to the wound
TGF-β 1
abrogation
TNF-α/Fas system
Granulation
breakdown
Apoptosis of
fibroblasts,
myofibroblasts,
and endothelial
cells
Impaired healing
F 5: Impact of hyperglycemia on granulation tissue biology. e onset of a systemic pro-in�ammatory program due to sustained
hyperglycemia is associated with the elevation of circulating levels of TNF-�. e cytokine release is further ampli�ed by the agonistic
interaction AGE�RAGE and the generation of R��. is preliminary triad ampli�es insulin receptor resistance and a multiorgan toxicity.
Excess TNF-� perpetuates the in�ammatory in�ltrate into the wound bed hindering the onset of the �broangiogenic phase in part by
abrogating TGF-�1 release. is TNF-� related growth factors de�cit within the wound bed may act as a vulnerability factor for granulation
tissue productive cells apoptosis.
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
signi�cantly reduce their proliferation rate and replicative
life span and were rendered more susceptible to commit
apoptosis [110]. �ther studies also con�rmed that hyperglycemic conditions abort keratinocytes’ proliferative ability
and their migratory response [111]. Aside from the glucosemediated direct cytotoxic effect on the keratinocytes, AGE
modi�cation of type-I collagen and other E�M 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 pro�le and order of released factors regulating keratinocytes proliferation and differentiation [113].
e 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 �eld 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-� 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 �-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 signi�cance. 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
BioMed Research International
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 GTPbinding proteins [123]. Recent experiments document the
negative effect of the Slug null mutation as a downstream
EGFR catalytic mediator for wound reepithelialization. us,
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. Skinreconstitution studies have shown that bone marrow stromal
cells (BMSCs), in addition to dermis-localized preadipocytes
and �broblasts distinctively promote epidermal regeneration
[126]. As diabetes courses with a de�cient 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 aer
wound contours surgical debridement keratinocytes migration resumes, for soon aer 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. e 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 �ourishing. 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 selfperpetuating loops, which include but are not limited to ROSlipid peroxidation, hyperin�ammation/disimmunity, AGERAGE toxicity, mitochondrial dysfunction, and nytrosilation
end-products accumulation. e concerted action of these
factors enforces �broblasts, 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. is 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
11
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. e
pathway to chroni�cation 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 chroni�cation. 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 �rst-line pharmaceuticals and smart devices
like engineered skin equivalents will certainly prevent and
reduce contemporary amputation �gures.
Abbreviations
GFs/TK-R’s:
MMPs:
ECM:
ROS:
OXPHOS:
TNF-�:
FOXO:
Growth factors/tyrosine kinase receptors
Matrix metalloproteases
Extracellular matrix
reactive oxygen species
oxidative phosphorylation
Tumor necrosis factor
Forkhead box O family of transcription
factors
E.R. Stress: Endoplasmic reticulum stress
AGE:
Advanced glycation end-products
RAGE:
receptor for the AGE.
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Hindawi Publishing Corporation
http://www.hindawi.com
Volume 2014
Volume 2014
Journal of
Obesity
Journal of
Ophthalmology
Hindawi Publishing Corporation
http://www.hindawi.com
Volume 2014
Evidence-Based
Complementary and
Alternative Medicine
Stem Cells
International
Hindawi Publishing Corporation
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Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com
Volume 2014
Journal of
Oncology
Hindawi Publishing Corporation
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Volume 2014
Hindawi Publishing Corporation
http://www.hindawi.com
Volume 2014
Parkinson’s
Disease
Computational and
Mathematical Methods
in Medicine
Hindawi Publishing Corporation
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Volume 2014
AIDS
Behavioural
Neurology
Hindawi Publishing Corporation
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Research and Treatment
Volume 2014
Hindawi Publishing Corporation
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Volume 2014
Hindawi Publishing Corporation
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Volume 2014
Oxidative Medicine and
Cellular Longevity
Hindawi Publishing Corporation
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Volume 2014