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8 Regeneration oftheSkin andPeripheral Nerves intheAdult
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128. Troxel K.Delay of skin wound contraction by porous collagenGAG matrices. 1994.
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Neuropeptides, Inflammation,
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andDiabetic Wound Healing: Lessons
fromExperimental Models andHuman
Subjects
LucasMota, FrankW.LoGerfo, AristidisVeves,
andLeenaPradhan-Nabzdyk
9
Abstract
Diabetic peripheral neuropathy and vascular disease,
along with trauma, have long been recognized as major
risk factors for the development of diabetic foot ulcerations (DFUs). More recently, chronic inammation,
abnormal extracellular matrix remodeling, and reduced
wound neovascularization, as a result of dysregulated cell
function with imbalanced secretion of cytokines, matrix
metalloproteinases, and growth factors, have been implicated in DFU failure to heal. Therefore, researchers are
now focusing their efforts on understanding the cellular
and molecular mechanisms of diabetes-associated
impaired wound healing, in an attempt to identify new
targets and novel potential therapeutic approaches for
DFUs, which remain a serious unmet clinical need.
Recent advances in technologies such as single-cell
sequencing are being implemented to further understand
the pathology underlying DFU and design precise therapeutics. A growing body of evidence suggests an important role of neuropeptides in skin repair, particularly in
L. Mota
Department of Surgery, Harvard Medical School, Beth Israel
Deaconess Medical Center, Boston, MA, USA
e-mail: lsouzamo@bidmc.harvard.edu
F. W. LoGerfo
Division of Vascular and Endovascular Surgery, Harvard Medical
School, Beth Israel Deaconess Medical Center, Boston, MA, USA
e-mail: ogerfo@bidmc.harvard.edu
A. Veves
The Rongxiang Xu, MD, Center for Regenerative Therapeutics,
Joslin-Beth Israel Deaconess Foot Center, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, MA, USA
e-mail: aveves@bidmc.harvard.edu
L. Pradhan-Nabzdyk (*)
Division of Vascular and Endovascular Surgery, Beth Israel
Deaconess Medical Center, Harvard Medical School,
Boston, MA, USA
e-mail: lpradhan@bidmc.harvard.edu
diabetes, where neuropeptide levels are diminished. On
the other hand, there is emerging interest in dissecting the
mechanisms of dysregulated inammation, namely, the
changes in immune cells, such as macrophages and mast
cells (MCs), in diabetic wound healing. Studies using
in vitro and in vivo models of diabetic wound healing
have considerably improved our understanding of the
healing process. However, the currently available models
have major caveats and are not ideal to study chronic,
complicated, and multifactorial wounds, such as DFUs.
In this chapter we summarize the involvement of neuropeptides and mast cells in diabetic wound healing, highlighting the most recent ndings. We also discuss the
benets and limitations of the current wound healing
models, emphasizing the need for conrmation and/or
validation in multiple models and/or tissue specimens
from human subjects.
Neuropeptides andDiabetic Wound Healing
Peripheral neuropathy is the most common complication of
diabetes, affecting approximately 50% of diabetics, and
being associated with up to 85% of DFUs [1, 2]. Diabetic
peripheral neuropathy is associated not only with loss of pain
sensitivity, especially at the lower extremities, rendering diabetic patients prone to disregard trauma in such areas, but
also with reduced levels of neuropeptides [3]. Neuropeptides
are secreted by the small nerve bers, both sensory and autonomic, as well as by dermal and epidermal cells [4, 5]. They
not only relay information such as pain signals to the central
nervous system but participate in the inammatory and proliferative phases of wound healing by binding to specic
receptors that are found in various skin cells, including
immune cells such as mast cells (MCs), as well as endothelial cells, broblasts, and keratinocytes [6]. In fact, neuropeptides can regulate the release of numerous cytokines and
growth factors that are pivotal for wound repair and imbal-
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
A. Veves et al. (eds.), The Diabetic Foot, Contemporary Diabetes, https://doi.org/10.1007/978-3-031-55715-6_9
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anced in diabetes, including IL-1, IL-6, IL-8, TNF-α, and
VEGF [4]. As a result, there is growing interest in the potential role of neuropeptides, namely, substance P (SP), neuropeptide Y (NPY), neurotensin (NT), calcitonin gene-related
peptide (CGRP), and alpha-melanocyte stimulating hormone
(α-MSH), in diabetic wound healing [3–5, 7]. As key players
in the bidirectional neuro-immune/neuro-inammatory axis
[4, 6], it is anticipated that neuropeptides are involved in the
healing of DFUs, where loss of sensory nerves, ineffective
immune and inammatory responses, and dysregulated
inammation are present. While focusing mostly on substance P (SP), our group has also investigated the roles of
neuropeptide Y (NPY) and neurotensin (NT) in diabetic
wound healing.
Substance P andDiabetic Wound Healing
Substance P (SP) is an 11-amino-acid peptide that belongs to
the tachykinin neuropeptide family, encoded by the
TAC1gene. SP is released by c-nociceptive bers in response
to injury and exerts its actions by activating three primary
types of neurokinin (NK) receptors—NK1R, NK2R, and
NK3R, with NK1R being the predominant and with highest
afnity, and is degraded by the enzyme neutral endopeptidase (NEP).
For decades, reports have suggested and/or demonstrated
that SP participates in acute noncomplicated wound healing.
SP is known to cause vasodilation [8, 9], to stimulate prolifera-
tion and migration of endothelial cells [10, 11], broblasts
[12–14], and keratinocytes [15], as well as to recruit and activate immune cells, particularly playing a crucial role in wound
site inltration of polymorphonuclear leukocytes [16–19].
Besides its trophic and chemoattractant effects, SP has also
proven to be pro-angiogenic invitro and invivo [20–22]. SP
has been also recently implicated in the mobilization of endothelial progenitor cells, accelerating wound healing via
improved angiogenesis [23, 24]. Based on such properties,
and on the fact that SP is promptly released following cutaneous injury, it was predicted to improve wound healing. In fact,
SP treatment was shown to ameliorate acute wound healing in
rodents [25]. Of interest, studies have reported reduced SP
expression in skin biopsies from diabetic subjects [26].
Therefore, emerging studies are starting to explore the involvement of SP in healing of diabetic wounds—in diabetic corneal
wounds [27, 28], where epithelial cells are the major effectors,
and also in diabetic cutaneous wounds [5, 29], which involve
a complex interplay between dermal and epidermal cells, and
are characterized by a chronic inammatory and highly proteolytic environment.
Studies by our group have shown reduced gene and protein expression of SP, as well as reduced gene expression of
the main SP receptor—neurokinin-1 receptor (NK1R)—in
the unwounded skin of diabetic rabbits when compared to
their nondiabetic counterparts [30–32]. This reduced SP skin
expression was accompanied by a local chronic inammatory state, indicated by an increased baseline proinammatory cytokine expression without further increase
in response to wounding, and resulted in delayed wound closure [5].
In acute noncomplicated healing, M1-activated macrophages are predominant during the inammatory phase, as
they initiate an acute inammatory response to injury, while
during the proliferative phase M2 macrophages take over to
promote angiogenesis and granulation tissue formation [31,
33–35]. However, diabetic rabbits showed an elevated base-
line skin M1/M2 macrophage ratio that persisted until the
later stages of wound healing (10days post-wounding), suggesting a chronic pro-inammatory environment.
In agreement with our diabetic rabbit ear model ndings,
we have shown reduced SP expression and increased NEP
expression in the skin of diabetic mice (Fig. 9.1) [19].
Importantly, in diabetic human subjects, the circulating levels of SP were reduced, the SP skin gene expression of SP
was reduced, and both the skin gene and protein expression
of NEP were increased when compared to healthy control
subjects, suggesting that SP bioavailability is severely
decreased in diabetes. In addition, skin gene expression of
the SP receptor NK1R was reduced in diabetes. Similarly, to
the diabetic rabbit, the diabetic murine wound healing model
was characterized by increased pro-inammatory cytokine
expression and elevated M1/M2 macrophage ratio at baseline, lack of a robust acute inammatory response at the earlier stages of healing (day 3 post-wounding), and defective
inammation resolution at the later stages (day 10 postwounding), with failure to switch from the pro-inammatory
M1 to the pro-regenerative M2 phenotype and delayed
wound closure. Moreover, genetically modied mice decient in SP and related tachykinins (TAC1KO mice) or in the
SP receptor NK1R (NK1RKO mice) also had delayed wound
closure compared to their wild-type (WT) controls and presented chronic low-grade inammation, with elevated baseline skin expression of pro-inammatory markers and
elevated M1/M2 ratio, similar to the diabetic animals.
Of note, the presence of diabetes did not further delay
wound healing in NK1R-decient (NK1RKO) mice [19].
Furthermore, using the rabbit sham/ischemic/neuroischemic
ear model, our group has shown that diabetes impairs wound
healing in both sham and ischemic conditions, but does not
have an additional negative impact on the healing of neu-
a
c
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Fig. 9.1 Substance P (SP) skin expression is reduced, and neutral
endopeptidase (NEP) skin expression is increased in diabetic mice. (a)
Skin gene expression of SP was reduced in wild-type mice with diabetes mellitus (WT DM) compared to their nondiabetic wild-type controls
(WT). (b) Skin gene expression of NEP was decreased in WT DM
mice, as well as in neurokinin 1 receptor knockout (NK1RKO) and
knockout (TAC1KO) mice at baseline (Day 0, d0). (c) Representative
images of NEP staining in the baseline (d0) skin from WT non-DM and
WT DM mice. DM mice have a higher NEP intensity in the epidermis
(red arrows) and a higher number of NEP-positive cells in dermis
roischemic wounds. Together, these results suggest loss of
neuropeptide (in particular SP) function as an important
component of diabetes-associated impaired wound healing.
Interestingly, neuroischemic wounds, which showed the
most delayed healing, had the highest baseline skin macrophage inltration and the highest wound M1/M2 ratio at later
stages of healing (day 10 post-wounding) [31].
Importantly, we have demonstrated that topical application of SP to the wounds of both diabetic mouse dorsum and
diabetic neuroischemic rabbit ear accelerates closure and
ameliorates healing (Fig. 9.2) [19]. SP topical treatment
induced the expression of pro-inammatory cytokines, such
as MCP-1, IL-6, and KC (mouse homolog of human IL-8),
as well as increased M1 macrophages during the early stages
(inammatory phase) of healing, whereas it reduced pro-
(black arrows) compared with WT non-DM mice at day 0. Scale bar:
100μm. NEP staining intensity was increased in the (d) epidermis and
(e) dermis of WT DM mice at baseline (d0). In WT non-DM mice, NEP
intensity increased at day 3 post-wounding but returned to baseline levels by day 10, whereas in WT DM mice, it remained elevated throughout the healing process. Data represent the mean±SEM. *p<0.05.
Copyright© Elsevier. Adapted from Leal, Carvalho, Tellechea et al.,
Substance P promotes wound healing in diabetes by modulating inammation and macrophage phenotype, American Journal of Pathology
2015 Jun;185 (6):1638–48 with permission from Elsevier
inammatory cytokine expression and polarized macrophages to the M2 phenotype during the later stages, therefore
allowing inammation resolution and progression to the proliferative phase (Fig.9.3). Topical SP has also been subsequently shown to also improve wound healing in murine
models through stimulating the proliferation and migrations
of dermal broblasts and epidermal keratinocytes, substantially improving healing times in these models [36–38].
In agreement with human studies that showed increased
MMP-9 levels in the skin of diabetic subjects [39], as well as
in diabetic and other chronic wounds [40–42], MMP-9
expression was increased in both unwounded skin and
wounds of our mouse model of diabetic wound healing [19].
Of interest, TAC1KO and NK1RKO mice also presented
increased baseline skin MMP-9 expression, whereas topical
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L. Mota et al.
Fig. 9.2 Substance P (SP) topical treatment accelerates wound healing
in mouse and rabbit models of diabetes. (a) Representative images of
mouse dorsal skin wounds at baseline (day 0) and day 10 of wild-type
nondiabetic (WT), WT diabetic (WT DM), and WT diabetic SP-treated
wounds (WT DM+ SP). (b) WT DM mice showed delayed healing
compared to WT non-DM mice. Topical SP accelerated wound closure
in both WT and WT DM mice. Data represent mean±SEM.*p<0.05,
compared to WT. †p<0.05, compared to DM. (c) Representative image
of the neuroischemic rabbit ear wounds at day 0. 1: untreated; 2:
SP treatment reduced MMP-9 expression post-wounding in
diabetic mice. This suggests that loss of SP function may
contribute to chronic elevated expression of MMP-9in diabetic skin and wounds, which is associated with impaired
healing [43], and that SP treatment may attenuate it.
More recently, other researchers have studied the effect of
systemically administered SP on cutaneous wound healing
vehicle- treated; 3: SP-treated; NI: neuroischemia. (d) Topical SP
improved wound healing in a DM NI rabbit wound healing model. Data
represent mean±SEM. **p<0.01 compared to untreated and vehicletreated wounds. Copyright© Elsevier. Adapted from Leal, Carvalho,
Tellechea etal., Substance P promotes wound healing in diabetes by
modulating inammation and macrophage phenotype, American
Journal of Pathology 2015 Jun;185 (6):1638–48 with permission from
Elsevier
in diabetic mice. As expected, their results conrmed that SP
accelerates diabetic wound closure and prevents the prolonged inammatory response to injury [44, 45]. Interestingly,
the effects of systemic SP observed in the serum of the diabetic mice were similar to the ones of topical SP in the wound
tissue at the later stages of healing, namely, elevated M2
monocytes in the peripheral blood mononuclear cell popula-
ab
9 Neuropeptides, Inammation, andDiabetic Wound Healing: Lessons fromExperimental Models andHuman Subjects
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157
Fig. 9.3 SP modulates skin macrophage phenotype during healing. (a)
Representative images of M1 and M2 macrophages in wild-type nondiabetic (WT non-DM) and diabetic (WT DM) mouse skin. Scale bar:
100μm. M1 (upper panel) and M2 (lower panel) are denoted by the
yellow-orange stain resulting from triple-positive stain with CD68,
TNFα and DAPI (for M1) or CD68, CD206 (for M2), and DAPI. (b)
M1/M2 ratio was higher in DM at day 0 (d0). In non-DM mice, M1/M2
peaked at day 3 (d3) and returned to baseline levels at day 10 (d10),
while it was persistently elevated in DM mice. SP increased M1/M2 at
d3 and reduced it at d10. (c) MCP-1 skin gene expression peaked at d3
tion/elevated M2 macrophages in the wound margins, and
reduced TNF-α circulating levels/reduced TNF-α wound
expression, respectively. Additionally, it appears that SP also
has a role in regulating adiponectin and fatty acid levels,
demonstrating that it not only plays a role as an antiinammatory peptide attenuating certain diabetic
complications, but it may have a direct role in pathophysiology of insulin resistance itself (Fig.9.4) [45].
The ability of SP to induce the switch from M1 to M2
macrophages has also recently been conrmed invitro [46,
47]. Of note, SP treatment promoted macrophage M2 polar-
ization and release of pro-inammatory factors via activation
of the NF-κB pathway in a co-culture model of broblasts
and resting macrophages [46], whereas it suppressed NF-κB
activation and reduced the production of pro-inammatory
cytokines and enzymes in LPS-stimulated murine macro-
and returned to pre-wounding levels at d10in WT non-DM mice, but
not remained elevated at d10in DM mice. In both WT non-DM and DM
mice, SP treatment further increased MCP-1 at d3 and reduced it at d10.
Data represent mean±SEM. *p<0.05; †p<0.05 compared to WT
non-DM d0. Copyright© Elsevier. Adapted from Leal, Carvalho,
Tellechea etal., Substance P promotes wound healing in diabetes by
modulating inammation and macrophage phenotype, American
Journal of Pathology 2015 Jun;185 (6):1638–48 with permission from
Elsevier
phages [47], again suggesting that it may act as pro- or antiinammatory depending on the environment/experimental
conditions.
In summary, SP acts as a modulator of the inammatory
response to injury and may be particularly important in the
treatment of diabetic cutaneous wounds, where it appears to
convert the chronic low-grade inammation into an early
acute inammatory response followed by inammation resolution with progression to the proliferative phase of healing.
NEP inhibitors have been suggested as a potential treatment
for DFUs, but their serious adverse effects, such as angioedema, impede its use [48, 49]. Alternatively, local delivery of
SP via biomaterials that gradually release SP [38, 50], protecting it from rapid degradation, or topical treatment with more
resistant SP analogs, have the potential to promote wound
healing in diabetes, without major off-target effects expected.
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Free fatty acid (FFA, mEq/L)
Leptin (pg/ml)
Time of SP injection
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400
350
300
250
200
150
Blood glucose (mg/dl)
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LETO+Saline
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OLETF+Saline
OLETF+SP
OLETF+SP+At
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60
T.CHO (mg/dl)
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Time of SP injection
L. Mota et al.
LETO+Saline
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OLETF+SP
OLETF+SP+At
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1400
200
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N=10/group. Adapted from Baek SM, Kim K, Kim S, Son Y, etal.,
SP prevents T2DM complications by immunomodulation. Sci Rep.
2020;10 (1):16753. Open Access. http://creativecommons.org/licenses/
by/4.0/. Theocharidis G, Thomas BE, Sarkar D, etal. Single cell tran-
scriptomic landscape of diabetic foot ulcers. Nature Communications.
2022 Jan;13 (1):181. Need this gure from Veves, OPEN ACCESS—
http://creativecommons.org/licenses/by/4.0/
Fig. 9.4 Substance P restores impaired glucose regulation by modulating serum obese/insulin resistance-related markers. Substance P (SP)
was injected into LETO or OLETF rats from postnatal 27weeks for
4weeks; (a) results of the glucose tolerance test (GTT) in LETO and
OLETF rats at postnatal 31 weeks were evaluated. (b–f) Serum biochemical and growth factors were quantied at 2 and 4weeks post SP
injection; p-values of less than 0.05 were considered statistically signicant (*p<0.05, **p <0.01, ***p<0.001). At: NK-1R antagonist.
Neuropeptide Y andDiabetic Wound Healing
binding to its G-protein-coupled receptors—Y1, Y2, Y4,
Y5, and y6—and is widely distributed through the central
The 36-amino-acid peptide neuropeptide Y (NPY) belongs
to the pancreatic polypeptide family and is one of the most
abundant neuropeptides in mammals [4]. NPY acts by
and peripheral nervous system but can also be found in
other tissues, including the gastrointestinal tract and the
skin [51–53].
9 Neuropeptides, Inammation, andDiabetic Wound Healing: Lessons fromExperimental Models andHuman Subjects
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Most studies on NPY are related to its neuroendocrine
effects on the central nervous system, where NPY acts as a
potent orexigenic peptide regulating appetite, energy metabolism, and body weight [54]. However, there is evidence that
NPY also inuences metabolic functions in peripheral tissues. NPY was shown to suppress lipolysis and promote adipogenesis [55, 56], suggesting benecial effects on lipid
uptake and storage in adipose tissue. Accordingly, mice decient in NPY or in its Y1 receptors developed insulin resistance and adipose tissue inammation following high-fat
diet feeding [57, 58]. Another study conrmed that NPY
modulates obesity-induced inammation, as loss of NPY
expression from hematopoietic cells increased the number of
adipose tissue macrophages and NPY receptor blockade
induced dendritic cell maturation and secretion of IL-6 and
TNFα [59]. Nonetheless, in a model of combined chronic
stress and diet-induced obesity, the expression of NPY and
Y2 receptor were associated with insulin resistance and
increased numbers of adipose tissue macrophages [60, 61].
Similar to obesity, diabetes affects NPY levels, distribu-
tion, and action in a complex fashion. Whereas elevated
plasma circulating levels of NPY have been associated with
type 2 diabetes [62, 63], and NPY has also been identied as
a minor autoantigen in type 1 diabetes [64, 65], the skin
expression of this neuropeptide is reduced in both type 1 and
type 2 diabetes [66, 67].
NPY has been reported to participate in angiogenesis as well
as in inammatory and immune responses both in vitro and
invivo. Namely, NPY induces proliferation, migration, and tube
formation of endothelial cells [68]; promotes angiogenesis [69–
71]; and regulates immune responses including leukocyte traf-
cking, macrophage function, phagocytosis, cytokine release
from macrophages and helper T cells, antigen presentation and
antibody production, and activation of natural killer cells [72–
75], all of which are important in wound healing. The effects of
NPY on immune cells are complex, and, similarly to SP, NPY
can induce either pro- or anti-inammatory activities. For example, NPY stimulated macrophage function in adult mice, but
suppressed the chemotactic and phagocytosis capacity of macrophages from aged mice [76, 77]. Likewise, NPY induced
nitric oxide (NO) release from LPS-stimulated peritoneal macrophages in young rats, but not from their older counterparts
[78]. Such ndings suggest that NPY may potentiate acute
inammatory responses while protecting against inammation
in chronic inammatory conditions.
The role of NPY in healing has been mostly studied in
ligament and tendon rupture [79–81], vascular remodeling
[82–84], and ischemic tissue regeneration [85, 86] and more
recently been shown to affect migration and angiogenesis
potential of human adipose-derived stromal cells in chronic
wounds [87]. Researches have also begun to explore its
involvement in cutaneous wound healing, through focusing
on its pro-angiogenic receptors (NPY-2R and NPY-5R).
Namely, mice decient in the Y2 receptor have delayed
wound healing and reduced skin neovascularization [88].
Studies by our group have shown that, similarly to SP, the
gene and protein expression of NPY is dysregulated in the
diabetic rabbit ear model [30, 31]. In particular, baseline
NPY skin protein expression was reduced in the diabetic animals, while its gene expression was reduced post-injury. In
addition, compared to their nondiabetic counterparts, NPY
gene expression was lower in diabetic ischemic and diabetic
neuroischemic wounds [31]. Interestingly, no differences
were observed in the gene expression of receptors Y2 and
Y5, which are known for their pro-angiogenic effects. The
above ndings suggest that NPY participates in the healing
of diabetic wounds. Additionally, some studies suggest that it
may be involved in neurogenesis and also play a role in the
pathophysiology of peripheral neuropathy itself, making it a
particularly interesting target in the management of DFU
[89–91]. However, further investigation is needed to unravel
the mechanistic pathways involved.
Neurotensin andDiabetic Wound Healing
Neurotensin (NT) is a 13-amino-acid bioactive peptide primarily distributed in the central nervous system and in the
gastrointestinal tract [92, 93]. NT mediates its functions
through the binding to two G-protein-coupled receptors—
neurotensin receptor 1 (NTR1), high-afnity and most predominant, and neurotensin receptor 2 (NTR2), low-afnity
receptor—and/or to an intracellular type I receptor, neurotensin receptor 3 (NTR3) [94].
NT displays pro-inammatory properties by stimulating
vasodilation, vascular permeability, immune cell migration,
and phagocytosis [95–97]. In addition, NT was able to induce
IL-8 expression via NF-ƙB and ERK pathways in human
colonocytes [98] and has been implicated in the pathophysiology of acute colonic inammation and intestinal angiogenesis [99–101]. However, NT also demonstrates protective
effects in inammatory conditions, as shown by its ability to
modulate intestinal inammation and stimulate healing following experimentally induced colitis [102, 103]. Together,
these ndings suggest an important immunomodulatory role
for this neuropeptide. Moreover, NT was found to promote
migration of microglial cells in an invitro cerebral wound
healing model [104]. Furthermore, the proliferative effects
of NT have been shown in both normal and malignant cells.
Most NT studies have focused on the central nervous system or gastrointestinal tract, and little is known about NT
signaling in diabetes, particularly in diabetic skin and/or
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wounds. Interestingly, studies have suggested a role for NT
in the development of diabetes. Fasting plasma levels of proneurotensin were associated with increased risk of diabetes
incidence in human subjects [105] and were also elevated in
both obese and insulin-resistant subjects [106]. In addition,
increased levels of NT were observed in the pancreas of
obese (ob/ob) mice and in the intestine of both ob/ob and
diabetic (db/db) mice and correlated with insulin deciency
[107, 108]. However, other studies reported no differences in
NT levels between nondiabetic, diabetic lean, and diabetic
obese subjects or between obese type 2 diabetic mice and
their respective lean controls [109, 110].
Recent studies have demonstrated that while inducing
inammation under homeostatic conditions, NT downregulates the inammatory responses of skin dendritic cells, macrophages, T cells, and broblasts when cells are previously
exposed to pro-inammatory and/or hyperglycemic conditions [111–113]. Such ndings highlight the role of NT as an
immune and inammatory modulator, particularly through its
role in downregulating inammatory pathways such as Janus
kinase (JNK) and nuclear factor kappa beta (NF-kB). In addition, NT treatment of cells previously exposed to LPS yielded
different results than pre-treatment with NT followed by LPS
treatment [111], suggesting that timely release of endogenous
NT is crucial for proper wound healing responses. Of interest,
hyperglycemic conditions reduced the expression of endogenous NT and its cell surface receptors NTR1 and NTR2in
mouse macrophages, and this was associated with a reduction
in macrophage migratory capacity, whereas NT treatment
was able to partially reverse the hyperglycemia-induced
impaired cell migration [112]. In addition, hyperglycemia
signicantly reduced NT and NTR expression in a human
keratinocyte cell line [114]. Recently treatment of keratinocytes and T cells with NT has also been shown to enhance cell
migration and reduce expression of inammatory markers
such as TNF-a and IL-18 [115]. Together these results suggest that NT may not only work through direct effect on keratinocytes but also via paracrine and/or autocrine effects on
macrophages, dendritic cells, and broblasts.
More importantly, invivo studies have shown that when
topically applied to diabetic (and/or nondiabetic) mouse
wounds, chitosan-, collagen-, and alginate-based biomaterials and other biodegradable nanobers delivering NT, either
alone or in combination with SP, signicantly accelerate
healing [50, 116–119]. Similar to SP treatment, NT treatment induced the expression of pro-inammatory cytokines
TNF-α, IL-6, and KC (mouse homolog of human IL-8) in
day 3 wounds of diabetic mice, while reducing it at day 10
post-wounding. In addition, NT treatment reduced MMP-9
gene and protein expression in the diabetic mouse wounds at
the later stages of healing, and this was associated with an
increase in broblast migration, as well as with an increase
in the expression and deposition of collagen [116, 117].
In summary, NT improves diabetic wound healing via
suppression of the prolonged and uncontrolled inammatory
response, and subsequent induction of the proliferative phase
of healing. This seems to be achieved by regulating the
timely expression of pro-inammatory cytokines, stimulating broblast migration, and modulating extracellular matrix
(ECM) remodeling, all of which are compromised in diabetic wounds and required for proper healing.
Calcitonin Gene-Related Peptide
andDiabetic Wound Healing
Calcitonin gene-related peptide (CGRP) is a 37-amino-acid
neuropeptide widely distributed in the central and peripheral
nervous systems and also present in non-neuronal tissues.
The receptors for CGRP and related peptides are calcitonin
receptor-like receptors (CLR) linked to an essential receptor
activity-modifying protein (RAMP), which is required for
full functionality. In the peripheral nervous system, CGRP is
co-localized with SP and released from capsaicin-sensitive
peripheral afferent neurons and therefore is implicated in
pain signaling. The relationship between SP and CGRP
appears to be synergistic for vasodilation of human skin,
with the co-application having a dose-dependent relationship
with long-lasting vasodilation [120]. In addition of being one
of the most potent peripheral microvascular vasodilators,
CGRP has also been shown to have cardioprotective effects
[121–123].
Most studies involving diabetes and CGRP focus on the
cardiovascular system. Diabetes was found to reduce the
expression of CGRP and its receptors in rodents and to
reduce CGRP-mediated vasodilation in rats [124–131]. In
addition, CGRP circulating levels were reduced in human
subjects with diabetes and cardiovascular disease [132]. In
support of such ndings, CGRP gene transfer was shown to
have protective effects in a diabetic mouse model of
ischemic- reperfusion injury [133] and play a modulatory
role decreasing autophagy in mice cardiac tissue [134].
However, studies have also shown that CGRP may play an
important role in the pathophysiology of diabetes, particularly type 2. A study in human obese nondiabetic subjects
detected a modest increase in CGRP levels compared to lean
controls [135], and a similar increase was also observed in
pre-obese Zucker rats [136]. Additionally, mice decient in
αCGRP are protected against diet-induced obesity, show
improved glucose tolerance and increased insulin sensitivity
9 Neuropeptides, Inammation, andDiabetic Wound Healing: Lessons fromExperimental Models andHuman Subjects
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161
[137]. Monoclonal therapy against CGRP has also shown to
improve glucose tolerance and insulin sensitivity, reduce adiposity, and assist in weight loss, suggesting that CGRP inhibition may prove benecial in the treatment of obesity and
insulin resistance. The impact of diabetes on CGRP and the
role of CGRP in diabetes is complex and requires further
investigation. Nonetheless, it is known that CGRP has an
important role in peripheral nerve regeneration, and the loss
of CGRP-containing sensory nerves has been implicated in
the pathophysiology of diabetic peripheral neuropathy
[138–141].
CGRP is released in the skin from sensory afferents and
can also be secreted by keratinocytes and immune cells,
including monocytes/macrophages, and Langerhans cells
[6]. There is evidence that CGRP participates in wound healing, as CGRP administration increased blood ow and ap
survival in a rat skin-ap model [142], and accelerated healing in a blister model of the rat hind footpad [143], whereas
deciency in CGRP, deciency in CGRP receptors, or pharmacological antagonism of CGRP-impaired wound healing
[144–147]. In a study, vacuum-assisted treated wounds in a
diabetic mice demonstrated a signicant increase in dermal
and epidermal bers, CGRP, and nerve growth factor expression [148]. The benecial effects of CGRP in wound healing
are likely related to vasodilation, induction of VEGF release
triggering the cAMP pathway, and angiogenesis [143, 147,
149, 150]. In addition, CGRP promoted wound healing of
human bronchial epithelial cells by stimulating cell survival,
proliferation, and migration via activation of PKC and
MAPK pathways [151]. The role of CGRP in inammation
and immunity is also complex, as it may increase the ow of
immune and inammatory cells to the site of injury following vasodilation and stimulate pro-inammatory cytokine
release, or suppress pro-inammatory-mediator release via
cAMP [145]. Namely, whereas CGRP can increase the
release of pro-inammatory cytokines including IL-1, IL-8,
IL-6, and TNF-α [152–155], as well as stimulate macrophage phagocytic activity [156], it also has the ability to
inhibit lymphocyte differentiation, proliferation, and IL-2
production [157–159]; modulate Langerhans cell antigenpresenting function [160]; suppress pro-inammatory Th1;
and induce regulatory Th2 responses [161]. Interestingly,
recent studies suggest that CGRP induces a regulatory phenotype in TLR4-stimulated macrophages and inhibits T-cell
proliferation [162, 163].
Together, these ndings clearly indicate a role for CGRP
as an immunomodulator and inammation regulator.
Although the properties of CGRP suggest a potential candidate for the treatment of diabetic wounds, the knowledge on
this subject remains very limited, and new studies are needed
to test this hypothesis.
Alpha-Melanocyte-Stimulating Hormone
andDiabetic Wound Healing
Alpha-melanocyte-stimulating hormone (α-MSH) is a tridecapeptide that belongs to the melanocortin family and derives
from the melanocortin precursor pro-opiomelanocortin
(POMC), which is mainly expressed in the pituitary gland
but can be found in a variety of tissues, including signicant
amounts in the human skin [164, 165]. Five melanocortin
receptors (MC1-5R) have been identied to date and shown
to be involved in the regulation of many physiological phenomena including skin pigmentation (MC1R), cortisol production (MC2R), food intake and energy metabolism (MC3R
and MC4R), and temperature regulation (MC5R) [166–172].
Various skin cell types, including melanocytes, keratinocytes, broblasts, and endothelial cells, produce α-MSH and
express melanocortin receptors [165, 173, 174]. Like
α-MSH, the enzyme that catalyzes its degradation—prolylcarboxypeptidase (PRCP)—is expressed in the central nervous system and in a variety of peripheral tissues, including
the skin.
In diabetes, α-MSH appears to have protective effects. In
particular, it reduces weight gain, adiposity, and hepatic fat
accumulation while stimulating muscle glucose uptake and
increasing energy expenditure in mouse models of obesity
[175, 176], as well as protects retinal vascular endothelial
cells against oxidative stress and apoptosis in a rat model of
diabetes [177]. Of interest, studies have shown reduced
expression of POMC in the hypothalamus and pituitary of
streptozotocin-induced diabetic rats [178, 179], increased
plasma levels of the enzyme PRCP in obese and/or diabetic
human subjects [180], and even suggested a role for α-MSH
deciency in the development of type 2 diabetes [181].
α-MSH is also known for its protective effects in inammatory conditions, including colitis, brain and pulmonary
inammation, transplantation, and skin inammatory diseases such as urticaria and psoriasis [182–192]. Its antiinammatory properties are vast and target multiple cells
such as lymphocytes, monocytes and macrophages, mast
cells, endothelial cells, broblasts, and keratinocytes, where
α-MSH inhibits the NF-ƙB pathway. Namely, α-MSH sup-
presses proliferation of stimulated lymphocytes and modulates its activity, inducing a regulatory phenotype [193, 194].
In addition, α-MSH inhibits monocyte adhesion to the vascular endothelium and reduces TNF-α release from LPSstimulated human monocytes in culture, while increasing
IL-10in both human peripheral blood monocytes and cultured monocytes [195–197]. Of note, such effects were
achieved with low doses (range of 10
also attenuates the expression of IFN-γ and nitic oxide in
LPS-stimulated murine macrophages [198, 199] and reduces
−10
to 10
−17
M). α-MSH
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