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Table 9.1 Summary of neuropeptide effects on angiogenesis and inammation, as well as on nondiabetic and diabetic cutaneous wound
healing
Neuropeptide effects Angiogenesis Inammation
Substance P (SP) Promotes angiogenesis
Neuropeptide Y (NPY) Promotes angiogenesis
Neurotensin (NT) Promotes angiogenesis
Calcitonin gene-related peptide (CGRP) Promotes angiogenesis
Alpha-melanocyte-stimulating hormone (α-MSH)
Somatostatin Inhibits angiogenesis [211] Suppresses inammation
Adrenomedullin Promotes angiogenesis
Secretoneurin Increase angiogenesis [216] Suppresses inammation
[20–22]
[68, 69]
[100, 101]
[143, 147, 149, 150]
Inhibits angiogenesis
[204]
[213]
Modulates inammation
[16–19, 44, 46, 47]
Modulates inammation
[72–78]
Modulates inammation
[95–99, 102, 103, 111–113]
Modulates inammation
[145, 152–162]
Suppresses inammation
[182–203]
[212]
Suppresses inammation
[214]
[219]
Nondiabetic skin
wound healing
Improves healing
[19, 25]
Needs investigation Needs investigation
Improves healing
[116]
Improves healing
[142, 143, 147]
Improves healing
[207]
Delays wound
healing [210]
Improves healing
[213]
Improves healing
[217]
Diabetic skin wound
healing
Improves healing
[19, 29, 44]
Improves healing
[116, 117]
Needs investigation
Needs investigation
Needs investigation
Needs investigation
Improves healing [216,
218]
L. Mota et al.
histamine, IL-1β, and TNF-α release from IgE-stimulated
bone marrow-derived mouse mast cells [200]. In human dermal broblasts and endothelial cells, it regulates the expression of IL-8 [201, 202] and in human keratinocytes increases
expression of anti-inammatory IL-10 [203].
A study has suggested that α-MSH inhibits angiogenesis
[204], which is crucial for proper wound healing. However,
α-MSH restored H2O2-induced inhibition of wound restitution in a rat intestinal epithelial cell scrape wound model,
suggesting a potential role in healing involving epithelial
cells [205]. In fact, topical application of the C-terminal tripeptide (KPV) sequence of α-MSH to a rabbit corneal wound
model conrmed positive healing outcomes [206]. More
importantly, a recent study showed that pre-treatment with
intraperitoneal α-MSH ameliorates cutaneous wound healing in adult mice [207]. This was accompanied by a reduction in the number of leucocytes and mast cells at days 3 and
7 post-wounding, and by a reduction of scar area as well as
improvement of dermal architecture at days 40 and 60 postwounding [207]. Additionally, α-MSH has also been demonstrated to signicantly inhibit Staphylococcus aureus colony
formation and decrease viability of Candida albicans, two
common organisms present in diabetic foot infections [208,
209]. Despite its apparent anti-angiogenic effect, the bene-
cial effects of α-MSH in acute noncomplicated wound healing, its protective effects against inammation and diabetes,
and its antimicrobial effects make it a promising candidate
for the treatment of diabetic wounds.
Recent studies also suggest a role of other neuropeptides
in cutaneous wound healing. Somatostatin has been shown to
inhibit keratinocyte and proliferation, delaying wound healing [210–212]. Topically delivered adrenomedullin has been
shown to accelerate granulation tissue formation and enhance
neovascularization, improving wound closure in a mouse
pressure ulcer model [213–215], while topical secretoneurin
has been shown to increase angiogenesis and accelerate
wound healing in a diabetic mice model [216–219].
The abovementioned studies indicate that neuropeptides
play an important role in wound healing, mostly by promoting angiogenesis and modulating the immune and inammatory responses to injury. Since diabetes reduces the
cutaneous expression of neuropeptides, impairs wound neovascularization, and causes wounds to become stalled in the
inammatory phase, these neuropeptides have great potential to ameliorate the healing of diabetic wounds. While the
benecial effects of some of these neuropeptides, such as SP
and NT, has been conrmed in experimental models of diabetic wound healing, others still require investigation
(Table9.1).
Inammation andDiabetic Wound Healing
Inammation is essential in the wound healing process, but
in order to achieve proper healing, the inammatory response
must be tightly regulated in time, space, and magnitude. In
9 Neuropeptides, Inammation, andDiabetic Wound Healing: Lessons fromExperimental Models andHuman Subjects
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physiologic conditions, skin injury causes a rapid onset of
acute and self-resolving inammation, with timely recruitment of immune cells from circulation—neutrophils and
monocyte-derived macrophages—as well as controlled activation of tissue-resident immune cells, such as mast cells
(MCs), T cells, and Langerhans cells [220–224]. Besides
controlling and ghting infection, acting as phagocytic
agents, and/or as antigen-presenting cells, immune cells also
release a cocktail of cytokines, chemokines, and growth factors, essential for broblast and endothelial cell proliferation
and migration, ECM production, granulation tissue formation, and angiogenesis [221, 225–230]. Therefore, this acute
inammatory cell inux is crucial for the progression to the
proliferative phase of healing, and pathological conditions
that interfere with this self-limited physiological process can
result in a chronic nonhealing wound.
Systemic and local chronic low-grade inammation,
altered extracellular matrix deposition, and impaired wound
neovascularization, as a result of an imbalanced secretion of
cytokines, matrix metalloproteinases (MMPs), and growth
factors, have been implicated in the pathophysiology of
DFUs [39, 231]. Diabetes is characterized by sustained
hyperglycemia and chronic elevation of pro-inammatory
mediators, leading to a chronic low-grade inammation,
with impaired cellular defense mechanisms that fail to mount
an acute response to injury. This delays the formation of
mature granulation tissue and reduces the wound tensile
strength [232]. Due to the chronic pro-inammatory environment, there is an imbalance between wound MMPs and
their inhibitors, which also contributes to poor formation of
new connective tissue [41, 233–235]. In addition, analysis of
the uid of diabetic wounds from both animal models and
human subjects has shown insulin-degrading activity, which
in turn has been correlated with the levels of hemoglobin
A1c (HbA1c) [236], suggesting a straight relationship
between sustained hyperglycemia and the wound proteolytic
environment. Moreover, macrophage efferocytosis is
impaired in diabetic wounds, resulting in increased apoptotic
burden and imbalanced inammatory status with higher levels of pro-inammatory cytokines and lower levels of antiinammatory mediators [237, 238]. As previously discussed,
macrophage polarization also plays a key role in diabetic
wounds. Through single-cell RNA sequencing of normal
lower extremity skin and DFUs, it has been demonstrated
that DFU have signicantly more M1 type macrophages,
which can be broadly described as pro-inammatory, compared to M2 macrophages, which play a larger role in the
regenerative phase of wound healing (Fig.9.5) [239]. The
importance of macrophages in wound healing has been further demonstrated in another study examining the impact of
alginate dressings containing murine macrophages on wound
healing [240]. At 16days following injury, this study demonstrated that wounds treated with macrophage (M1 or M2)
containing alginate dressings had a signicant improvement
in wound closure compared to control dressings, with M2
macrophages displaying the best effect in wound closure
(Fig.9.6). Neutrophils also show reduced chemotactic and
phagocytic activities, rendering the wounds more prone to
infection [241, 242]. In fact, diabetic patients have over 50%
higher risk of wound infection and are also more likely to
develop biolms compared to nondiabetic subjects [243–
246]. Naturally, infection and biolm formation further hin-
der the healing process.
In summary, contrasting with normal wound healing,
where inammation occurs in a sequential, regulated and
self-resolving manner, in diabetic wounds, the immune and
inammatory responses are prolonged and noneffective. As a
consequence, diabetic wounds become stalled in a chronic
inammatory state and fail to progress to the proliferative
and reparative phases of healing. Further understanding of
this process could help identify and develop new therapeutic
strategies.
As emphasized earlier in this chapter, whereas neuropeptides play an important role in wound healing, namely, by
regulating the inammatory response to injury, their skin
expression is reduced in diabetes, and therefore exogenous
application of neuropeptides may be a benecial strategy in
the treatment of diabetic wounds. However, due to the highly
proteolytic environment of the diabetic wound, neuropeptide
delivery should ensure protection against rapid inactivation.
For example, biomaterials such as alginate-, chitosan-, and
collagen-based materials serve as vehicles for sustained
delivery of neuropeptides to the wounds and prevent rapid
neuropeptide degradation at the wound site [50, 116, 117].
An alternative strategy in the treatment of diabetic wounds is
to directly target immune cells to (i) prevent increased baseline skin inammation, (ii) promote the acute inammatory
response, and/or (iii) contribute to proper resolution of the
inammatory phase and progression to the proliferative
phase.
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a
b
c
d
e
f
ghi
Fig. 9.5 Comparative analysis of transcriptome proles of foot samples in
the different clinical groups, elucidating differences in cell type composition, gene expression, and biological pathways. (a) UMAP dimensionality
reduction embedding of foot cells from DFU-Healers, DFU-Non-healers,
Healthy subjects, and non-DFU DM patients. The cellular clusters depicting signicant enrichment in the healers are marked with blue asterisks.
Dotted lines mark cell groups of similar lineages. Comparative analysis
depicted b HE-Fibro, c M1 macrophages, and d SMC2 cellular enrichment
in the foot sample from DFU healers. Data represent the mean and SEM
values from n=9 Healthy, n=6 Diabetic, n=7 Healer, and n= 4 Nonhealer subjects. Two-sided Welch’s t-test was used; p=0.013 for Healthy
vs Healers, p=0.007 for Diabetes vs Healers and p=0.006 for Healers vs
Non-healers in (b); p=0.026 for Healthy vs Healers, p=0.017 for Diabetes
vs Healers and p=0.042 for Healers vs Non-healers in (c); p=0.005 for
Healthy vs Healers, p=0.002 for Diabetes vs Healers and p= 0.02 for
Healers vs Non- healers in (d). (e) Stacked bar plots showing the proportions of different cell types across the different clinical groups (green:
Healthy subjects, orange: DFU-Healers, red: DFU-Non-healers, purple:
non-DFU DM patients). (f) Venn diagram analysis to compare genes that
are differentially expressed between M1 and M2 macrophages and between
Healers vs. Non-healers. The comparison identied 195 genes that are differentially expressed in M1 macrophages from DFU-Healers. Volcano plot
showing the genes that are signicantly differentially expressed (red dots)
in M1 macrophages of Healers (Benjamini–Hochberg corrected P-value
<0.00001, FC>1). (g) Selected biological pathways that are signicantly
(P value <0.01) affected in the healing associated M1 macrophages. Each
bar represents a pathway with signicance of enrichment determined using
the one-tail Fisher’s exact t-test (−log10 P value is shown on primary
x-axis). The directionality of each pathway is depicted using a pseudo color
(red for activated, blue for inhibited). Regulators that are signicantly activated (h) and inhibited (i) in the M1 macrophages from Healers. The activation and inhibition of pathways was measured based on Z-score
calculation using the IPA platform. Adapted from Theocharidis G,
Thomas BE, Sarkar D, etal. Single cell transcriptomic landscape of diabetic foot ulcers. Nature Communications. 2022 Jan;13(1):181. Open
Access: http://creativecommons.org/licenses/by/4.0/
cd
Wounds Bandages
Wound closure
M2
9 Neuropeptides, Inammation, andDiabetic Wound Healing: Lessons fromExperimental Models andHuman Subjects
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165
a
Day 0: Wound Creation
& Bandage Placement
b
Ctrl M0 M1 M2a M2c
e
Ctrl M0
g
g
Ctrl M0
i
CD11b / YM1/DAP
Day 3: Bandage Replacement
& Wound Measurement
D0
D16
Bandages left undisturbed from Day 3 to Day 16
Ctrl M0
M1
M1
M1 M2a
M2a
M2a
Immunohistochemistry (IHC
Immunofluorescence (IF)
Day 16 : Wound Measurements
& Sample Collection
M2c
M2c
M2c
100
80
60
40
20
0
% Open wound from Day 0
2
2
2
Ctrl
f
800
600
400
200
RFP + cells / mm
0
h
800
600
400
200
F4/80+ cells / mm
j
500
400
300
200
100
Cd11b/Ym1+ cells / mm
M1
M0
Ctrl
M0
0
Ctrl
M0
0
M0
Ctrl
M2c
M2a
M1
M2c
M2a
M1
M2c
M2a
a
M1
M2c
Fig. 9.6 Application of macrophage-loaded bandages to diabetic
mouse wounds enhanced healing after 16days. (a) Experimental design
for wound healing experiment: two 6-mm biopsy punch wounds were
inicted on the back of 16-week old male db/db mice and control (Ctrl)
or macrophage-loaded bandages were placed onto the wounds.
Treatment groups included M0, M1, M2a and M2c polarized macrophages. The bandages were changed on day 3 post-wounding and
wounds were collected for analyses on day 16. (b) Representative
image of db/db mouse with bandage covered dorsal wounds (right,
zoomed in). (c) Representative macroscopic views of wounds on day 0
(D0) and day 16 (D16) for the different treatment groups. (d)
Quantication of wound closure on D16 expressed as % of open wound
compared to D0, n=10–12 wounds. (e) Representative immunouorescence staining for RFP+cells (red) in D16 wounds. Nuclei are stained
with DAPI (blue). (f) Quantication of RFP+cells, n=6–11 wounds.
(g) Representative images from D16 wounds of immunohistochemical
staining for macrophage marker F4/80. (h) Quantication of F4/80+
cells, n=10–12 wounds. (i) Representative immunouorescence staining of D16 wound sections for pan-macrophage marker CD11b (green)
and M2-associated marker YM1 (red). Nuclei are stained with DAPI
(blue). (j) Quantication of CD11b and YM1 double positive cells,
n = 10–12 wounds. Data are represented as mean ± s.d., *P <0.05;
**P < 0.01; ***P < 0.001, one-way ANOVA with Tukey’s post-hoc
test. Scale bars represent 10 mmx2009; (b), 2mm (c), 100μm (e, g, i).
Insets on (e, g, i) are 4X zoom. Copyright© Elsevier. Adapted from
Theocharidis G, Rahmani S, Lee S, Li Z, Lobao A, Kounas K, et al.
Murine macrophages or their secretome delivered in alginate dressings
enhance impaired wound healing in diabetic mice. Biomaterials 2022
Sep; 288:121692 with permission from Elsevier
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Mast Cells andDiabetic Wound Healing
Mast cells (MCs) are immune cells that originate from hematopoietic pluripotent stem cells in the bone marrow [247–
250]. Committed MC progenitors are released into the
bloodstream and subsequently home to virtually every organ
in the body, where they differentiate and mature under the
inuence of tissue-specic growth factors and cytokines,
giving rise to distinct phenotypes in different tissues [248,
249, 251, 252]. Mature MCs are more abundant in tissues
interfacing with the external environment, including the
skin, the respiratory, and gastrointestinal tracts, as well as in
proximity with blood vessels [253–255].
A particular feature of mature MCs, especially those
residing in the skin and other connective tissues, is that their
cytoplasm is lled with numerous granules, where preformed
mediators are stored [256]. MC activation results in rapid
degranulation (5–30min) [257] with exocytosis of various
preformed mediators, including biogenic amines: mostly
histamine and serotonin; enzymes: beta-hexosaminidase,
tryptase, and chymase; proteoglycans: serglycin proteoglycan (SGPG), heparin, chondroitin sulfate, and hyaluronic
acid; and the preformed cytokine TNF-α [258, 259]. MC
activation also induces de novo synthesis and delayed release
(12–24h later) of various cytokines and chemokines [258,
260], including interferon-α (IFN-α), TNF-α, several inter-
leukins (IL-1β, IL-3, IL-4, IL-5, IL-6, IL-8, IL-13), and chemokine MCP-1, as well as growth factors such as stem cell
factor (SCF), granulocyte-macrophage colony-stimulating
factor (GM-CSF), nerve growth factor (NGF), and VEGF
[258, 261]. MCs are often thought to act as rst responders
to metabolic and immunologic changes [262]. Of note, some
of these biological mediators can be secreted to the extracellular environment through selective or differential release, a
process that occurs independently from degranulation [263].
MCs are mostly known as the effector cells in immunoglobulin E (IgE)-mediated allergic responses [264–267].
However, they also participate in several other physiological
and pathophysiological processes, and there is growing
interest in the role of MCs in nonallergic immune and
inammatory responses, including cutaneous wound healing
[254, 268–277]. In the skin, MCs are abundant and strategically located in the vicinity of blood vessels and sensory
nerves [278]. In fact, there is a bidirectional communication
between MCs and primary sensory neurons, as neuropeptides such as substance P (SP) [279] and neurotensin (NT)
[280] stimulate/activate MCs, and MC mediators in turn
regulate neuropeptide release [281]. Unlike the IgE-mediated
process, neuropeptide-induced MC activation occurs through
different G-protein-coupled receptors [282, 283], and also
via receptor-independent mechanisms by direct or indirect
activation of G proteins [284–286]. Interestingly, neuropeptides such as corticotropin-releasing hormone (CRH) and SP
can cause selective release of VEGF from MCs without
degranulation [287, 288].
Skin MC activation occurs soon after tissue injury, but the
exact mechanisms are not fully understood [289, 290].
Pathogens, LPS, other pathogen products, and cytokines
[254, 291], as well as pain signals, such as SP [292] and
mechanical stress [293], may be involved. MCs have been
implicated in all phases of wound healing [261, 276, 290,
293–296]. More specically, MCs induce vascular permea-
bility and participate in the brin clot formation but also prevent excessive clotting, as they secrete tryptase-heparin
complexes that degrade the excess brinogen [297, 298]. In
addition, MCs contribute to inammation by recruiting neutrophils to the wound site [223, 299–301], as well as releasing chemokines, cytokines, histamine, and other mediators
that activate tissue-resident macrophages [302]. Moreover,
MCs promote the proliferative phase of wound healing as
they stimulate proliferation and migration of several skin cell
types, namely, broblasts, endothelial cells, and keratinocytes [299, 303–305]; induce vascular growth and angiogenesis [306–309]; and participate in ECM remodeling [294].
Finally, MCs participate in the maturation phase of healing
by stimulating wound contraction [310–316] and scarring
[317–319]. The role of MCs in scar formation has been suggested in a study where MC-decient (Kit
w/w-v
) mice fetuses
had signicant less scar formation when compared to their
wild-type (Kit
+/+
) counterparts [296].
While most in vivo studies have reported an important
role for MCs in normal wound healing [293, 294, 299, 301,
306], other studies failed to conrm it [320–322]. Although
the reasons for such discrepancy are not clear, the use of different mouse models or different wound healing models may
play a role. Given the heterogeneity of mature MCs, it is possible that some models of MC deciency do not lack the full
spectrum of MCs, whereas others may have additional
underlying defects. On the other hand, the use of splinted
wounds [321] may explain the contradictory results observed.
Another study that employed splinting has shown robust
abnormalities in important parameters of wound healing,
9 Neuropeptides, Inammation, andDiabetic Wound Healing: Lessons fromExperimental Models andHuman Subjects
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167
including cell proliferation, angiogenesis, tissue granulation,
and collagen maturation, despite a lack of difference in
wound closure [293]. Moreover, other authors have raised
concerns that splinting may alter the wound healing phenotype due to mechanical changes [323].
Despite the large number of reports on mast cells and normal wound healing, few studies have investigated the role of
MCs in diabetic wound healing. Interestingly, however, MCs
have been implicated in insulin-induced lipoatrophy [324],
obesity, and type 2 diabetes [325, 326], namely, through IL6
and IFN-γ release. Additionally, elevated plasma levels of
MC proteases and MC degranulation activator IgE have been
suggested as inammatory markers and risk factors of human
prediabetes and diabetes [327]. Other studies have suggested
a role for MCs in the pathophysiology of type 1 diabetes.
More specically, MCs have been implicated in both the
development of type 1 diabetes in the spontaneously diabetic
BioBreeding rat [328] and the immune-mediated beta cell
alterations that occur in human type 1 diabetes [329]. While
CD8+ and macrophages have been described as the most
prominent inltrating type cells in islets devoid of insulinproducing ß cells, MCs are also thought to play a role in
activating T cells via antigen presentation, as well as stimulating T-cell migration into these sites via chemokine production [330, 331]. However, MCs can also have protective
effects against streptozotocin-induced diabetes in mice,
namely, by increasing the pool of regulatory T cells and
decreasing IL-17 producing T cells in pancreatic lymph
nodes [332], whereas MC deciency has been shown to
worsen type 1 diabetes and its complications [331, 332].
Additionally, conicting ndings have also been reported
regarding the role of MC in obesity/type 2 diabetes. Some
studies have suggested that the absence of MC may not affect
inammatory markers, metabolic dysregulation, weight
gain, or insulin resistance [333, 334].
A recent study used streptozotocin-induced diabetic
mice and nondiabetic mice to create excisional wounds and
evaluate the healing process with focus on MC numbers
[335]. Despite not having found signicant differences in
wound closure between the diabetic and nondiabetic
groups, perhaps due to the short duration of diabetes
(4weeks), neovascularization in the proliferative phase and
vascular regression in the remodeling phase were impaired
in the diabetic mice. Of interest, MC accumulation in the
diabetic wounds was delayed when compared to their nondiabetic counterparts [335].
Previous studies have reported increased MC degranulation in unwounded forearm and foot skin from diabetic
human subjects, which was associated with increased local
and systemic inammation (Fig. 9.7) [336]. Similarly,
increased MC degranulation was also observed in the dorsal
skin from streptozotocin-induced diabetic mice, when compared to their nondiabetic controls. In addition, the study
shows that whereas nondiabetic mouse skin MCs undergo
considerable degranulation after injury, an observation previously reported by others [289, 290, 293, 301], in diabetic
mice MC degranulation does not further increase postwounding (Fig.9.8). This failure to induce MC degranulation post-wounding is consistent with the inability to mount
an acute inammatory response to injury observed in diabetic animals [19, 31] and may contribute to poor healing.
More importantly, treatment with the MC degranulation
inhibitor disodium cromoglycate (DSCG) prior to wounding
was able to signicantly accelerate wound closure in diabetic
mice, achieving an outcome comparable to that of nondiabetic mice (Fig.9.9). The observed improvement in wound
healing was associated with M2 macrophage polarization,
with stimulation of wound neovascularization, as well as
with an elevation in VEGF (known to be reduced in diabetic
wounds), and a reduction in MMP-9 (elevated in chronic
wounds) local expression at day 10 post-wounding [336].
Additionally, topical application of mast cell stabilizer was
also able to demonstrate comparable accelerated wound
healing as to that achieved using a systemic mast cell stabilizer [337]. Such ndings suggest that blocking pre- wounding
MC degranulation ameliorates diabetic wound healing by
suppressing the chronic inammation observed in diabetes
and promoting angiogenesis.
Of interest, whereas topical treatment with SP accelerated
wound healing in both nondiabetic and diabetic mice, conrming previous ndings [19], it did not affect wound closure in MC-decient mice [336], suggesting that the
benecial effects of SP in wound healing are at least partly
mediated by MCs.
In summary, the results emerging from this study suggest
that strategies to prevent/inhibit chronic MC degranulation
may ameliorate wound healing in diabetes. In light of the
recent controversy regarding the role of MCs in normal, noncomplicated wound repair, further studies evaluating MC
function in diabetic wound healing are of major importance
to test the validity of the above mentioned ndings and to
further examine the underlying mechanisms.
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a
de
Total MC
Degranulated MC Degranulated MC Degranulated MC
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L. Mota et al.
Non-DM
DM
Forearm skinFoot skin
Non-DM
DM
bc
30
20
10
(number/biopsy)
fgh
0
Non-DM
200
150
100
50
0
Dermis Inflammatory Cells
Forearm skin
0
100
80
60
40
20
Degranulated MC
r=0.27,
p<0.05
0
DM
20
40
Fig. 9.7 Skin mast cell (MC) degranulation is increased in patients with
diabetes and is associated with inammation. (a) Representative images
of toluidine blue-stained MCs in the forearm skin (top panel) and of tryptase-immunostained MCs in foot skin specimens (bottom panel) from
subjects without (non-DM) and with diabetes mellitus (DM). Scale bar:
10μm. Black arrows show non-degranulated MCs, and red arrows show
degranulated MCs. Degranulated MCs were in proximity with inammatory cells (blue arrows). The (b) total number and (c) percentage of
degranulated MCs stained with toluidine blue were increased in forearm
Forearm skin
Non-DM
160
120
80
IL-6
60
DM
40
0
0
20
Degranulated MC
(%)
100
50
0
40
skin specimens from patients with diabetes. (d) MC degranulation was
also increased in foot skin specimens from subjects with diabetes stained
with tryptase, while (e) the total number of MCs was not different.
*P<0.05, **P <0.01, and ****P<0.0001. A positive correlation was
observed between degranulated MCs and (f) the dermis inammatory
cells and the serum levels of (g) IL-6 and (h) TNF-α. Copyright©
American Diabetes Association. Reprinted from Tellechea et al., Mast
Cells Regulate Wound Healing in Diabetes, Diabetes 2016 Jul; 65
(7):2006–2019, with permission from The American Diabetes Association
DM
Non-DM
r=0.34,
p<0.01
Foot skin
60
Non-DM
Foot skin
r=0.31,
P<0.02
40
DM
60
30
20
10
Total MC (number/hpf)
DM
25
20
15
TMFa
10
5
0
0
0
20
9 Neuropeptides, Inammation, andDiabetic Wound Healing: Lessons fromExperimental Models andHuman Subjects
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a
169
bcd
efg
Fig. 9.8 Mast cell (MC) degranulation is increased in the unwounded skin
of diabetic (DM) mice and fails to further increase after wounding. (a)
Representative images of nondegranulated (black arrows) and degranulated
(red arrows) MCs in day 0 skin biopsy specimens from WT C57BL/6J
nondiabetic and diabetic (DM) mice, untreated and pre- treated with the
MC degranulation inhibitor DSCG.Scale bar: 100μm. (b) MC degranulation was increased in diabetic mice at day 0 and DSCG pre-treatment
reduced it. (c) Nondiabetic mice showed increased MC degranulation at
day 10 when compared with day 0, but there were no changes in DM mice
between days 0 and 10. As a result, there were no differences among the
various groups at day 10. (d) When the difference between days 10 and 0
was calculated, a signicant increase was noticed in nondiabetic mice, irrespective of treatment. No difference was observed in the DM mice, but this
difference was restored in the DSCG-treated diabetic mice. (e–g) Similar
results were observed in a different set of nondiabetic and diabetic mice that
were studied at day 3 post-wounding. Thus, (e) MC degranulation was also
increased in the skin in diabetic mice on day 0, (f) but was not different at
day 3, resulting in (g) failure to increase MC degranulation from day 0 to
day 3 post-wounding in diabetic mice. *P< 0.05. Copyright© American
Diabetes Association. Reprinted from Tellechea etal., Mast Cells Regulate
Wound Healing in Diabetes, Diabetes 2016 Jul; 65 (7):2006–2019, with
permission from The American Diabetes Association
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L. Mota et al.
Fig. 9.9 Blocking pre-wounding mast cell (MC) degranulation with
DSCG accelerates wound closure in diabetic (DM) mice. (a) Wound healing progress over a 10-day period in wild-type (WT) C57BL6 nondiabetic
and diabetic (DM) mice, non-treated and DSCG pre-treated. Wound healing was delayed in DM mice compared and DSCG pre- treatment accelerated it from days 6 to 10 post-wounding. DSCG had no effect on
In Vivo Models ofDiabetic Wound Healing:
Focus onNeuropeptides andMast Cells
nondiabetic mouse wound healing. (b) At day 10 post- wounding, pretreatment with DSCG in diabetic mice achieved similar wound closure to
nondiabetic mice. Data represent mean±SEM. *p<0.05. Copyright©
American Diabetes Association. Adapted from Tellechea etal., Mast Cells
Regulate Wound Healing in Diabetes, Diabetes 2016 Jul; 65 (7):2006–
2019, with permission from The American Diabetes Association
from patients with DFUs, we created a model where the
response of inammatory cells to proposed treatment can be
better evaluated (Fig.9.10) [339]. Although promising, fur-
The previous sections of this chapter highlighted the role of
neuropeptides and mast cells in wound healing. Both invitro
systems and animal models are useful tools to evaluate their
potential as therapeutic targets for diabetic wound healing,
as they help us understand their mechanisms of actions and
test their efcacy.
Wound repair is a highly complex and dynamic process,
encompassing a series of coordinated and overlapping phenomena, which involve multiple cell types with autocrine
and paracrine effects, and are affected by the extracellular
environment. Therefore, it cannot be recapitulated by simple
single-cell assays. In an effort to overcome some of the limitations of such assays, invitro organotypic models have been
fabricated, including a 3D full-thickness skin equivalent that
is composed of not only epidermis and dermis but also a
layer of hypodermis containing blood vessels, nerves, and
broblasts, which provide support to the epidermis and dermis [338]. Whereas such models enable exploration of the
crosstalk between different cell types—mostly broblasts
and keratinocytes—they still lack the complexity of in vivo
models, namely, the immune and inammatory components
of wound healing. With these limitations in mind, we have
also explored the creation of 3D disease models of the human
skin that mimics the diabetic tissue microenvironment.
Incorporating monocytes and primary broblast isolated
ther studies are still needed to assess the models ability to
incorporate other cell types that play a role in DFUs that are
not currently included. Ex vivo skin explants have also been
developed and optimized to study wound healing processes
[340–342]. But once again they fail to fully recreate the
wound environment present in their invivo counterparts and
cannot identify potential off-target or systemic effects of the
therapeutics tested.
Rodents are the most widely used in vivo models of diabetic wound healing, due to their economic feasibility, easy
manipulation, and relatively short reproduction times.
Another advantage of rodents, particularly mice, is the availability of genetically modied models, which allow studying
the role of a particular cell type or molecule. Mice and rats
can be rendered diabetic via streptozotocin (STZ), or, less
commonly, alloxan monohydrate, drugs that destroy the beta
cells in the pancreas and cause type 1 diabetes. The leptin
receptor-decient (db/db) mouse is another commonly used
model for diabetic wound healing [343, 344]. Db/db mice
spontaneously develop obesity and subsequently type 2 diabetes at 4–6weeks of age, with hyperinsulinemia and hyperlipidemia. It is important that chronic diabetes and its
complications, namely, diabetic peripheral neuropathy—the
most common complication of diabetes affecting approximately 50% of the patients [345–347] and a major risk factor
ab
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ef
9 Neuropeptides, Inammation, andDiabetic Wound Healing: Lessons fromExperimental Models andHuman Subjects
https://t.me/med1917
171
Fig. 9.10 Development of an HSE tissue. (a) An acellular collagen
layer is placed at the bottom of a 24-mm transwell membrane. (b)
Fibroblasts with or without macrophages are mixed with bovine type 1
collagen and seeded on top. (c) The broblasts remodel and contract the
collagen over the course of 1week. (d) Neonatal human keratinocytes
are seeded on top of the collagen matrix, where (e) they proliferate and
begin differentiation. (f) The tissue is brought to an air-liquid interface
for DFU in humans [1, 348]—have been established prior to
creating cutaneous wounds, especially when studying neuropeptides. The minimum duration of diabetes depends on the
animal model and the features of neuropathy to be studied. In
the case of the STZ-induced diabetic rat, nerve conduction
velocity slowing and impaired sensory responses occur as
early as 2–4weeks post-STZ treatment, in the STZ-diabetic
to catalyze epithelial cornication. HSE, human skin equivalent.
Adapted from Smith A, Watkins T, Theocharidis G, Lang I, etal. A
Novel Three-Dimensional Skin Disease Model to Assess Macrophage
Function in Diabetes. Tissue Eng Part C Methods. 2021 Feb;27 (2):49–
58. Mary Ann Liebert, Inc. publishers does not require authors of the
content being used to obtain a license for their personal reuse of full
article, gures/tables or text excerpt
mice such changes occur within 2–8 weeks of diabetes,
whereas in the db/db mice, they start at 4–8weeks of diabetes
and progress over time [349]. Nonobese diabetic (NOD) mice
and Akita mice, two genetically modied mouse models of
type 1 diabetes, have also been used in wound healing studies
[350–353]; however, there is inconsistent information about
the neuropathy status in these models [349]. Despite being
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