ORIGINAL ARTICLE

Proinsulin C-Peptide Prevents Impaired Wound

Healing by Activating Angiogenesis in Diabetes
Young-Cheol Lim1, Mahendra Prasad Bhatt1, Mi-Hye Kwon1, Donghyun Park1, SungHun Na2,
Young-Myeong Kim1 and Kwon-Soo Ha1
Diabetes mellitus disrupts wound repair and leads to the development of chronic wounds, likely due to impaired
angiogenesis. We previously demonstrated that human proinsulin C-peptide can protect against vasculopathy in
diabetes; however, its role in impaired wound healing in diabetes has not been studied. We investigated the
potential roles of C-peptide in protecting against impaired wound healing by inducing angiogenesis using
streptozotocin-induced diabetic mice and human umbilical vein endothelial cells. Diabetes delayed wound
healing in mouse skin, and C-peptide supplement using osmotic pumps significantly increased the rate of skin
wound closure in diabetic mice. Furthermore, C-peptide induced endothelial cell migration and tube formation
in dose-dependent manners, with maximal effect at 0.5 nM. These effects were mediated through activation of
extracellular signalregulated kinase 1/2 and Akt, as well as nitric oxide formation. C-peptide-enhanced
angiogenesis in vivo was demonstrated by immunohistochemistry and Matrigel plug assays. Our findings
highlight an angiogenic role of C-peptide and its ability to protect against impaired wound healing, which may
have significant implications in reparative and therapeutic angiogenesis in diabetes. Thus, C-peptide replacement
is a promising therapy for impaired angiogenesis and delayed wound healing in diabetes.
Journal of Investigative Dermatology (2015) 135, 269278; doi:10.1038/jid.2014.285; published online 7 August 2014

INTRODUCTION
Human proinsulin C-peptide is secreted into circulation in
equimolar concentrations with insulin by pancreatic b-cells
(Ido et al., 1997; Wahren et al., 2007; Hills et al., 2010;
Wahren et al., 2012). C-peptide prevents diabetic complications, including neuropathy, nephropathy, vascular dysfunctions, and inflammation, in animal models of diabetes and
in type 1 diabetes mellitus (DM) patients (Ido et al., 1997;
Wahren et al., 2007; Hills et al., 2010; Wahren et al., 2012).
In diabetes, increased intracellular reactive oxygen species
levels are recognized as the most common cause of diabetic
vascular complications (Giacco and Brownlee, 2010). We
previously demonstrated that C-peptide can ameliorate endothelial dysfunction through inhibiting intracellular reactive
oxygen species-mediated apoptosis of endothelial cells

Department of Molecular and Cellular Biochemistry and Institute of Medical

Science, Kangwon National University School of Medicine, Chuncheon,
Kangwon-do, Korea and 2Department of Obstetrics and Gynecology, Kangwon
National University School of Medicine, Chuncheon, Kangwon-do, Korea
Correspondence: Kwon-Soo Ha, Department of Molecular and Cellular
Biochemistry and Institute of Medical Science, Kangwon National University
School of Medicine, Chuncheon, Kangwon-do 200-701, Korea.
E-mail: ksha@kangwon.ac.kr
Abbreviations: DM, diabetes mellitus; ERK1/2, extracellular signalregulated
kinase 1/2; HUVEC, human umbilical vein endothelial cell; L-NAME, L-NGnitroarginine methyl ester; NO, nitric oxide; PECAM-1, platelet endothelial cell
adhesion molecule-1; VEGF, vascular endothelial growth factor
Received 30 October 2013; revised 5 June 2014; accepted 13 June 2014;
accepted article preview online 9 July 2014; published online 7 August 2014

& 2015 The Society for Investigative Dermatology

by activating AMP-activated protein kinase a (Bhatt et al.,

2013a,b). Recently, we also demonstrated the protective role
of C-peptide against diabetic retinopathy by amelioration
of retinal microvascular leakage (Lim et al., 2013). The multifaceted effects of C-peptide in diabetic animals and in
patients are attributed to the activation of multiple cell
signaling pathways, involving p38 mitogen-activated protein
kinase, extracellular signalregulated kinase 1/2 (ERK1/2), Akt,
and endothelial nitric oxide (NO) production (Kitamura et al.,
2002; Wallerath et al., 2003; Zhong et al., 2005). However,
the mechanism(s) by which C-peptide prevents and/or
ameliorates diabetic complications is not fully understood.
C-peptide deficiency is a typical feature of type 1 DM and
the later stages of type 2 DM (Wahren et al., 2007; MassiBenedetti and Orsini-Federici, 2008). DM is one of the major
contributors of impaired wound healing; it leads to apoptosis
and abnormal angiogenesis (Hansen et al., 2003; Graves et al.,
2007). Impaired wound healing can result in diabetic
complications, including chronic open wounds, infections,
and ulcers, and even amputation (Ferringer and Miller, 2002;
Jeffcoate and Harding, 2003; Hirsch et al., 2008). Wound
healing is a dynamic and interactive process involving
angiogenesis, coagulation, inflammation, tissue formation,
and tissue remodeling controlled by growth factors (Li et al.,
2007; Monroe and Hoffman, 2012; Newman and Hughes,
2012). Angiogenesis, the formation of new blood vessels from
preexisting vessels, is a crucial process for wound healing (Sun
et al., 2011). Therefore, understanding the mechanism(s)
involved in the prevention of impaired wound healing is
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C-Peptide and Wound Healing in Diabetes

necessary to ameliorate diabetic complications (Eming et al.,

2007; Kolluru et al., 2012). C-peptide treatment has been
implicated in the prevention of diabetes complications. However, the beneficial role of C-peptide in preventing against
impaired wound healing and angiogenesis due to diabetes is
not clear.
In the present study, we aimed to elucidate the physiological functions of C-peptide with regard to impaired wound
healing in diabetes. We hypothesized that C-peptide might
exert a beneficial effect on impaired wound healing by
stimulating angiogenesis and inhibiting inflammation. We
investigated the role of C-peptide in therapeutic angiogenesis
and excisional skin wound repair using streptozotocininduced diabetic mice. We further examined the potential
role of C-peptide and its underlying mechanisms in the
process of vascular angiogenesis through the activation of
ERK1/2, Akt, and NO production using human umbilical
vein endothelial cells (HUVECs) in vitro. We assessed neovascularization using the Matrigel plug assay and immunohistochemistry. In addition, we demonstrated that C-peptide
normalizes increased inflammatory cytokine levels and
infiltrated inflammatory cells in diabetic mice.
RESULTS
C-peptide prevents impaired wound healing in diabetic mice

To investigate whether C-peptide could normalize impaired

wound healing in diabetic mice, C-peptide was continuously
supplemented using subcutaneously implanted osmotic
pumps in streptozotocin-induced diabetic mice. Wounds of
4-mm diameter were created on the dorsal surface of the
hindlimbs of normal, diabetic, and C-peptidesupplemented
diabetic mice. Diabetic mice exhibited a significantly slower
healing rate compared with normal mice (Figure 1a). However, C-peptide supplement accelerated wound closure in
diabetic mice. This effect was quantitatively analyzed by
measuring wound diameter (Figure 1b). Wound closure
was much slower in diabetic mice compared with normal
mice (n 10, Po0.001); however, C-peptide supplementation
attenuated this effect (n 10, Po0.01). To further study the
C-peptidemediated stimulation of wound closure, we
analyzed hematoxylin and eosin-stained transverse sections
of the wound beds (Supplementary Figure S1 online). As
suggested by macroscopic observation, wound closure was
dramatically stimulated by C-peptide at 10 days (Figure 1c).
Similar results were obtained by measuring length and area
of hyper-proliferative epithelium at 6 days (Figure 1d,e). Our
results indicate that C-peptide prevents against impaired
wound healing including wound closure in diabetic mice.
C-peptide activation of endothelial cell migration, proliferation,
and tube formation

Because angiogenesis is a crucial process for wound healing

(Sun et al., 2011), we investigated whether C-peptide could
activate endothelial cell migration, proliferation, and tube
formation. C-peptide treatment induced a dose-dependent
increase in endothelial cell chemotactic migration, with a
maximal effect at 0.5 nM (Figure 2a and b). However, heatinactivated C-peptide had no effect on endothelial cell
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Journal of Investigative Dermatology (2015), Volume 135

migration (data not shown). C-peptideinduced cell migration

was further investigated with wound-healing assays. C-peptide
(0.5 nM) significantly stimulated cell migration to the wounded
area (Po0.01, Figure 2c and d), with cell proliferation demonstrated by increase in cell viability (Po0.05, Figure 2e).
As reported previously (Caldwell et al., 2003; Lamalice
et al., 2007), vascular endothelial growth factor (VEGF) also
stimulated chemotactic migration, wound healing, and proliferation in endothelial cells (Figures 1 and 2).
Consistent with the cell migration assay results, C-peptide
stimulated tube formation in a dose-dependent manner, with
maximal effect at 0.5 nM (Figure 3a and b). VEGF induced
similar tube formation (Figure 3c). Thus, it was confirmed
that C-peptide activates endothelial cell migration, proliferation, and tube formation, which are essential for in vitro
angiogenesis.
C-peptide stimulation of angiogenesis through pathways
involving ERK1/2, PI3K/Akt, and NO production

To understand the mechanism by which C-peptide activates

angiogenesis, we investigated the effect of C-peptide on
upstream pathways of angiogenesis, including ERK1/2 and
Akt phosphorylation and NO production. C-peptide increased
ERK1/2 phosphorylation in a time-dependent manner, with
maximal stimulation at 15 minutes, and this ERK1/2 activation was maintained until 60 minutes (Figure 4a). Similarly,
C-peptide induced time-dependent Akt phosphorylation, with
maximal effect at 15 minutes (Figure 4b). VEGF also induced
ERK1/2 and Akt phosphorylation in a time-dependent manner
(Figure 4a and b). Because Akt phosphorylation increases NO
production via VEGF, which is an important angiogenesismediating factor (Dimmeler et al., 1999), we investigated
whether C-peptide could stimulate NO production (Figure 4c).
Treatment of HUVECs with C-peptide significantly elevated
intracellular NO (Po0.05). A higher level of intracellular
NO was produced with VEGF treatment (Po0.01). These
results suggest that signaling pathways involving ERK1/2, Akt,
and NO production might be involved in C-peptideinduced
angiogenesis.
The roles of ERK1/2, PI3K/Akt, and NO in C-peptide
induced angiogenesis were examined using PD98059, wortmannin, and L-NG-nitroarginine methyl ester (L-NAME),
respectively (Figure 3d). C-peptidestimulated tube formation
of HUVECs was inhibited by PD98059, the inhibitor of
ERK1/2, in a dose-dependent manner, with maximal inhibition at 10 mM. The PI3K/Akt inhibitor wortmannin and the
NO synthase inhibitor L-NAME also dose dependently inhibited C-peptideinduced tube formation. As expected, VEGFinduced tube formation was prevented by treatment
with PD98059, wortmannin, or L-NAME (Figure 3e). Our
results indicate that C-peptide stimulates angiogenesis via
signaling pathways involving ERK1/2, PI3K/Akt, and NO
production.
C-peptide activation of blood vessel formation in diabetic mice

To investigate the effect of C-peptide on blood vessel formation in the wound, the inner surface of dorsal skins from
normal, diabetic, and C-peptidesupplemented diabetic mice

Y-C Lim et al.

C-Peptide and Wound Healing in Diabetes

2d

6d

12 d

10 d

20 d

Diabetes

Diabetes
+C-pep

Normal

0d

100

**
**

80

**
**

60

**

40
20

Normal mice
Diabetic
Diabetic+C-peptide

**

**
4
3
2
1
0

0
0

8
6
Day

10

12

6d

14

10 d

Endpoint

35,000

**

**

5,000
4,000
3,000
2,000
1,000

Area of hyper-proliferative
epithelium (m2)

6,000
Length of hyper-proliferative
epithelium (m)

Normal mice
Diabetic
Diabetic+C-peptide

Wound diameter (mm)

Wound area by
macroscopic imaging (%)

120

**

30,000

**

25,000
20,000
15,000
10,000
5,000
0

0
Normal

Diabetic Diabetic
+CP

Normal

Diabetic Diabetic
+CP

Figure 1. C-peptide prevention against impaired wound healing in diabetic mice. (a, b) Wounds of 4-mm diameter were created on the dorsal skin of the
hindlimbs of normal (n 10), diabetic (n 10), and C-peptidesupplemented diabetic mice (Diabetic C-peptide; n 10), and wound closure was
photographically monitored for 14 days. (a) Representative macroscopic images in each group. (b) Wound area was determined by measuring the diameter of
open wounds. (ce) Hematoxylin and eosin (H&E)-stained transverse sections of the wounds (n 6) were observed at the indicated time using an inverted
microscope. Endpoints are 10, 20, and 12 days for normal, diabetic, and C-peptidesupplemented diabetic mice, respectively. (c) Wound diameter was calculated
using the distance of open wounds. (d, e) Length and area of hyper-proliferative epithelium were measured histologically from the images (6 days). **Po0.01.

was photographed. Blood vessels were obvious in the subcutaneous tissue surrounding the wounds in normal mice but
not in the wound of diabetic mice (Figure 5a). Blood vessel
formation was apparent in C-peptidesupplemented diabetic
mice.
C-peptideinduced blood vessel formation was also investigated by confocal microscopy of the inner surface of the
dorsal skins from three groups of mice after platelet endothelial cell adhesion molecule-1 (PECAM-1) staining of blood
vessels (Figure 5b). PECAM-1 immunohistochemistry revealed

increased blood vessel formation in normal and C-peptide

supplemented diabetic mice compared with diabetic mice
(Figure 5b and c), indicating that C-peptide activated blood
vessel formation in the skin of diabetic mice. C-peptide
induced formation of blood vessels was supported by the
Matrigel plug assay. One week after implantation in C57BL/6
mice, Matrigel containing C-peptide exhibited significantly
greater angiogenesis compared with control, and this result
was similar to that observed following VEGF treatment
(Figure 5d).
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C-pep (0.1)

C-pep (0.5)

C-pep (1.0)

500

200

**

400
300

**

200

**

**

*
100
0

0.
1

O
N
VE
nM GF
C
0.
-p
2
n M ep
C
0.
-p
5
n M ep
C
-p
1
n M ep
C
-p
ep

C-pep (0.2)

VEGF

Number of migrated cells per field

CON

Number of migrated cells per field

C-Peptide and Wound Healing in Diabetes

CON

CON

VEGF

0h

C-pep

24 h

**

150
125
100
75
50
25
0
CON

200

Cell viability (%)

**

175

150

VEGF

C-pep

100

50

0
CON

VEGF

C-pep

Figure 2. C-peptide activation of endothelial cell migration and proliferation. (a, b) Transwell migration assays; human umbilical vein endothelial cells (HUVECs)
were incubated in the presence of 10 ng ml  1 vascular endothelial growth factor (VEGF) or the indicated concentrations of C-peptide (C-pep). Migrated cells
on the lower surface of the filters were imaged (a) and counted (n 3) (b). Bar 20 mm. (c, d) Wound-healing assays; confluent cell layers were wounded
and incubated with 10 ng ml  1 VEGF or 0.5 nM C-pep. Images were obtained by confocal microscopy (c), and cells that migrated to the scratched area were
counted (d) (n 3). Bar 300 mm. (e) Cell viability assay; HUVECs were incubated for 24 hours in the presence of 10 ng ml  1 VEGF or 0.5 nM C-pep (n 3).
*Po0.05, **Po0.01; for VEGF or C-pep versus control.

C-peptide activation of blood vessel formation was further

confirmed by confocal microscopy of transverse sections of
wound beds after immunohistochemistry with anti-PECAM-1
and a-smooth muscle actin antibodies (Figure 6a). The number
of blood vessels was significantly lower in diabetic mice
compared with normal mice (Po0.01), and this effect was
normalized in C-peptide supplementation (Figure 6a and b),
indicating that C-peptide activates neovascularization in the
wound bed of diabetic mice. Thus, C-peptide normalizes
impaired wound healing by activating angiogenesis in diabetic
mice.
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Journal of Investigative Dermatology (2015), Volume 135

C-peptide inhibition of inflammation in diabetic mice

To investigate C-peptide effect on inflammation in the

wounds, we performed immunohistochemistry on transverse
sections of wound beds from normal (10 days), diabetic (20
days), and C-peptidesupplemented diabetic mice (12 days)
and determined the levels of inflammatory cytokines by
confocal microscopy. Diabetic mice demonstrated significantly higher levels of IL-1b, IL-6, and tumor necrosis factora (TNF-a) compared with normal mice, which were normalized by C-peptide supplementation (Po0.01; Figure 6c),
indicating that C-peptide inhibits inflammation in diabetic

Y-C Lim et al.

C-Peptide and Wound Healing in Diabetes

CON

0.1 nM

0.2 nM

0.5 nM

1 nM

2 nM

250
Relative tube length (%)

Relative tube length (%)

250

200

150

100

C-pep

50

CON

250

*
**

**

**

100
50

Relative tube length (%)

Relative tube length (%)

150

VEGF

100

0.5
1.0
1.5
2.0
C-peptide concentration (nM)

250
200

**

150

0
0.0

**

200

200
150

**

**

**

100
50

0
C-pep + + + + + + + + + +

0
VEGF

PD98059 2.5 5 10

PD98059

Wortmannin 25 50100

Wortmannin

L-NAME 0.5 1 2

L-NAME

Figure 3. C-peptide and vascular endothelial growth factor (VEGF) stimulation of tube formation and its inhibition by various inhibitors. (ac) C-peptide
stimulation of tube formation. (a) Tube formation was imaged by confocal microscopy. Bar 300 mm. (b) Dose-dependent tube formation induced by C-peptide.
(c) Tube formation induced by 10 ng ml  1 VEGF or 0.5 ng ml  1 C-peptide. **Po0.01; VEGF or C-peptide versus control (CON). (d) Human umbilical vein
endothelial cells (HUVECs) were incubated for 2430 hours with 0.5 nM C-peptide in the presence of the indicated concentrations of PD98058 (mM), wortmannin
(nM), or L-NG-nitroarginine methyl ester (L-NAME) (mM). (e) HUVECs were incubated with 10 ng ml  1 VEGF in the presence of 10 mM PD98058, 100 nM
wortmannin, or 2 mM L-NAME. Results are represented as meanSD of three independent experiments. *Po0.05, **Po0.01; for inhibitors versus C-peptide
or VEGF.

mice. In addition, C-peptide reversed the increased number of

infiltrated inflammatory cells (CD11b- and Ly6c-positive cells)
in diabetic mice (Po0.05; Figure 6d). Taken together, our data
indicate that C-peptide normalizes impaired wound healing
by activating angiogenesis as well as inhibiting inflammation
in diabetic mice.
DISCUSSION
C-peptide has emerged as a physiologically active peptide for
ameliorating diabetes-induced complications including vascular dysfunction and inflammation (Ido et al., 1997; Wahren
et al., 2007; Hills et al., 2010; Wahren et al., 2012; Lim et al.,
2013; Bhatt et al., 2013a,b). In this study, we focused on the

potential angiogenic role of C-peptide and its ability to

ameliorate impaired wound healing in diabetes (Figures 5
and 6). Impaired wound healing in diabetes is a serious
complication that leads to systemic infection, ulceration, and
amputation (Werner and Grose, 2003; Nabuurs-Franssen
et al., 2005; Barrientos et al., 2008). Although hyperglycemia alone can cause alterations in body homeostasis,
the deficiency or absence of circulating insulin and C-peptide
may contribute to the development and progression of
hyperglycemia-induced complications.
Experiments with wound animal models have demonstrated
that secretion of growth factors and reductions in inflammatory and oxidative responses are underlying mechanisms of
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C-Peptide and Wound Healing in Diabetes

C-peptide (minutes)
CON 5

VEGF (minutes)

10 15 30 60

15 30

p-ERK1/2
t-ERK1/2

ERK phosphorylation (fold)

6
5
4
3
2
1
0
CON 5

C-peptide (minutes)
CON 5

10 15 30 60

C-peptide
(minutes)

VEGF
(minutes)

VEGF (minutes)

10 15 30 60

15

15 30

CON

VEGF

C-pep

30
p-Akt
t-Akt
3.0
Relative NO level (fold)

AKT phosphorylation (fold)

5
4
3
2
1
0

**

2.5

2.0
1.5
1.0
0.5
0.0

CON 5

10 15 30 60

15 30

CON

VEGF

C-pep

C-peptide (minutes) VEGF (minutes)

Figure 4. C-peptide activation of extracellular signalregulated kinase 1/2 (ERK1/2) and Akt phosphorylation and nitric oxide (NO) production in human
umbilical vein endothelial cells (HUVECs). (a, b) HUVECs were treated with 10 ng ml  1 vascular endothelial growth factor (VEGF) or 0.5 nM C-peptide for
the indicated times. Phosphorylation of (a) ERK1/2 and (b) Akt was determined by western blot analyses. The levels of protein phosphorylation were normalized
using the total amount of the proteins and expressed relative to unstimulated control levels (CON). (c) HUVECs were treated with 10 ng ml  1 VEGF or
0.5 nM C-peptide for 2 hours, and levels of intracellular NO were determined by confocal microscopy as described in the Methods. Bar 100 mm. Results are
expressed as meanSD of three independent experiments. *Po0.05, **Po0.01; for VEGF or C-peptide versus control.

angiogenic factor treatment (Werner and Grose, 2003).

Granulation tissue formation and enhanced re-epithelialization are associated with increased angiogenesis and an
increase in fibroblast number (Fadini et al., 2010). Our
results demonstrate the preventive role of C-peptide against
impaired wound healing through stimulating angiogenesis and
inhibiting inflammation in streptozotocin-induced diabetic
mice. There is a report that C-peptide had no effect on
diabetic wound healing; however, this controversy might be
caused by using non-physiological C-peptide concentration
(over 10 nM in plasma) (Langer et al., 2002). C-peptide
replacement therapy has been investigated in animal models
of diabetic neuropathy and nephropathy but not in diabetesimpaired wound healing. We systemically administered
C-peptide to diabetic mice to investigate the protective role
of C-peptide against diabetes-impaired wound healing. The
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Journal of Investigative Dermatology (2015), Volume 135

most important finding of the present study is that C-peptide

treated diabetic mice showed significantly improved wound
healing.
We further investigated the underlying mechanisms of
C-peptide protection in the process of vascular angiogenesis
through the activation of ERK1/2, Akt, and NO production;
this effect was demonstrated in parallel with VEGF-induced
angiogenesis. ERK1/2 has a distinct role in tube formation by
endothelial cells and tube structure maintenance during
angiogenesis, whereas NO stimulates endothelial cell proliferation and migration (Yang et al., 2004). We demonstrated
increases in endothelial cell proliferation, migration, and tube
formation following C-peptide treatment, with a maximum
effect observed within its physiological concentration (0.5 nM);
these effects were similar to those observed following VEGF
treatment. In parallel with VEGF, C-peptide also stimulated

Y-C Lim et al.

C-Peptide and Wound Healing in Diabetes

10
8

**

6
4
2

d)
(1

ep

(2
tic

(1
al

-p

be

ia

be

tic

ia

or
N

d)

0
0

Transmitted

12

+C

Diabetic + C-pep
(12 d)

Diabetic + C-pep
(12 d)

d)

Diabetic
(20 d)

PECAM-1

Normal
(10 d)

Diabetic
(20 d)

Total vessel length per field (mm)

Normal
(10 d)

CON

VEGF

C-pep

Figure 5. C-peptide activation of blood vessel formation in diabetic mice. (a, b) Dorsal skin tissues were excised from the hindlimbs of normal (n 6),
diabetic (n 6), and C-peptidesupplemented diabetic (Diabetic C-peptide, n 6) mice at 10, 20, and 12 days after wounding, respectively, and were
immediately subjected to immunohistochemistry with a platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody. The inner surface of the dorsal
skin from each group of mice was photographed (a), and blood vessels were observed by confocal microscopy (b). Arrows indicate vessels. Bar 200 mm.
(c) Total vessel length (mm) per field was determined using the images in b. **Po0.01. (d) In vivo the Matrigel plug assay; mice were injected subcutaneously
for 7 days with 0.5 ml Matrigel containing vehicle (CON), 100 ng ml  1 vascular endothelial growth factor (VEGF), or 5 nM C-peptide (n 8 per group).

ERK1/2 and Akt phosphorylation and NO production in

endothelial cells. Blocking the ERK1/2, PI3K/Akt, or endothelial NO species pathway using specific inhibitors suppressed in vitro tube formation induced by both C-peptide and
VEGF. Moreover, the in vivo Matrigel plug assay results
demonstrated a significant increase in angiogenesis upon
C-peptide treatment, similar to that induced by VEGF
treatment. Thus, C-peptide has significant VEGF-mimetic
angiogenic potency both in vitro and in vivo.
We investigated whether C-peptidemediated acceleration
of wound repair was mediated via its effect on wound site.
C-peptide supplementation normalized increased levels of
inflammatory cytokines and CD11b- and Ly6C-positive cells

in the wound bed of diabetic mice. The normal woundhealing process is initiated by migration of keratinocytes and
fibroblasts. In diabetes, increased inflammatory cytokines,
such as IL-1b, IL-6, and TNF-a, induce impaired wound
healing by suppressing keratinocyte and fibroblast migration
(Xu et al., 2013). C-peptide induced migration of NIH3T3 cells
in vitro (Supplementary Figure S2 online), suggesting that
C-peptide can directly activate fibroblasts for accelerating
wound repair. However, C-peptide did not activate the HaCaT
cell migration (data not shown), indicating that C-peptide may
indirectly reverse diabetes-induced suppression of keratinocyte migration through inhibiting inflammatory cytokines.
PECAM-1 immunohistochemistry demonstrated new blood
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C-Peptide and Wound Healing in Diabetes

-SMA

PECAM-1

Merged

16

##

12
8

**

d)
tic

ep

(2

(1

0
(1
al
ia

+C

-p

be

m
or

ia

be

tic

Diabetes+
C-pep (12 d)

Normal (10 d)
Diabetic (20 d)
Diabetic+C-pep (12 d)

**

5
4

**

**

##

##

##

50
Inflammation cells per field

CD11b
Ly6C

**

40
30

20
#
10

d)
2

0
(2
D

ia

+C

-p

be

ep

tic

(1
al
m
or

ia

be

(1

TNF-

tic

IL-6

IL-1

d)

d)

Cytokine expression (fold)

d)

0
d)

Diabetes
(20 d)

Normal
(10 d)

Microvascular density per field

20

Figure 6. C-peptide activation of neovascularization and inhibition of inflammation in the wound beds. Wound beds were excised from the hindlimbs at 10 days
(normal, n 6), 20 days (Diabetic, n 6), and 12 days (Diabetic C-peptide, n 6) after wounding. Immunohistochemistry was performed using platelet
endothelial cell adhesion molecule-1 (PECAM-1), a-smooth muscle actin (a-SMA), 4,6-diamidino-2-phenylindole (DAPI), CD11b, Ly6C, IL-1b, IL-6, and
tumor necrosis factor-a (TNF-a) antibodies, and stained sections were observed using confocal microscopy. (a) Confocal microscopy of PECAM-1- and
a-SMA-positive blood vessels (arrows) with DAPI staining. Bar 50 mm. (b) Microvascular density per field was determined by measuring the number of
blood vessels from the images in a. (c) Levels of inflammatory cytokines were determined by confocal microscopy. (d) Inflammatory cells were determined by
counting CD11b- and Ly6C-positive cells. *Po0.05, **Po0.01; for diabetic versus normal. #Po0.05, ##Po0.01; for diabetic C-peptide versus diabetic.

vessel formation in the wounded dorsal skin. Blood vessels

from intact vessels and loops of new capillaries gave the
matrix a red granular appearance. Thus, C-peptide has a
beneficial role in preventing diabetes-impaired wound healing
via its effect on anti-inflammation and neovascularization.
In conclusion, our study reveals an angiogenic role of
C-peptide exerted via pathways involving ERK1/2 and Akt
phosphorylation and NO production. C-peptide supplement
therapy improved diabetes-impaired wound healing by
inhibiting inflammation and stimulating angiogenesis. Thus,
C-peptide replacement is a promising therapeutic strategy for
diabetes-impaired wound healing.
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Journal of Investigative Dermatology (2015), Volume 135

MATERIALS AND METHODS

Animals
Six-week-old male C57BL/6 mice were obtained from KOSTECH
(Pyeongtaek, Korea). Mice were maintained in temperature-controlled
clean racks with a 12-hour-light/dark cycle. All experiments
were performed in accordance with the guidelines of the Institutional
Animal Care and Use Ethics Committee of Kangwon National
University.

Diabetic mouse model

Diabetic mice were generated by a single intraperitoneal injection of
streptozotocin (150 mg per kg body weight) as previously described

Y-C Lim et al.

C-Peptide and Wound Healing in Diabetes

(Bhatt et al., 2013a). After 1 week, mice with non-fasting blood

glucose levels greater than 16 mM, polyuria, and glucosuria were
defined as diabetic. Two weeks after the streptozotocin injection, one
group of diabetic mice was subcutaneously implanted with ALZET
mini-osmotic pumps 2004 (DIRECT, Cupertino, CA) containing
C-peptide in phosphate-buffered saline with a delivery rate of
35 pmol kg  1 per minute for 2 weeks. The other diabetic and
normal groups underwent sham operations.

Skin excision wound and quantitative assessment of wound

healing
The dorsal skin of the vertebral column was shaved with Veet cream
before creating a wound. Full-thickness skin wounds, 4 mm in
diameter, were created on the dorsal surface of the hindlimbs with
a biopsy punch, and closure was monitored with a digital camera.
Open-wound size was measured by tracing the wound margin using
a vernier caliper every other day.

Cell culture
HUVECs were isolated from the human umbilical cord vein according to the Declaration of Helsinki as previously described (Park et al.,
2009). Cells were grown at 37 1C in a humidified 5% CO2 incubator
in M199 culture media supplemented with 20% fetal bovine
serum, 3 ng ml  1 basic fibroblast growth factor, 5 U ml  1 heparin,
100 U ml  1 penicillin, and 100 mg ml  1 streptomycin. For experiments, cells were incubated for 6 hours in low-serum medium (M199
supplemented as above), but with only 1% fetal bovine serum. Cell
viability was accessed by the Cell Counting Kit-8 (Enzo Life Sciences,
Farmingdale, NY).

Transwell migration assay

manufacturers instructions. HUVECs were seeded onto a layer of

Matrigel at a density of 4  105 cells per well and treated with VEGF
or C-peptide at 37 1C for 2430 hours. The degree of tube formation
was quantified by measuring tube length from the images using
FV-300 software (Olympus).

Western blot analyses

HUVECs were scraped off with ice-cold lysis buffer (50 mM Tris-HCl,
pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, 10 mg ml  1 aprotinin, and 10 mg ml  1
leupeptin) and centrifuged at 18,000g for 10 minutes at 4 1C. The
resulting cell lysates were separated by SDS-PAGE and transferred to
polyvinylidene fluoride membranes. Protein bands were visualized
using a chemiluminescent substrate (Pierce, Rockford, IL).

Measurement of intracellular NO production

Intracellular NO levels were measured using diaminofluorescein-FM
diacetate (Molecular Probes, Eugene, OR) as previously described
(Namkoong et al., 2009). Briefly, HUVECs were treated with VEGF or
C-peptide in phenol red-free low-serum media for the indicated
times and incubated with 2 mM diaminofluorescein-FM diacetate for
the final 60 minutes. Intracellular NO levels were determined by
comparing the fluorescence intensities of treated cells with those of
untreated control cells.

Whole-mount immunostaining
Skin tissues were excised using scissors and were immediately fixed
overnight with 4% paraformaldehyde in phosphate-buffered saline.
For immunohistochemistry, tissue samples were blocked with 2%
BSA in tris-buffered saline containing 0.1% Triton X-100 and
incubated with goat-polyclonal PECAM-1 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) for 2 days, followed by probing with
Alexa 546-conjugated rabbit anti-goat IgG overnight and observed
under the confocal microscope (FV-300). The length of blood vessel
was determined using FV-300 software (Olympus).

Migration assays were performed as previously described (Lee et al.,

2006). Briefly, HUVEC chemotactic motility was assayed using
Transwell Permeable Supports (Costar, Corning, NY). The lower
surface of the filters was coated with 5 ml 2% gelatin. Low-serum
media containing VEGF or C-peptide were placed in the lower wells.
HUVECs were seeded in the upper wells in a volume of 200 ml lowserum media containing 1  105 cells per well and incubated at 37 1C
for 12 hours. Cells were fixed with 100% methanol for 15 minutes
and then stained with hematoxylin and eosin. The average number of
migrated cells in three randomly chosen fields per insert was taken
to quantify the extent of migration using a fluorescence inverted
microscope (Olympus, Tokyo, Japan).

In vivo Matrigel plug assays were performed as previously described

(Min et al., 2007). Briefly, mice were injected subcutaneously with
0.5 ml Matrigel containing 100 ng ml  1 VEGF or 5 nM C-peptide and
10 U heparin. After 7 days, the Matrigel plugs were removed from the
subcutaneous region and photographed with a camera (Sony, Tokyo,
Japan).

Wound-healing assay

Histology and immunohistochemistry

Confluent cell layers were starved with low-serum media for 6 hours,
stained with 1 mM calcein-AM for 30 minutes, and wounded with
a plastic scraper. Cell debris was removed and replaced with 2 ml
low-serum media containing VEGF or C-peptide. Cells were then
incubated at 37 1C for 24 hours, and the number of migrated cells
was counted from images obtained using a confocal microscope
(FV-300, Olympus).

Full-skin thickness samples of wound tissue from mice were excised

using scissors, fixed immediately in 4% paraformaldehyde in phosphate-buffered saline for overnight, and then embedded in paraffin.
In total, 8-mm paraffin skin sections were cut (Leica, Nussloch,
Germany). Hematoxylin and eosin staining was performed in Harris
hematoxylin solution (Sigma, St Louis, MO) for 5 minutes, followed
by 0.5% eosin (Sigma) for 2 minutes. The wound diameter and hyperproliferative epithelium length and area were determined using
FV-300 software (Olympus, Tokyo, Japan).
Immunohistochemistry was performed using goat-polyclonal
PECAM-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA),
rabbit-polyclonal a-smooth muscle actin and CD11b antibodies
(Santa Cruz Biotechnology), rat-polyclonal Ly6C (BD Biosciences,

Tube formation assay

The formation of capillary-like networks by HUVECs on growth
factorreduced Matrigel (BD Biosciences, Franklin Lakes, NJ) was
achieved as previously described (Namkoong et al., 2009). Briefly,
24-well culture plates were coated with Matrigel according to the

In vivo Matrigel plug assay

www.jidonline.org

277

Y-C Lim et al.

C-Peptide and Wound Healing in Diabetes

Franklin Lakes, NJ), IL-1b, IL-6, and TNF-a antibodies (Abcam,

Cambridge, MA), followed by probing with Alexa 546-conjugated
rabbit anti-goat IgG, FITC-conjugated goat anti-rabbit IgG, or FITCconjugated goat anti-rat IgG for 2 hours, and stained samples were
observed using confocal microscopy (FV-300). The number of blood
vessel and inflammatory cells was counted and the level of inflammatory cytokines was determined from the images.

Statistical analysis
Data processing was performed using Origin 6.1 (OriginLab,
Northampton, MA). Statistical significance was determined using
Students t-tests and analysis of variance. A P-value less than 0.05
was considered statistically significant.
CONFLICT OF INTEREST
There is a PCT patent application with a filing date of 23 July 2013 and
international application number PCT/KR2013/006571. We confirm that this
does not alter our adherence to the Journal of Investigative Dermatology
policies on sharing the data and materials of the manuscript.

ACKNOWLEDGMENTS
This work was supported in part by grants from the Korea Research Foundation
of Korea (2013-008193) and by the Ministry of Health and Welfare through the
National R&D Program for Cancer Control (1020420).
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at http://
www.nature.com/jid

REFERENCES

Ido Y, Vindigni A, Chang K et al. (1997) Prevention of vascular and neural

dysfunction in diabetic rats by C-peptide. Science 277:5636
Jeffcoate WJ, Harding KG (2003) Diabetic foot ulcers. Lancet 361:154551
Kitamura T, Kimura K, Jung BD et al. (2002) Proinsulin C-peptide activates
cAMP response element-binding proteins through the p38 mitogenactivated protein kinase pathway in mouse lung capillary endothelial
cells. Biochem J 366:73744
Kolluru GK, Bir SC, Kevil CG (2012) Endothelial dysfunction and diabetes:
effects on angiogenesis, vascular remodeling, and wound healing. Int J
Vasc Med 2012:918267
Lamalice L, Le Boeuf F, Huot J (2007) Endothelial cell migration during
angiogenesis. Circ Res 100:78294
Langer S, Born F, Breidenbach A et al. (2002) Effect of C-peptide on wound
healing and microcirculation in diabetic mice. Eur J Med Res 7:5028
Lee SJ, Namkoong S, Kim YM et al. (2006) Fractalkine stimulates angiogenesis
by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal
pathways. Am J Physiol Heart Circ Physiol 291:H283646
Li J, Chen J, Kirsner R (2007) Pathophysiology of acute wound healing.
Clin Dermatol 25:918
Lim YC, Bhatt MP, Kwon MH et al. (2013) Prevention of VEGF-mediated
microvascular permeability by C-peptide in diabetic mice. Cardiovasc Res
101:15564
Massi-Benedetti M, Orsini-Federici M (2008) Treatment of type 2 diabetes with
combined therapy: what are the pros and cons? Diabetes Care 31(Suppl
2):S1315
Min JK, Cho YL, Choi JH et al. (2007) Receptor activator of nuclear factor (NF)kappaB ligand (RANKL) increases vascular permeability: impaired permeability and angiogenesis in eNOS-deficient mice. Blood 109:1495502
Monroe DM, Hoffman M (2012) The clotting systema major player in wound
healing. Haemophilia 18(Suppl 5):116

Barrientos S, Stojadinovic O, Golinko MS et al. (2008) Growth factors and

cytokines in wound healing. Wound Repair Regen 16:585601

Nabuurs-Franssen MH, Huijberts MS, Nieuwenhuijzen Kruseman AC et al.

(2005) Health-related quality of life of diabetic foot ulcer patients and
their caregivers. Diabetologia 48:190610

Bhatt MP, Lim YC, Hwang J et al. (2013a) C-peptide prevents hyperglycemiainduced endothelial apoptosis through inhibition of reactive oxygen
species-mediated transglutaminase 2 activation. Diabetes 62:24353

Namkoong S, Kim CK, Cho YL et al. (2009) Forskolin increases angiogenesis

through the coordinated cross-talk of PKA-dependent VEGF expression
and Epac-mediated PI3K/Akt/eNOS signaling. Cell Signal 21:90615

Bhatt MP, Lim YC, Kim YM et al. (2013b) C-peptide activates AMPKalpha and
prevents ROS-mediated mitochondrial fission and endothelial apoptosis
in diabetes. Diabetes 62:385162

Newman AC, Hughes CC (2012) Macrophages and angiogenesis: a role for

Wnt signaling. Vasc Cell 4:13

Caldwell RB, Bartoli M, Behzadian MA et al. (2003) Vascular endothelial

growth factor and diabetic retinopathy: pathophysiological mechanisms
and treatment perspectives. Diabetes Metab Res Rev 19:44255
Dimmeler S, Fleming I, Fisslthaler B et al. (1999) Activation of nitric oxide
synthase in endothelial cells by Akt-dependent phosphorylation. Nature
399:6015
Eming SA, Brachvogel B, Odorisio T et al. (2007) Regulation of angiogenesis:
wound healing as a model. Prog Histochem Cytochem 42:11570

Park JY, Jung SH, Jung JW et al. (2009) A novel array-based assay of in situ
tissue transglutaminase activity in human umbilical vein endothelial cells.
Anal Biochem 394:21722
Sun G, Zhang X, Shen YI et al. (2011) Dextran hydrogel scaffolds enhance
angiogenic responses and promote complete skin regeneration during
burn wound healing. Proc Natl Acad Sci USA 108:2097681
Wahren J, Ekberg K, Jornvall H (2007) C-peptide is a bioactive peptide.
Diabetologia 50:5039
Wahren J, Kallas A, Sima AA (2012) The clinical potential of C-Peptide
replacement in type 1 diabetes. Diabetes 61:76172

Fadini GP, Albiero M, Menegazzo L et al. (2010) The redox enzyme p66Shc
contributes to diabetes and ischemia-induced delay in cutaneous wound
healing. Diabetes 59:230614

Wallerath T, Kunt T, Forst T et al. (2003) Stimulation of endothelial nitric oxide

synthase by proinsulin C-peptide. Nitric Oxide 9:95102

Ferringer T, Miller F 3rd (2002) Cutaneous manifestations of diabetes mellitus.

Dermatol Clin 20:48392

Werner S, Grose R (2003) Regulation of wound healing by growth factors and

cytokines. Physiol Rev 83:83570

Giacco F, Brownlee M (2010) Oxidative stress and diabetic complications. Circ

Res 107:105870

Xu F, Zhang C, Graves DT (2013) Abnormal cell responses and role of TNFalpha in impaired diabetic wound healing. Biomed Res Int 2013:754802

Graves DT, Liu R, Oates TW (2007) Diabetes-enhanced inflammation and

apoptosis: impact on periodontal pathosis. Periodontol 2000 45:12837

Yang B, Cao DJ, Sainz I et al. (2004) Different roles of ERK and p38 MAP
kinases during tube formation from endothelial cells cultured in
3-dimensional collagen matrices. J Cell Physiol 200:3609

Hansen SL, Myers CA, Charboneau A et al. (2003) HoxD3 accelerates wound
healing in diabetic mice. Am J Pathol 163:242131
Hills CE, Brunskill NJ, Squires PE (2010) C-peptide as a therapeutic tool in
diabetic nephropathy. Am J Nephrol 31:38997

278

Hirsch T, Spielmann M, Zuhaili B et al. (2008) Enhanced susceptibility to

infections in a diabetic wound healing model. BMC Surg 8:5

Journal of Investigative Dermatology (2015), Volume 135

Zhong Z, Davidescu A, Ehren I et al. (2005) C-peptide stimulates ERK1/2 and

JNK MAP kinases via activation of protein kinase C in human renal
tubular cells. Diabetologia 48:18797

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