Ari 2 Diabetik Dpp4 Inhibitor

7/26/2019 Ari 2 Diabetik Dpp4 Inhibitor

1/11

Proinsulin C-Peptide Prevents Impaired Wound

Healing by Activating Angiogenesis in DiabetesYoung-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 impairedangiogenesis. We previously demonstrated that human proinsulin C-peptide can protect against vasculopathy indiabetes; however, its role in impaired wound healing in diabetes has not been studied. We investigated thepotential roles of C-peptide in protecting against impaired wound healing by inducing angiogenesis usingstreptozotocin-induced diabetic mice and human umbilical vein endothelial cells. Diabetes delayed woundhealing in mouse skin, and C-peptide supplement using osmotic pumps significantly increased the rate of skinwound 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 ofextracellular signalregulated kinase 1/2 and Akt, as well as nitric oxide formation. C-peptide-enhancedangiogenesis in vivo was demonstrated by immunohistochemistry and Matrigel plug assays. Our findingshighlight an angiogenic role of C-peptide and its ability to protect against impaired wound healing, which mayhave significant implications in reparative and therapeutic angiogenesis in diabetes. Thus, C-peptide replacementis 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

INTRODUCTIONHuman proinsulin C-peptide is secreted into circulation inequimolar 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 complica-tions, including neuropathy, nephropathy, vascular dysfunc-tions, and inflammation, in animal models of diabetes andin type 1 diabetes mellitus (DM) patients (Ido et al., 1997;Wahren et al., 2007;Hillset al., 2010;Wahrenet al., 2012).In diabetes, increased intracellular reactive oxygen specieslevels are recognized as the most common cause of diabeticvascular complications (Giacco and Brownlee, 2010). Wepreviously demonstrated that C-peptide can ameliorate endo-thelial dysfunction through inhibiting intracellular reactiveoxygen species-mediated apoptosis of endothelial cells

by activating AMP-activated protein kinase a (Bhatt et al.,2013a,b). Recently, we also demonstrated the protective roleof C-peptide against diabetic retinopathy by amelioration

of retinal microvascular leakage (Limet al., 2013). The multi-faceted effects of C-peptide in diabetic animals and inpatients are attributed to the activation of multiple cellsignaling pathways, involving p38 mitogen-activated proteinkinase, 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/orameliorates diabetic complications is not fully understood.

C-peptide deficiency is a typical feature of type 1 DM andthe later stages of type 2 DM (Wahren et al., 2007; Massi-Benedetti and Orsini-Federici, 2008). DM is one of the majorcontributors of impaired wound healing; it leads to apoptosis

and abnormal angiogenesis (Hansenet al., 2003;Graveset al.,2007). Impaired wound healing can result in diabeticcomplications, including chronic open wounds, infections,and ulcers, and even amputation (Ferringer and Miller, 2002;

Jeffcoate and Harding, 2003; Hirsch et al., 2008). Woundhealing is a dynamic and interactive process involvingangiogenesis, 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 frompreexisting vessels, is a crucial process for wound healing (Sunet al., 2011). Therefore, understanding the mechanism(s)involved in the prevention of impaired wound healing is

ORIGINAL ARTICLE

1Department of Molecular and Cellular Biochemistry and Institute of MedicalScience, Kangwon National University School of Medicine, Chuncheon,Kangwon-do, Korea and2Department of Obstetrics and Gynecology, KangwonNational University School of Medicine, Chuncheon, Kangwon-do, Korea

Correspondence: Kwon-Soo Ha, Department of Molecular and CellularBiochemistry and Institute of Medical Science, Kangwon National UniversitySchool of Medicine, Chuncheon, Kangwon-do 200-701, Korea.E-mail:[email protected]

Received 30 October 2013; revised 5 June 2014; accepted 13 June 2014;accepted article preview online 9 July 2014; published online 7 August 2014

Abbreviations: DM, diabetes mellitus; ERK1/2, extracellular signalregulatedkinase 1/2; HUVEC, human umbilical vein endothelial cell; L-NAME, L-NG-nitroarginine methyl ester; NO, nitric oxide; PECAM-1, platelet endothelial celladhesion molecule-1; VEGF, vascular endothelial growth factor

&2015 The Society for Investigative Dermatology www.jidonline.org 269
http://dx.doi.org/10.1038/jid.2014.285mailto:[email protected]://www.jidonline.org/http://www.jidonline.org/mailto:[email protected]://dx.doi.org/10.1038/jid.2014.285


2/11

necessary to ameliorate diabetic complications (Eming et al.,2007; Kolluru et al., 2012). C-peptide treatment has beenimplicated in the prevention of diabetes complications. How-ever, the beneficial role of C-peptide in preventing againstimpaired wound healing and angiogenesis due to diabetes isnot clear.

In the present study, we aimed to elucidate the physio-logical functions of C-peptide with regard to impaired woundhealing in diabetes. We hypothesized that C-peptide mightexert a beneficial effect on impaired wound healing bystimulating angiogenesis and inhibiting inflammation. Weinvestigated the role of C-peptide in therapeutic angiogenesisand excisional skin wound repair using streptozotocin-induced diabetic mice. We further examined the potentialrole of C-peptide and its underlying mechanisms in theprocess of vascular angiogenesis through the activation ofERK1/2, Akt, and NO production using human umbilicalvein endothelial cells (HUVECs) in vitro. We assessed neo-vascularization using the Matrigel plug assay and immuno-

histochemistry. In addition, we demonstrated that C-peptidenormalizes increased inflammatory cytokine levels andinfiltrated inflammatory cells in diabetic mice.

RESULTSC-peptide prevents impaired wound healing in diabetic miceTo investigate whether C-peptide could normalize impairedwound healing in diabetic mice, C-peptide was continuouslysupplemented using subcutaneously implanted osmoticpumps in streptozotocin-induced diabetic mice. Wounds of4-mm diameter were created on the dorsal surface of thehindlimbs of normal, diabetic, and C-peptidesupplementeddiabetic mice. Diabetic mice exhibited a significantly slower

healing rate compared with normal mice (Figure 1a). How-ever, C-peptide supplement accelerated wound closure indiabetic mice. This effect was quantitatively analyzed bymeasuring wound diameter (Figure 1b). Wound closurewas much slower in diabetic mice compared with normalmice (n 10,Po0.001); however, C-peptide supplementationattenuated this effect (n10, Po0.01). To further study theC-peptidemediated stimulation of wound closure, weanalyzed hematoxylin and eosin-stained transverse sectionsof the wound beds (Supplementary Figure S1 online). Assuggested by macroscopic observation, wound closure wasdramatically 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). Ourresults indicate that C-peptide prevents against impairedwound healing including wound closure in diabetic mice.

C-peptide activation of endothelial cell migration, proliferation,and tube formationBecause angiogenesis is a crucial process for wound healing(Sun et al., 2011), we investigated whether C-peptide couldactivate endothelial cell migration, proliferation, and tubeformation. C-peptide treatment induced a dose-dependentincrease in endothelial cell chemotactic migration, with amaximal effect at 0.5nM (Figure 2a and b). However, heat-inactivated C-peptide had no effect on endothelial cell

migration (data not shown). C-peptideinduced cell migrationwas further investigated with wound-healing assays. C-peptide(0.5nM) significantly stimulated cell migration to the woundedarea (Po0.01,Figure 2c and d), with cell proliferation demon-strated 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) alsostimulated chemotactic migration, wound healing, and proli-feration in endothelial cells (Figures 1 and 2).

Consistent with the cell migration assay results, C-peptidestimulated tube formation in a dose-dependent manner, withmaximal effect at 0.5nM (Figure 3a and b). VEGF inducedsimilar tube formation (Figure 3c). Thus, it was confirmedthat C-peptide activates endothelial cell migration, prolifera-tion, and tube formation, which are essential for in vitroangiogenesis.

C-peptide stimulation of angiogenesis through pathwaysinvolving ERK1/2, PI3K/Akt, and NO production

To understand the mechanism by which C-peptide activatesangiogenesis, we investigated the effect of C-peptide onupstream pathways of angiogenesis, including ERK1/2 andAkt phosphorylation and NO production. C-peptide increasedERK1/2 phosphorylation in a time-dependent manner, withmaximal stimulation at 15minutes, and this ERK1/2 activa-tion was maintained until 60 minutes (Figure 4a). Similarly,C-peptide induced time-dependent Akt phosphorylation, withmaximal effect at 15 minutes (Figure 4b). VEGF also inducedERK1/2 and Akt phosphorylation in a time-dependent manner(Figure 4a and b). Because Akt phosphorylation increases NOproduction via VEGF, which is an important angiogenesis-mediating factor (Dimmeler et al., 1999), we investigated

whether C-peptide could stimulate NO production (Figure 4c).Treatment of HUVECs with C-peptide significantly elevatedintracellular NO (Po0.05). A higher level of intracellularNO was produced with VEGF treatment (Po0.01). Theseresults suggest that signaling pathways involving ERK1/2, Akt,and NO production might be involved in C-peptideinducedangiogenesis.

The roles of ERK1/2, PI3K/Akt, and NO in C-peptideinduced angiogenesis were examined using PD98059, wort-mannin, and L-NG-nitroarginine methyl ester (L-NAME),respectively (Figure 3d). C-peptidestimulated tube formationof HUVECs was inhibited by PD98059, the inhibitor ofERK1/2, in a dose-dependent manner, with maximal inhibi-

tion at 10 mM. The PI3K/Akt inhibitor wortmannin and theNO synthase inhibitor L-NAME also dose dependently inhi-bited C-peptideinduced tube formation. As expected, VEGF-induced tube formation was prevented by treatmentwith PD98059, wortmannin, or L-NAME (Figure 3e). Ourresults indicate that C-peptide stimulates angiogenesis viasignaling pathways involving ERK1/2, PI3K/Akt, and NOproduction.

C-peptide activation of blood vessel formation in diabetic miceTo investigate the effect of C-peptide on blood vessel forma-tion in the wound, the inner surface of dorsal skins fromnormal, diabetic, and C-peptidesupplemented diabetic mice

Y-C Limet al.C-Peptide and Wound Healing in Diabetes

270 Journal of Investigative Dermatology (2015), Volume 135


3/11

was photographed. Blood vessels were obvious in the sub-cutaneous tissue surrounding the wounds in normal mice butnot in the wound of diabetic mice (Figure 5a). Blood vesselformation was apparent in C-peptidesupplemented diabeticmice.

C-peptideinduced blood vessel formation was also inves-tigated by confocal microscopy of the inner surface of thedorsal skins from three groups of mice after platelet endothe-lial cell adhesion molecule-1 (PECAM-1) staining of bloodvessels (Figure 5b). PECAM-1 immunohistochemistry revealed

increased blood vessel formation in normal and C-peptidesupplemented diabetic mice compared with diabetic mice(Figure 5b and c), indicating that C-peptide activated bloodvessel formation in the skin of diabetic mice. C-peptideinduced formation of blood vessels was supported by theMatrigel plug assay. One week after implantation in C57BL/6mice, Matrigel containing C-peptide exhibited significantlygreater angiogenesis compared with control, and this resultwas similar to that observed following VEGF treatment(Figure 5d).

****

**

**

****

**

Normal miceDiabeticDiabetic+C-peptide

Norma

l

Diabe

tes

Diabe

tes

+C-pep

Normal miceDiabeticDiabetic+C-peptide

6

6 d 10 d Endpoint

Day

5

4

3

2

1

06 8 10 12 14420

Woun

dareab

y

macroscop

icimag

ing

(%)

Woun

ddiame

ter(

mm

)

120

100

80

60

40

20

0

** ** ** **

Areao

fhyper-prol

ifera

tive

ep

ithe

lium

(m

2)

Leng

tho

fhyper-pro

lifera

tive

ep

ithe

lium

(m

)

0

5,000

15,000

25,000

35,000

20,000

30,000

10,000

6,000

5,000

4,000

3,000

2,000

1,000

0

0 d 2 d 6 d 10 d 12 d 20 d

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 (n10), diabetic (n10), and C-peptidesupplemented diabetic mice (DiabeticC-peptide; n10), 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 (n6) 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.


www.jidonline.org 271
http://www.jidonline.org/http://www.jidonline.org/


4/11

C-peptide activation of blood vessel formation was furtherconfirmed by confocal microscopy of transverse sections ofwound beds after immunohistochemistry with anti-PECAM-1anda-smooth muscle actin antibodies (Figure 6a). The numberof blood vessels was significantly lower in diabetic micecompared with normal mice (Po0.01), and this effect wasnormalized in C-peptide supplementation (Figure 6a and b),indicating that C-peptide activates neovascularization in thewound bed of diabetic mice. Thus, C-peptide normalizesimpaired wound healing by activating angiogenesis in diabeticmice.

C-peptide inhibition of inflammation in diabetic miceTo investigate C-peptide effect on inflammation in thewounds, we performed immunohistochemistry on transversesections of wound beds from normal (10 days), diabetic (20days), and C-peptidesupplemented diabetic mice (12 days)and determined the levels of inflammatory cytokines byconfocal microscopy. Diabetic mice demonstrated signifi-cantly higher levels of IL-1b, IL-6, and tumor necrosis factor-a (TNF-a) compared with normal mice, which were norma-lized by C-peptide supplementation (Po0.01; Figure 6c),indicating that C-peptide inhibits inflammation in diabetic

Num

bero

fm

igra

tedcellsper

fie

ld

Ce

llv

iability

(%)

0

25

50

75

100

125150

175

200

0

50

100

150

200 CON C-pepVEGF

CON C-pepVEGF

CON

0 h 24 h

**

VEGF C-pep (0.1)

****

Num

bero

fmigra

tedce

llsper

fie

ld 500

400

300

200

100

0

CON

VEGF

0.1

nMC-p

ep

0.2

nMC-p

ep

0.5

nMC-p

ep

1nM

C-p

ep

**

**

** ***

CON CON VEGF C-pep

C-pep (1.0)C-pep (0.2) C-pep (0.5)

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 ml1 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 (n3) (b). Bar20mm. (c, d) Wound-healing assays; confluent cell layers were wounded

and incubated with 10 ng ml1 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) (n3). Bar 300mm. (e) Cell viability assay; HUVECs were incubated for 24 hours in the presence of 10 ng ml1 VEGF or 0.5nM C-pep (n3).

*Po0.05, **Po0.01; for VEGF or C-pep versus control.




5/11

mice. In addition, C-peptide reversed the increased number ofinfiltrated inflammatory cells (CD11b- and Ly6c-positive cells)in diabetic mice (Po0.05;Figure 6d). Taken together, our dataindicate that C-peptide normalizes impaired wound healingby activating angiogenesis as well as inhibiting inflammationin diabetic mice.

DISCUSSIONC-peptide has emerged as a physiologically active peptide forameliorating diabetes-induced complications including vascu-lar dysfunction and inflammation (Ido et al., 1997; Wahrenet al., 2007;Hillset al., 2010;Wahrenet al., 2012;Limet al.,2013;Bhatt et al., 2013a,b). In this study, we focused on the

potential angiogenic role of C-peptide and its ability toameliorate impaired wound healing in diabetes (Figures 5and 6). Impaired wound healing in diabetes is a seriouscomplication that leads to systemic infection, ulceration, andamputation (Werner and Grose, 2003; Nabuurs-Franssenet al., 2005; Barrientos et al., 2008). Although hyper-glycemia alone can cause alterations in body homeostasis,the deficiency or absence of circulating insulin and C-peptidemay contribute to the development and progression ofhyperglycemia-induced complications.

Experiments with wound animal models have demonstratedthat secretion of growth factors and reductions in inflamma-tory and oxidative responses are underlying mechanisms of

Re

lative

tube

leng

th(%)

Re

lative

tube

leng

th(%)

0

50

100

150

200

250

Re

lative

tube

leng

th(%)

0

50

100

150

200

250

**** **

******

** *

C-peptide concentration (nM)

0.5 1.00.0 1.5 2.0

C-pep

PD98059

Wortmannin

L-NAME

VEGF

PD98059

Wortmannin

L-NAME

+ + + + + + + + + +

2.5 5 10

25 50100 +

+ + + +

+

+ 0.5 1 2

100

150

200

250

Relative

tube

leng

th(%) ****

0

50

100

150

200

250

CON C-pepVEGF

CON 0.1 nM 0.2 nM

2 nM1 nM0.5 nM

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. Bar300mm. (b) Dose-dependent tube formation induced by C-peptide.

(c) Tube formation induced by 10 ngml1 VEGF or 0.5ngml1 C-peptide. **Po0.01; VEGF or C-peptide versus control (CON). (d) Human umbilical vein

endothelial cells (HUVECs) were incubated for 2430hours with 0.5 nMC-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 ml1 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.




6/11

angiogenic factor treatment (Werner and Grose, 2003).Granulation tissue formation and enhanced re-epithelializa-tion are associated with increased angiogenesis and an

increase in fibroblast number (Fadini et al., 2010). Ourresults demonstrate the preventive role of C-peptide againstimpaired wound healing through stimulating angiogenesis andinhibiting inflammation in streptozotocin-induced diabeticmice. There is a report that C-peptide had no effect ondiabetic wound healing; however, this controversy might becaused by using non-physiological C-peptide concentration(over 10nM in plasma) (Langer et al., 2002). C-peptidereplacement therapy has been investigated in animal modelsof diabetic neuropathy and nephropathy but not in diabetes-impaired wound healing. We systemically administeredC-peptide to diabetic mice to investigate the protective roleof C-peptide against diabetes-impaired wound healing. The

most important finding of the present study is that C-peptidetreated diabetic mice showed significantly improved woundhealing.

We further investigated the underlying mechanisms ofC-peptide protection in the process of vascular angiogenesisthrough the activation of ERK1/2, Akt, and NO production;this effect was demonstrated in parallel with VEGF-inducedangiogenesis. ERK1/2 has a distinct role in tube formation byendothelial cells and tube structure maintenance duringangiogenesis, whereas NO stimulates endothelial cell prolif-eration and migration (Yang et al., 2004). We demonstratedincreases in endothelial cell proliferation, migration, and tubeformation following C-peptide treatment, with a maximumeffect observed within its physiological concentration (0.5 nM);these effects were similar to those observed following VEGFtreatment. In parallel with VEGF, C-peptide also stimulated

C-peptide (minutes)

CON

p-Akt

t-Akt

**

*

VEGF (minutes)

p-ERK1/2

t-ERK1/2

ERKp

hosp

hory

lation

(fold

)

AKTp

hosp

hory

lation

(fo

ld)

Re

lative

NOleve

l(fo

ld)

6

5

4

3

2

1

0

5

4

3

2

1

0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

5 15 305 1510 6030

CON 5 15 305 1510 6030

5 15 30CON 5 1510 6030

5 15 30CON 5 1510 6030

C-peptide(minutes)

C-peptide (minutes)

C-peptide (minutes)

VEGF(minutes)

VEGF (minutes)

VEGF (minutes)CON VEGF C-pep

CON VEGF C-pep

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 ngml1 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 ngml1 VEGF or

0.5nM C-peptide for 2 hours, and levels of intracellular NO were determined by confocal microscopy as described in the Methods. Bar100mm. Results are

expressed as meanSD of three independent experiments. *Po0.05, **Po0.01; for VEGF or C-peptide versus control.




7/11

ERK1/2 and Akt phosphorylation and NO production inendothelial cells. Blocking the ERK1/2, PI3K/Akt, or endo-thelial NO species pathway using specific inhibitors suppres-sed in vitro tube formation induced by both C-peptide andVEGF. Moreover, the in vivo Matrigel plug assay resultsdemonstrated a significant increase in angiogenesis uponC-peptide treatment, similar to that induced by VEGFtreatment. Thus, C-peptide has significant VEGF-mimeticangiogenic potency both in vitroand in vivo.

We investigated whether C-peptidemediated accelerationof wound repair was mediated via its effect on wound site.C-peptide supplementation normalized increased levels ofinflammatory cytokines and CD11b- and Ly6C-positive cells

in the wound bed of diabetic mice. The normal wound-healing process is initiated by migration of keratinocytes andfibroblasts. In diabetes, increased inflammatory cytokines,such as IL-1b, IL-6, and TNF-a, induce impaired woundhealing by suppressing keratinocyte and fibroblast migration(Xuet al., 2013). C-peptide induced migration of NIH3T3 cellsin vitro (Supplementary Figure S2 online), suggesting thatC-peptide can directly activate fibroblasts for acceleratingwound repair. However, C-peptide did not activate the HaCaTcell migration (data not shown), indicating that C-peptide mayindirectly reverse diabetes-induced suppression of keratino-cyte migration through inhibiting inflammatory cytokines.PECAM-1 immunohistochemistry demonstrated new blood

PE

CAM-1

Transm

itted

Normal(10 d)

Diabetic(20 d)

Diabetic + C-pep(12 d)

Normal(10 d)

Diabetic(20 d)

Diabetic + C-pep(12 d)

VEGFCON C-pep

To

talvesse

lleng

thper

fie

ld(mm

)12

10

8

6

4

2

0

**

Norm

al(1

0d)

Diab

etic

(20d

)

Diab

etic+

C-pe

p(1

2d)

Figure 5. C-peptide activation of blood vessel formation in diabetic mice.(a, b) Dorsal skin tissues were excised from the hindlimbs of normal (n6),

diabetic (n 6), and C-peptidesupplemented diabetic (DiabeticC-peptide, n6) 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. Bar200mm.

(c) Total vessel length (mm) per field was determined using the images in b. **Po0.01. (d)In vivothe Matrigel plug assay; mice were injected subcutaneously

for 7 days with 0.5 ml Matrigel containing vehicle (CON), 100 ng ml1 vascular endothelial growth factor (VEGF), or 5 nM C-peptide (n8 per group).




8/11

vessel formation in the wounded dorsal skin. Blood vesselsfrom intact vessels and loops of new capillaries gave thematrix a red granular appearance. Thus, C-peptide has abeneficial role in preventing diabetes-impaired wound healingvia its effect on anti-inflammation and neovascularization.

In conclusion, our study reveals an angiogenic role ofC-peptide exerted via pathways involving ERK1/2 and Aktphosphorylation and NO production. C-peptide supplementtherapy improved diabetes-impaired wound healing byinhibiting inflammation and stimulating angiogenesis. Thus,C-peptide replacement is a promising therapeutic strategy fordiabetes-impaired wound healing.

MATERIALS AND METHODSAnimalsSix-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 modelDiabetic mice were generated by a single intraperitoneal injection of

streptozotocin (150 mg per kg body weight) as previously described

Cy

tokineexpress

ion

(fo

ld)

Inflamma

tionce

llsper

fie

ld

6

5

4

3

2

1

0

CD11b

Ly6C**

**

**

**

## ####

#

#

*

50

40

30

20

10

0

Norm

al(10

d)

Diab

etic

(20d)

Diab

etic+

C-pe

p(12d

)

Norma

l

(10d)

Diabe

tes

(20d)

Diabe

tes+

C-pep

(12d)

-SMAPECAM-1 Merged

**

##

20

16

12

8

4

0Microvascu

lardens

ityper

fie

ld

Norm

al(1

0d)

Diab

etic

(20

d)

Diab

etic+

C-pe

p(12

d)

IL-6IL-1 TNF-

Normal (10 d)

Diabetic (20 d)

Diabetic+C-pep (12 d)

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, n6), 20 days (Diabetic, n6), and 12 days (DiabeticC-peptide, n6) 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. Bar50mm. (b) Microvascular density per field was determined by measuring the number of

blood vessels from the images ina. (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 diabeticC-peptide versus diabetic.




9/11

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

glucose levels greater than 16mM, 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

35pmolkg1

per minute for 2 weeks. The other diabetic andnormal groups underwent sham operations.

Skin excision wound and quantitative assessment of woundhealingThe 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 accord-ing to the Declaration of Helsinki as previously described (Parket al.,

2009). Cells were grown at 37 1C in a humidified 5% CO2 incubator

in M199 culture media supplemented with 20% fetal bovine

serum, 3ngml1 basic fibroblast growth factor, 5 U ml 1 heparin,

100Uml1 penicillin, and 100mg ml1 streptomycin. For experi-

ments, 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 assayMigration assays were performed as previously described (Leeet al.,

2006). Briefly, HUVEC chemotactic motility was assayed usingTranswell Permeable Supports (Costar, Corning, NY). The lower

surface of the filters was coated with 5ml 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 low-

serum media containing 1105 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).

Wound-healing assay

Confluent cell layers were starved with low-serum media for 6 hours,stained with 1mM 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 24hours, and the number of migrated cells

was counted from images obtained using a confocal microscope

(FV-300, Olympus).

Tube formation assayThe 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

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 2430hours. The degree of tube formation

was quantified by measuring tube length from the images using

FV-300 software (Olympus).

Western blot analysesHUVECs were scraped off with ice-cold lysis buffer (50 mM Tris-HCl,

pH 7.5, 1% Triton X-100, 150 mM NaCl, 1mM EDTA, 0.1 mM

phenylmethylsulfonyl fluoride, 10mg ml 1 aprotinin, and 10mg ml1

leupeptin) and centrifuged at 18,000g for 10minutes 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 productionIntracellular NO levels were measured using diaminofluorescein-FM

diacetate (Molecular Probes, Eugene, OR) as previously described

(Namkoonget al., 2009). Briefly, HUVECs were treated with VEGF or

C-peptide in phenol red-free low-serum media for the indicatedtimes and incubated with 2mM 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 immunostainingSkin 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 observedunder the confocal microscope (FV-300). The length of blood vessel

was determined using FV-300 software (Olympus).

In vivoMatrigel plug assayIn vivoMatrigel plug assays were performed as previously described

(Min et al., 2007). Briefly, mice were injected subcutaneously with

0.5ml Matrigel containing 100 ng ml1 VEGF or 5nMC-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).

Histology and immunohistochemistry

Full-skin thickness samples of wound tissue from mice were excisedusing scissors, fixed immediately in 4% paraformaldehyde in phos-

phate-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 hyper-

proliferative 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,




10/11

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 FITC-

conjugated 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 inflam-

matory cytokines was determined from the images.

Statistical analysisData 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 INTERESTThere is a PCT patent application with a filing date of 23 July 2013 andinternational application number PCT/KR2013/006571. We confirm that thisdoes not alter our adherence to the Journal of Investigative Dermatologypolicies on sharing the data and materials of the manuscript.

ACKNOWLEDGMENTSThis work was supported in part by grants from the Korea Research Foundationof Korea (2013-008193) and by the Ministry of Health and Welfare through theNational 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

Barrientos S, Stojadinovic O, Golinko MS et al. (2008) Growth factors andcytokines in wound healing. Wound Repair Regen16:585601

Bhatt MP, Lim YC, Hwang J et al. (2013a) C-peptide prevents hyperglycemia-

induced endothelial apoptosis through inhibition of reactive oxygenspecies-mediated transglutaminase 2 activation. Diabetes62:24353

Bhatt MP, Lim YC, Kim YMet al. (2013b) C-peptide activates AMPKalpha andprevents ROS-mediated mitochondrial fission and endothelial apoptosisin diabetes.Diabetes62:385162

Caldwell RB, Bartoli M, Behzadian MA et al. (2003) Vascular endothelialgrowth factor and diabetic retinopathy: pathophysiological mechanismsand treatment perspectives. Diabetes Metab Res Rev19:44255

Dimmeler S, Fleming I, Fisslthaler B et al. (1999) Activation of nitric oxidesynthase in endothelial cells by Akt-dependent phosphorylation. Nature399:6015

Eming SA, Brachvogel B, Odorisio T et al.(2007) Regulation of angiogenesis:wound healing as a model. Prog Histochem Cytochem42:11570

Fadini GP, Albiero M, Menegazzo L et al. (2010) The redox enzyme p66Shccontributes to diabetes and ischemia-induced delay in cutaneous wound

healing. Diabetes59:230614

Ferringer T, Miller F 3rd (2002) Cutaneous manifestations of diabetes mellitus.Dermatol Clin20:48392

Giacco F, Brownlee M (2010) Oxidative stress and diabetic complications. CircRes107:105870

Graves DT, Liu R, Oates TW (2007) Diabetes-enhanced inflammation andapoptosis: impact on periodontal pathosis. Periodontol 200045:12837

Hansen SL, Myers CA, Charboneau A et al. (2003) HoxD3 accelerates woundhealing in diabetic mice.Am J Pathol163:242131

Hills CE, Brunskill NJ, Squires PE (2010) C-peptide as a therapeutic tool indiabetic nephropathy.Am J Nephrol31:38997

Hirsch T, Spielmann M, Zuhaili B et al. (2008) Enhanced susceptibility toinfections in a diabetic wound healing model. BMC Surg8:5

Ido Y, Vindigni A, Chang K et al. (1997) Prevention of vascular and neuraldysfunction in diabetic rats by C-peptide. Science277:5636

Jeffcoate WJ, Harding KG (2003) Diabetic foot ulcers.Lancet361:154551

Kitamura T, Kimura K, Jung BD et al. (2002) Proinsulin C-peptide activatescAMP response element-binding proteins through the p38 mitogen-activated protein kinase pathway in mouse lung capillary endothelialcells. Biochem J366:73744

Kolluru GK, Bir SC, Kevil CG (2012) Endothelial dysfunction and diabetes:effects on angiogenesis, vascular remodeling, and wound healing. Int JVasc Med2012:918267

Lamalice L, Le Boeuf F, Huot J (2007) Endothelial cell migration duringangiogenesis. Circ Res100:78294

Langer S, Born F, Breidenbach A et al. (2002) Effect of C-peptide on woundhealing and microcirculation in diabetic mice. Eur J Med Res7:5028

Lee SJ, Namkoong S, Kim YMet al. (2006) Fractalkine stimulates angiogenesisby activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signalpathways. Am J Physiol Heart Circ Physiol291:H283646

Li J, Chen J, Kirsner R (2007) Pathophysiology of acute wound healing.Clin Dermatol25:918

Lim YC, Bhatt MP, Kwon MH et al. (2013) Prevention of VEGF-mediatedmicrovascular permeability by C-peptide in diabetic mice. Cardiovasc Res101:15564

Massi-Benedetti M, Orsini-Federici M (2008) Treatment of type 2 diabetes withcombined therapy: what are the pros and cons? Diabetes Care31(Suppl2):S1315

Min JK, Cho YL, Choi JHet al.(2007) Receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) increases vascular permeability: impaired perme-ability and angiogenesis in eNOS-deficient mice.Blood109:1495502

Monroe DM, Hoffman M (2012) The clotting systema major player in woundhealing. Haemophilia 18(Suppl 5):116

Nabuurs-Franssen MH, Huijberts MS, Nieuwenhuijzen Kruseman AC et al.(2005) Health-related quality of life of diabetic foot ulcer patients andtheir caregivers. Diabetologia48:190610

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

through the coordinated cross-talk of PKA-dependent VEGF expressionand Epac-mediated PI3K/Akt/eNOS signaling. Cell Signal21:90615

Newman AC, Hughes CC (2012) Macrophages and angiogenesis: a role forWnt signaling. Vasc Cell4:13

Park JY, Jung SH, Jung JW et al. (2009) A novel array-based assay of in situtissue transglutaminase activity in human umbilical vein endothelial cells.Anal Biochem394:21722

Sun G, Zhang X, Shen YI et al. (2011) Dextran hydrogel scaffolds enhanceangiogenic responses and promote complete skin regeneration duringburn wound healing. Proc Natl Acad Sci USA 108:2097681

Wahren J, Ekberg K, Jornvall H (2007) C-peptide is a bioactive peptide.Diabetologia50:5039

Wahren J, Kallas A, Sima AA (2012) The clinical potential of C-Peptidereplacement in type 1 diabetes. Diabetes61:76172

Wallerath T, Kunt T, Forst T et al.(2003) Stimulation of endothelial nitric oxidesynthase by proinsulin C-peptide.Nitric Oxide9:95102

Werner S, Grose R (2003) Regulation of wound healing by growth factors andcytokines. Physiol Rev83:83570

Xu F, Zhang C, Graves DT (2013) Abnormal cell responses and role of TNF-alpha in impaired diabetic wound healing.Biomed Res Int2013:754802

Yang B, Cao DJ, Sainz I et al. (2004) Different roles of ERK and p38 MAPkinases during tube formation from endothelial cells cultured in3-dimensional collagen matrices. J Cell Physiol200:3609

Zhong Z, Davidescu A, Ehren I et al.(2005) C-peptide stimulates ERK1/2 andJNK MAP kinases via activation of protein kinase C in human renaltubular cells. Diabetologia48:18797


http://www.nature.com/jidhttp://www.nature.com/jidhttp://www.nature.com/jidhttp://www.nature.com/jid


11/11

C o p y r i g h t o f J o u r n a l o f I n v e s t i g a t i v e D e r m a t o l o g y i s t h e p r o p e r t y o f N a t u r e P u b l i s h i n g G r o u p

a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v w i t h o u t

t h e c o p y r i g h t h o l d e r ' s e x p r e s s w r i t t e n p e r m i s s i o n . H o w e v e r , u s e r s m a y p r i n t , d o w n l o a d , o r

e m a i l a r t i c l e s f o r i n d i v i d u a l u s e .

Documents

Ari 2 Diabetik Dpp4 Inhibitor