Diabetic mice are characterized by an altered expression pattern of VEGF, and impaired vasculogenesis during healing. We investigated the effects of porcine derived relaxin in diabetes-related wound healing defects in genetically diabetic mice. An incisional wound model was produced on the back of female diabetic C57BL/KsJm+/+ Leptdb (db+/db+) mice and their normal littermates (db+/+m). Animals were treated daily with RLX (25μg mouse/day subcutaneously) or its vehicle. Mice were killed on days 3, 6 and 12 after skin injury for measurements of vascular-endothelial-growth-factor (VEGF) mRNA and protein synthesis. Furthermore, we evaluated wound-breaking strength, histological changes, and angiogenesis at day 12. At day 6, RLX administration resulted in an increase in VEGF mRNA expression and protein wound content. Furthermore the histological evaluation indicated that RLX improved the impaired wound healing, and increased wound breaking strength at day 12 in diabetic mice. Immunohistochemistry showed that RLX in diabetic animals augmented new vessel formation. These data strongly suggest that RLX may have a potential application in diabetes-related wound disorders

Altavilla, Domenica

Bitto, Alessandra

Caccia, Paolo

Irrera, Natasha

Minutoli, Letteria

Squadrito, Francesco

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ITALIAN JOURNAL OF ANATOMY AND EMBRYOLOGY
IJAE 
Vo l .  118 ,  n .  1  (Supp l . ) :  6 6 -70 ,  2013
Porcine derived relaxin stimulates new vessel 
formation and improves the disturbed wound healing 
of the genetically diabetic mice
Francesco Squadrito1,*, Alessandra Bitto1, Natasha Irrera1, Letteria Minutoli1, Paolo Caccia2, 
Domenica Altavilla3
1Department of Clinical and Experimental Medicine, University of Messina, Italy; 2Institut Biochimique IBSA, 
Lugano, Switzerland; 3Department of Pediatric, Gynaecological, Microbiological and Biomedical Sciences, 
University of Messina, Italy.
Summary
Diabetic mice are characterized by an altered expression pattern of VEGF, and impaired vascu-
logenesis during healing. We investigated the effects of porcine derived relaxin in diabetes-re-
lated wound healing defects in genetically diabetic mice. 
An incisional wound model was produced on the back of female diabetic C57BL/KsJ-
m+/+Leptdb (db+/db+) mice and their normal littermates (db+/+m). Animals were treated daily 
with RLX (25µg mouse/day subcutaneously) or its vehicle. Mice were killed on days 3, 6 and 
12 after skin injury for measurements of vascular-endothelial-growth-factor (VEGF) mRNA and 
protein synthesis. Furthermore, we evaluated wound-breaking strength, histological changes, 
and angiogenesis at day 12. 
At day 6, RLX administration resulted in an increase in VEGF mRNA expression and pro-
tein wound content. Furthermore the histological evaluation indicated that RLX improved the 
impaired wound healing, and increased wound breaking strength at day 12 in diabetic mice. 
Immunohistochemistry showed that RLX in diabetic animals augmented new vessel formation. 
These data strongly suggest that RLX may have a potential application in diabetes-related 
wound disorders. 
Key words
Angiogenesis, diabetes, relaxin, wound healing
Healing impairment in diabetics is characterized by delayed cellular infiltra-
tion and granulation tissue formation, decreased collagen organization and, more 
interestingly, reduced angiogenesis. As far as angiogenesis is concerned an altered 
expression pattern of VEGF mRNA during skin repair, has been shown in diabet-
ic mice (Altavilla et al., 2001). Relaxin has been shown to induce VEGF expres-
sion and angiogenesis selectively at wound sites in an experimental in vitro mod-
el (Unemori et al., 2000). However the effects of relaxin in the diabetes impaired 
wound healing have not been fully investigated. We therefore investigated the 
effects of a porcine derived relaxin in an incisional wound healing model in the 
genetically diabetic mice (Altavilla et al., 2001). 
* Corresponding Author: Department of Clinical and Experimental Medicine, Torre Biologica 5th floor, c/o AOU Policlinico G. Mar-
tino, Via C. Valeria Gazzi, 98125, Messina, ITALY; Tel. +39 090 2213648; Fax +39 090 2213300; E-mail: Francesco.Squadrito@unime.it.
67Relaxin effects in diabetic wound healing
All animal procedures were in accordance with the Principles of Laboratory Ani-
mal Care (NIH publication no.85-23, revised 1985) and authorised by our National 
Institution. Genetically diabetic female (30-35g) C57BL/KsJ-m+/+Leptdb mice (db+/
db+) and their normal littermates (22-25g) (db+/+m) were obtained from Jackson Lab-
oratory (Bar Harbor, USA). After general anesthesia with sodium pentobarbital (80 
mg/kg/i.p.), hair on the back was shaved and two parallel 4-cm incisions were pro-
duced with the use of a scalpel (Figure 1A), on the back of all mice as previously 
described (Altavilla et al., 2011). Normoglycaemic and diabetic mice, received either 
RLX (25µg/mouse/day s.c.) or its vehicle (6µl/mouse/day of 0.9% NaCl) for up to 
12 days. Ten animals from each strain were killed after 3, 6 and 12 days after surgery, 
and the wounds removed by using a scalpel to cut the shape of an ellipse around the 
lesion. Skin was used for molecular, functional and histological analysis.
Wounds obtained at each time point were analysed for VEGF mRNA content by 
Real-Time PCR using b-actin as endogenous control. The results were expressed as 
the n-fold difference relative to normal controls (relative expression levels). The 
amount of VEGF in wounds was determined at 3, 6 and 12 days, by a commercially 
available VEGF-specific ELISA assay kit (R&D Systems, USA) as previously described 
(Galeano et al., 2006). The amount of VEGF was expressed as picograms per mil-
ligram of protein. 
For the histological analysis wound samples were fixed in 10% neutral buffered 
formalin, and processed as previously described (Altavilla et al., 2011). All slides 
were examined by a pathologist blinded to the previous treatment, and the follow-
ing parameters were evaluated and scored: re-epithelialization, dermal matrix depo-
sition and regeneration, granulation tissue formation and remodelling, according to 
the literature data concerning wound healing in experimental models (Altavilla et al., 
2011; Galeano et al., 2008; Galeano et al., 2011). For the immunohistochemical study 
paraffin-embedded tissues were sectioned (5 mm), re-hydrated, and antigen retrieval 
was performed using 0.05M sodium citrate buffer. Slides were incubated overnight 
with primary antibody to detect CD-31, VEGFR-1 (all from Abcam, UK), and VEG-
FR-2 (Cell Signaling, USA) as previously described (Altavilla et al., 2011). DAB (3-3’ 
Diaminobenzidine, Sigma, USA) was used to reveal the reaction and counterstain 
was performed with haematoxylin where needed. To assess new blood vessel forma-
tion, microvessel density (MVD) was estimated after CD-31 staining. To assess posi-
tive VEGFR-1 and VEGFR-2 staining six areas per section were randomly selected in 
the dermis and the positive endothelial cells were counted per high-power field. 
At day 12, wound breaking strength was evaluated by the maximum load toler-
ated by wounds using a calibrated tensometer (Sans, Italy) as previously described 
(Altavilla et al., 2011). The ends of the skin strip were pulled at a constant speed (20 
cm min-1), and breaking strength was expressed as the mean maximum level of ten-
sile strength in newton (N) before separation of wounds. 
All data were expressed as means and standard deviations (mean ± SD). Com-
parisons between different treatments were analysed by one-way ANOVA followed 
by Tukey’s multiple comparison test. In all cases, a probability error of less than 0.05 
was selected as the criterion for statistical significance. Graphs were drawn using 
GraphPad Prism (version 5.0 for Windows).
The results obtained showed that non-diabetic animals, have a strong induction 
of VEGF mRNA and protein at day 3 and 6, that decline at day 12. In this animals 
68 Francesco Squadrito, Alessandra Bitto, Natasha Irrera, Letteria Minutoli, Paolo Caccia, Domenica Altavilla
administration of RLX enhanced VEGF mRNA and protein content. At day 3 and 6 
wounds from diabetic mice showed a markedly reduced expression and protein 
levels of VEGF when compared with non-diabetic ones, and were still significantly 
detectable at day 12. Daily treatment with RLX enhanced VEGF expression and pro-
tein levels at days 3 and 6, thus restoring the disturbed pattern of VEGF secretion.
The histological scores depicted in the Figure show that normoglycemic animals 
completed the remodeling and wound closure process at day 12, however the admin-
istration of RLX qualitatively improved wound healing. By contrast, diabetic wounds 
of mice administered with vehicle at day 12 showed poor-to mild reepithelialization, 
with partially organized granulation tissue. Wounds of diabetic mice treated with 
RLX, showed moderate to complete reepithelialization and well-formed granulation 
tissue. CD-31 immunostaining was investigated at day 12 to confirm neo-vessel for-
mation. In fact, this protein represents an highly specific marker for endothelial cells. 
Positive staining was present in the wounds from vehicle administered non diabetic 
animals. This staining appeared markedly reduced in the wounds from diabetic ani-
mals injected with vehicle. Administration of RLX augmented CD-31 immunostaining 
in both strains of mice. The effect was remarkable in diabetic wounds where the posi-
tive staining was mostly localized in small vessels and capillaries. To confirm neo-an-
giogenesis, VEGF-R1 immunostaining was also studied at day 12. Positive staining 
was evident in the wounds from vehicle injected non diabetic animals. This staining 
was significantly reduced in the wounds from diabetic animals injected with vehicle 
and increased by relaxin. In RLX-treated diabetics a marked staining in the sub-ep-
ithelial layer was observed, VEGF-R1 in fact exist also in soluble form and the dif-
fuse staining suggest an enhanced production. At day 12, immunostaining was per-
formed also for VEGFR-2 that identify endothelial precursors cells. These cells are not 
Figure 1. Histological scores in wound samples collected from either normoglycaemic (db+/+m) and diabetic 
mice (db+/db+) given either RLX (25µg mouse/day/s.c.) or vehicle (6µl mouse/day of 0.9 % NaCl) at day 12. 
Each bar represents the mean ± S.D. of six animals. *p<0.001 vs db+/db+ + vehicle.
69Relaxin effects in diabetic wound healing
only circulating and bone-marrow derived, but they are also present in subcutaneous 
adipose tissue. A slight staining for VEGFR-2 was observed in both strains of mice 
administered with vehicle. Injection of RLX significantly increased VEGFR-2 staining 
in both non-diabetic and diabetic animals. Using the images obtained from the immu-
noistochemichal staining, the tissue samples were assessed for micro-vessel density 
and angiogenesis quantification (Figure). 
At day 12 the wound breaking strength of diabetic mice was significantly lower 
than that of normoglycaemic animals. Administration of RLX did not significantly 
change this parameter in non diabetic animals. The breaking strength of incision-
al diabetic wounds of mice treated with RLX was higher than that of diabetic mice 
treated with vehicle.
The present findings indicate that porcine relaxin improved the altered healing 
process, augmented new vessel formation and increased wound breaking strength 
in obese-diabetic animals. Relaxin also ameliorated the disturbed pattern of VEGF 
expression. Interestingly, relaxin stimulated also the expression of CD31, VEGFR-1 
and VEGFR-2 in new vessels close to the wound site in genetically obese-diabetic 
mice. In diabetes, the need of neovascularization arises from the inadequate VEGF 
production and release in wounds: thus, the classic angiogenetic process is extremely 
delayed and poor and, as a consequence, therapeutically valuable approaches have 
been addressed to the aim of stimulating the expression of the impaired angiopoietic 
factor. Our results clearly suggest that relaxin efficiently induces active angiogenesis, 
in turn improving the disturbed healing process. 
In conclusion, results from this study demonstrate that relaxin administration can 
ameliorate the altered diabetic wound healing, by accelerating new vessel formation. 
Since a recombinant form of human relaxin has been studied in Phase II/III clinical trials 
and demonstrated promising results, particularly in the treatment of acute heart failure 
(Teerlink et al., 2009), that is more likely to occur in diabetic patients, we believe that our 
study might shed light on the possible use of relaxin in diabetic patients with peripheral 
artery diseases and local circulatory insufficiency like foot ulcers.
Acknowledgements
This work was supported in part by a liberal donation from IBSA. Dr Paolo Cac-
cia has a duality of interest as employee of IBSA. All others authors have no potential 
conflict of interest. 
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Porcine derived relaxin stimulates new vessel formation and improves the disturbed wound healing of the genetically diabetic mice

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Porcine derived relaxin stimulates new vessel formation and improves the disturbed wound healing of the genetically diabetic mice

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