Research Article: Insulin treatment prevents wounding associated changes in tissue and circulating neutrophil MMP-9 and NGAL in diabetic rats

Date Published: February 9, 2017

Publisher: Public Library of Science

Author(s): Maryam Abdollahi, Taria Shin Yi Ng, Alireza Rezaeizadeh, Sarah Aamidor, Stephen M. Twigg, Danqing Min, Susan V. McLennan, Marco Meloni.


Neutrophils are important for wound repair, but their persistence can impair the healing process. Neutrophils express matrix metalloproteinases including MMP-9 and its regulator neutrophil gelatinase associated lipocalin (NGAL). Whether wounding affects neutrophil MMP-9 and NGAL in diabetic animals is not known. Skin wound tissue MMP-9 and NGAL was examined by qRT-PCR and immunohistochemistry in control, diabetic and insulin treated diabetic rats. The temporal expression of MMP-9 and NGAL mRNA, MMP-9 activity and the NGAL/MMP-9 complex was also investigated in an implant model and their circulating neutrophils. The cellular localisation of MMP-9 and NGAL was confirmed by immunofluorescence and the ability of glucose to regulate these factors was examined in isolated neutrophils. In skin wound tissue compared with control, diabetes increased neutrophil infiltration, NGAL mRNA and MMP-9 protein (P<0.05). Diabetes significantly increased implant neutrophil NGAL and MMP-9 protein as well as NGAL mRNA, wound fluid NGAL/MMP-9 complex and MMP-9 activity (all <0.05). Circulating neutrophil MMP-9 and NGAL was also increased in these diabetic animals (P<0.05). These changes were prevented by insulin treatment. Ex vivo, high glucose (25mM) increased neutrophil NGAL and MMP-9 (both by 2 fold, P<0.05). NGAL and MMP-9 are increased in wound and circulating neutrophils in diabetic rodents. These changes and the association between higher NGAL and increased wound fluid MMP-9 activity suggest that increased neutrophil NGAL may contribute to increased MMP-9 in poorly healing diabetic wounds. Whether targeting neutrophil NGAL or MMP-9 can improve diabetic wound healing remains to be investigated.

Partial Text

Delayed healing of wounds is a poorly understood complication that affects 15–30% of all persons with diabetes [1, 2]. It is also associated with much morbidity and the escalating number of people with diabetes in an ageing population will increasingly add to the economic and human burden of this diabetic complication. For these reasons understanding the pathophysiology of poor wound healing in diabetes is of critical importance.

Delayed wound healing represents a major health problem and is a common experience for many people with diabetes [22]. Whilst intensive research effort has focussed on some complications of diabetes the pathophysiology of poor wound healing has received less attention. We and others have shown that the concentration and activity of MMP-9 is increased in fluids from poorly healing wounds [9] and can predict delayed healing [11]. In this study we have focussed on MMP-9 expressed by neutrophils and examined the expression and activities of one of its regulators NGAL, in two different wound models. Our results have shown a diabetes related increase in neutrophil number in the circulation as well as in the cellular fraction obtained from the implant. Additionally in these acute inflammatory models, we show an increase in wound MMP-9 activity, increased NGAL mRNA and the formation and persistence of NGAL/MMP-9 complex in the diabetic animals. These changes occur in association with an increase in activation of circulating neutrophils, as identified by increased NGAL and MMP-9 at both the gene and protein level. Moreover insulin treatment from the onset of diabetes can ameliorate some but not all of these changes suggesting that glycaemic control, or at least the anabolic effects of insulin therapy, are important but are not sufficient to improve neutrophil function and wound healing. Additionally our cell culture study suggests that high glucose concentration is in part responsible for these changes.

This work identifies novel diabetes related differences in circulating and wound tissue neutrophil NGAL and MMP-9. These results show for the first time persistence of the NGAL/MMP-9 complex in wound fluids. As this complex can stabilise MMP-9 this increase suggests a possible mechanism by which MMP-9 levels are maintained at high levels in poorly healing and chronic ulcers. Further as neutrophils are critical for the initiation and progression of wound repair a better understanding of the effect of diabetes on their function at the various phases of wound repair is required. Whether these changes are exacerbated clinically in wounds in people with diabetes where healing is often more prominently delayed and infection risk is high remains to be studied.