ABSTRACT
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.
Subject(s)
Acute-Phase Proteins/metabolism , Diabetes Complications/drug therapy , Insulin/therapeutic use , Lipocalins/metabolism , Matrix Metalloproteinase 9/metabolism , Neutrophils/metabolism , Proto-Oncogene Proteins/metabolism , Skin/injuries , Wound Healing , Acute-Phase Proteins/genetics , Animals , Diabetes Complications/metabolism , Insulin/pharmacology , Lipocalin-2 , Lipocalins/genetics , Male , Matrix Metalloproteinase 9/genetics , Neutrophils/drug effects , Proto-Oncogene Proteins/genetics , Rats , Rats, Sprague-Dawley , Skin/drug effects , Skin/metabolismABSTRACT
Connective tissue growth factor (CTGF), also known as CCN-2, is a cysteine-rich secreted protein that is involved in a range of biological processes, including regulation of cell growth and differentiation. Our previous in vitro studies have shown that CCN-2 inhibits adipocyte differentiation, although whether CCN-2 is regulated in vivo in adipogenesis is undetermined and was investigated in this study. C57BL/6 male mice were fed either standard laboratory chow (ND) or a diet high in fat (HFD; 45% fat) for 15 or 24 wk. HFD animals that gained >5 g in weight (termed HFD-fat) were insulin resistant and were compared with HFD-fed animals, which failed to gain weight (termed HFD-lean). HFD-fat mice had significantly increased CCN-2 mRNA levels in both the subcutaneous and epididymal fat pads, whereas CCN-2 mRNA was not induced in the epididymal site in HFD-lean mice. Also in HFD-fed animals, epididymal CCN-2 mRNA correlated positively with key genes involved in adipocyte differentiation, adiponectin and PPARγ (P < 0.001 and P < 0.002, respectively). Additionally, epididymal CCN-2 mRNA correlated positively with two markers of tissue turnover, PAI-1 in HFD-fat mice only and TIMP-1, but only in the HFD-lean mice. Collectively, these findings suggest that CCN-2 plays a role in adipocyte differentiation in vivo and thus in the pathogenesis of obesity linked with insulin resistance.
