Increased matrix metalloproteinase-9 (MMP-9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer.

BACKGROUND: The pathology of chronic wounds is often characterized by elevated levels of proinflammatory cytokines [e.g. tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta], proteases [e.g. matrix metalloproteinases (MMPs)] and neutrophil elastase. MMPs specifically have been implicated by a number of studies as the major protease family responsible for the degradation of key factors critical to the ulcer's ability to heal. OBJECTIVES: To assess individual MMPs in chronic wound fluid (CWF) in order to develop improved treatments for chronic ulcers. METHODS: Collagen type I and IV zymography, immunoprecipitation followed by a substrate activity assay, and an indirect enzyme-linked immunosorbent assay were all used to analyse MMP levels in CWF. RESULTS: Our studies demonstrate that there is excessive protease activity in CWF compared with both human serum and acute wound fluid (AWF), which can be specifically attributed to MMPs as determined through a MMP-inhibitor study. Multiple MMPs were immunoprecipitated from the CWF samples and MMP-9 was identified as the predominant protease in CWF, with significantly elevated activity levels in CWF compared with AWF. In addition, the clinical status of the ulcer is directly associated with the amounts of MMP-9 present in the wound fluid. Therefore, this study suggests that higher levels of MMP-9 in chronic wound fluid correlate with a clinically worse wound. CONCLUSIONS: In view of these results, it is hypothesized that a specific inhibitor of MMP-9 could potentially be more therapeutically effective than general MMP inhibitors in modulating chronic ulcers towards a healing state.

Development of an enhanced proteomic method to detect prognostic and diagnostic markers of healing in chronic wound fluid.

BACKGROUND: Chronic venous leg ulcers are a significant cause of pain, immobility and decreased quality of life for patients with these wounds. In view of this, research efforts are focused on multiple factors in the wound environment to obtain information regarding the healing of ulcers. OBJECTIVES: Chronic wound fluid (CWF), containing a complex mixture of proteins, is an important modulator of the wound environment, and therefore we hypothesized that these proteins may be indicators of the status of wounds and their potential to heal or otherwise. To explore this we developed and validated a proteomic approach to analyse CWF. METHODS: In this study, pooled CWF was depleted of high abundant proteins using immunoaffinity chromatography. The flow-through and bound fractions were collected, concentrated, desalted and analysed using a range of techniques. Each fraction was further separated using two-dimensional (2D) gel electrophoresis and 2D liquid chromatography and analysed using mass spectrometry (MS). RESULTS: Western blot analysis against three high abundant proteins confirmed the selective removal of these proteins from CWF. Critically, one-dimensional and 2D gel electrophoresis indicated that subsequent removal of these proteins enhanced the ability to detect proteins in low abundance in CWF. Further, MS demonstrated that depletion of these abundant proteins increased the detection of other proteins in these samples. CONCLUSIONS: Results obtained indicate that this approach significantly improves separation of proteins present in low concentrations in CWF. This will facilitate the identification of biomarkers in samples collected from patients with ulcers and lead to improved patient therapies and wound care approaches.

Insulin-like growth factor I and its binding proteins: a study of the binding interface using B-domain analogues.

The biological activity of the insulin-like growth factors (IGF-I and IGF-II) is regulated by six IGF binding proteins (IGFBPs 1-6). To examine the surface of IGF-I that associates with the IGFBPs, we created a series of six IGF-I analogues, [His(4)]-, [Gln(9)]-, [Lys(9)]-, [Ser(16)]-, [Gln(9),Ser(16)]-, and [Lys(9),Ser(16)]IGF-I, that contained substitutions for residues Thr(4), Glu(9), or Phe(16). Substitution of Ser for Phe(16) did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe(16) is functionally important for IGFBP association. While His(4) or Gln(9) substitutions had little effect on IGFBP affinity, changing the negative charge of Glu(9) to a positive Lys(9) selectively decreased the affinities of IGFBP-2 and -6 by 140- and 30-fold, respectively. Furthermore, the effects of mutations to both residues 9 and 16 appear to be additive. The analogues are biologically active in rat L6 myoblasts and they retain native structure as assessed by their far-UV circular dichroism (CD) profiles. We propose that Phe(16) and adjacent hydrophobic residues (Leu(5) and Leu(54)) form a functional binding pocket for IGFBP association.

Secondary structure determination of 15N-labelled human Long-[Arg-3]-insulin-like growth factor 1 by multidimensional NMR spectroscopy.

Insulin-like growth factors (IGFs) are a group of proteins that promote cell growth and differentiation. Long-[Arg-3]-IGF-I (Francis et al. (1992) J. Mol. Endocrinol. 8, 213-223), a potent analogue of IGF-I, which has a Glu-3 to Arg-3 substitution and a hydrophobic, thirteen amino acid N-terminal extension, has been studied by 1H,15N NMR spectroscopy. All the backbone 1H and 15N assignments and most of the 1H sidechain assignments have been completed. The secondary structure elements were identified by determining the sequential and medium range NOEs from sensitivity-enhanced 15N-NOESY-HSQC and sensitivity-enhanced 15N-HSQC-NOESY-HSQC spectra. The IGF-I domain of Long-[Arg-3]-IGF-I was found to have an almost identical structure to IGF-I. The N-terminal seven amino acid residues of the extension have very few medium range or long range NOEs but the next five amino acids form a turn-like structure that is spatially close to the beginning of helix 1 in the IGF-I domain. Hydrogen-deuterium exchange experiments show that all the slowly exchanging backbone amide protons in the IGF-I domain are either in the helical or the extended structural elements. Many of the amide protons in the N-terminal extension are also protected from the solvent although the residues in this part of the extension do not have any identifiable secondary structure. The results are interpreted in terms of the increased biological potency of Long-[Arg-3]-IGF-I and the decreased binding to insulin-like growth factor binding proteins.

Insulin-like growth factor (IGF)-I A- and B-domain analogues with altered type 1 IGF and insulin receptor binding specificities.

Insulin-like growth factor-I (IGF-I) analogues were produced with the aim of identifying IGF-I residues that contribute to the specificity of binding to the type 1 IGF receptor as opposed to the insulin receptor. Receptor binding properties of a series of A- and B-domain analogues were compared using rat L6 myoblasts, soluble human IGF type 1 receptors and soluble human insulin receptor isoforms HIR-A (-Ex11) and HIR-B (+Ex11). IGF-I analogues, [Leu8] IGF-I and [Phe59] IGF-I, were shown to exhibit respectively, a 28- and 17-fold decrease in affinity for the HIR-A with only a 6- and 5-fold decrease in affinity for the human IGF type 1 receptor. In contrast, the analogue [His4] IGF-I was equipotent to IGF-I in binding to the soluble type 1 IGF receptor while showing 7-fold and 4-fold increases in HIR-A and HIR-B binding respectively. Furthermore, [Leu62] IGF-I was 8-fold less potent than IGF-I in soluble IGF type 1 receptor binding but only showed a 2-fold decrease in HIR-A and HIR-B binding. Our study supports the conclusion that the co-evolution of the IGF-I and insulin receptor/ligand systems has resulted in subtle structural differences in the A- and B-regions of each ligand important for defining receptor binding specificity.