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Moor AN, Vachon DJ, Gould LJProteolytic activity in wound fluids and tissues derived from chronic venous leg ulcers. Wound Repair Regen 17:832-839

Department of Surgery, University of South Florida, Tampa, Florida, USA.
Wound Repair and Regeneration (Impact Factor: 2.75). 11/2009; 17(6):832-9. DOI: 10.1111/j.1524-475X.2009.00547.x
Source: PubMed

ABSTRACT

Venous leg ulcers affect approximately 1% of the general population and 3.6% of those over the age of 65. The goal of the research described herein is to shorten the time to healing by developing wound care alternatives that are based on a comprehensive understanding of the venous ulcer wound environment. The proteolytic and inflammatory components in wound fluids and tissue biopsy samples were characterized in subjects with documented long-standing venous ulcers that had showed resistance to standard therapy. All wounds showed polymicrobial colonization with greater than 10(6) CFU/g. Myeloperoxidase, a measure of leukocyte infiltration, was also markedly elevated in these wounds. Zymography revealed the presence of both pro-matrix metalloproteinase (MMP)-2 and pro-MMP-9 in wound fluids and to a lesser extent in tissue biopsies. Using an immunocapture activity assay we reveal a sevenfold excess of MMP-9 in wound fluid as compared to tissue, with 73% in the activated form. In contrast, MMP-8 total protein levels were nearly equal in wound fluids and biopsies. Fibronectin, a critical component of the extracellular matrix, was shown to be degraded in both wound fluids and biopsy samples. Finally, the potential of a novel wound dressing to neutralize several constituents of this hostile wound environment is shown.

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    • "The analysis of wound fluid (WF) provides a direct insight into the local extracellular microenvironment of a wound [2]. In the last years, several proteins, especially inflammatory mediators, growth factors and cytokines [3] [4], proteases [5] [6], and oxidative stress related proteins [7] [8], were identified in WF which can be used to characterize and classify the effect on different phases of the wound healing process and thereby can be used as predictive and diagnostic tools. Nevertheless, the underlying mechanisms in wound healing disturbance are not completely understood and so far none of the potential biomarkers for dysregulated wound healing was established obtained desorbates were highly concentrated, contained up to 100 micrograms of total protein, required no further purification, and were sufficient for a reproducible analysis of more than 500 proteins by in-gel separation and proteolytic digestion followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). "
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    ABSTRACT: Wound healing of soft tissue and bone defects is a complex process in which cellular differentiation and adaption are regulated by internal and external factors, among them are many different proteins. In contrast to insights into the significance of various single proteins based on model systems, the knowledge about the processes at the actual site of wound healing is still limited. This is caused by a general lack of methods that allow sampling of extracellular factors, metabolites, and proteins in situ. Sampling of wound fluids in combination with proteomics and metabolomics is one of the promising approaches to gain comprehensive and time resolved data on effector molecules. Here, we describe an approach to sample metabolites by microdialysis and to extract proteins simultaneously by adsorption. With this approach it is possible (i) to collect, enrich, and purify proteins for a comprehensive proteome analysis; (ii) to detect more than 600 proteins in different defects including more than 100 secreted proteins, of which many proteins have previously been demonstrated to have diagnostic or predictive power for the wound healing state; and (iii) to combine continuous sampling of cytokines and metabolites and discontinuous sampling of larger proteins to gain complementary information of the same defect.
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    • "The vast majority of cutaneous chronic wounds clinically present as venous, pressure, and diabetic ulcers (Mustoe, 2004). Chronic wounds show persistent inflammation devoid of a proliferative phase, poor deposition of matrix components, and failure to heal in a defined time line (Moor et al., 2009; Palolahti et al., 1993; Wilkinson et al., 1993). The pathogenesis of chronic wounds is multifactorial and mostly associated to underlying conditions related to age and diabetes or both, for example, reduced gene transcription with increasing age causes reduced protein synthesis and nonenzymatic glycosylation of proteins plus microvascular dysfunction in diabetes can retard growth factor function and alter vascularization, respectively (Algenstaedt et al., 2003; Sen, 2009; Wheaton et al., 1996). "
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    ABSTRACT: Following injury, vascular damage results in the loss of perfusion and consequent low oxygen tension (hypoxia) which may be exacerbated by a rapid influx of inflammatory and mesenchymal cells with high metabolic demands for oxygen. Changes in systemic and cellular oxygen concentrations induce tightly regulated response pathways that attempt to restore oxygen supply to cells and modulate cell function in hypoxic conditions. Most of these responses occur through the induction of the transcription factor hypoxia-inducible factor-1 (HIF-1) which regulates many processes needed for tissue repair during ischemia in the damaged tissue. HIF-1 transcriptionally upregulates expression of metabolic proteins (GLUT-1), adhesion proteins (integrins), soluble growth factors (TGF-β and VEGF), and extracellular matrix components (type I collagen and fibronectin), which enhance the repair process. For these reasons, HIF-1 is viewed as a positive regulator of wound healing and a potential regulator of organ repair and tissue fibrosis. Understanding the complex role of hypoxia in the loss of function in scarring tissues and biology of chronic wound, and organ repair will aid in the development of pharmaceutical agents that can redress the detrimental outcomes often seen in repair and scarring.
    Full-text · Article · Dec 2012 · International review of cell and molecular biology
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    • "Moor et al.[33] reported that wound fluid myeloperoxidase levels were associated with nonhealing and infected wounds. Studies are also on to determine if wound analysis for volatile organic compounds (e.g., various esters, alcohols, and organic compounds) may help in prognostication.[34] "
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    ABSTRACT: Researchers have identified several of the cellular events associated with wound healing. Platelets, neutrophils, macrophages, and fibroblasts primarily contribute to the process. They release cytokines including interleukins (ILs) and TNF-α, and growth factors, of which platelet-derived growth factor (PDGF) is perhaps the most important. The cytokines and growth factors manipulate the inflammatory phase of healing. Cytokines are chemotactic for white cells and fibroblasts, while the growth factors initiate fibroblast and keratinocyte proliferation. Inflammation is followed by the proliferation of fibroblasts, which lay down the extracellular matrix. Simultaneously, various white cells and other connective tissue cells release both the matrix metalloproteinases (MMPs) and the tissue inhibitors of these metalloproteinases (TIMPs). MMPs remove damaged structural proteins such as collagen, while the fibroblasts lay down fresh extracellular matrix proteins. Fluid collected from acute, healing wounds contains growth factors, and stimulates fibroblast proliferation, but fluid collected from chronic, nonhealing wounds does not. Fibroblasts from chronic wounds do not respond to chronic wound fluid, probably because the fibroblasts of these wounds have lost the receptors that respond to cytokines and growth factors. Nonhealing wounds contain high levels of IL1, IL6, and MMPs, and an abnormally high MMP/TIMP ratio. Clinical examination of wounds inconsistently predicts which wounds will heal when procedures like secondary closure are planned. Surgeons therefore hope that these chemicals can be used as biomarkers of wounds which have impaired ability to heal. There is also evidence that the application of growth factors like PDGF will help the healing of chronic, nonhealing wounds.
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