Epithelial tissue-type plasminogen activator expression, unlike that of urokinase, its receptor, and plasminogen activator inhibitor-1, is increased in chronic venous ulcers.

BACKGROUND: The plasminogen activation system represents a potent mechanism of extracellular proteolysis and is an essential component of normal wound healing. It has also been implicated in the pathogenesis of chronic, nonhealing ulcers. Traditionally, urokinase-type plasminogen activator (uPA) has been associated with pericellular proteolytic activity involved in tissue remodelling processes, and tissue-type plasminogen activator (tPA) mainly with intravascular fibrinolysis. OBJECTIVES: The present study was conducted to characterize the spatial distribution of the various plasminogen activation system components in chronic ulcers and acute, well-granulating wounds. METHODS: The expression of uPA, tPA, urokinase receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and vitronectin was investigated by immunohistochemical staining, in addition to uPA, tPA and PAI-1 expression by in-situ hybridization, in samples from eight chronic venous ulcers, five decubitus ulcers, five well-granulating acute wounds and five normal skin samples. RESULTS: In chronic venous leg ulcers tPA mRNA was detected in basal and suprabasal keratinocytes at the leading wound edge, while in well-granulating wounds and in decubitus ulcers tPA mRNA was expressed only in a few keratinocytes. However, tPA was widely expressed in fibroblast- and macrophage-like cells in the stroma of well-granulating wounds, while less tPA was detected in the granulation tissue of chronic ulcers. tPA mRNA and protein were localized in the superficial granular layers in normal skin. Although no qualitative differences in expression of uPA, PAI-1 or uPAR in the wound edge keratinocytes in chronic ulcers vs. normally granulating wounds were found, their expressions were more pronounced in the granulation tissue of well-granulating wounds. CONCLUSIONS: These results suggest that in poorly healing venous leg ulcers, the pattern of tPA expression is altered in keratinocytes at the leading edge of the wound, and the patterns of tPA, uPA and PAI-1 expression are altered in the granulation tissue.

Differential effects of acute and chronic wound fluids on urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and tissue-type plasminogen activator in cultured human keratinocytes and fibroblasts.

The effect of wound fluids collected from acute well-healing wounds and chronic nonhealing venous leg ulcers on the plasminogen activation system of keratinocyte and fibroblast cell cultures was studied in a simplified wound-healing model. Acute wound fluid was collected from donor sites of split skin grafts at different time points representing the progressive healing of the wound. Urokinase-type plasminogen activator, tissue-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor 1 expression were studied. The methods used were immunocapture assay and immunocytochemistry. The results indicated that the later the acute wound fluid was collected, the greater the urokinase-type plasminogen activator and the lower the plasminogen inhibitor-1 level in treated cells. In contrast, the level of urokinase-type plasminogen activator receptor remained stable irrespective of wound fluid treatment. Immunostaining for urokinase-type plasminogen activator of acute wound fluid-treated cells showed a disseminated punctate pattern over the cell surface, but with chronic wound fluid, urokinase-type plasminogen activator was localized to focal contacts. Our findings support the view that in the acute wound environment the plasminogen activator system is proteolytically active and that in chronic leg ulcers urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor may also be organized for cell adhesion and migration.

Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds.

The present study was carried out to characterize the patterns of expression of matrix metalloproteinases or their tissue inhibitor (TIMP-1) in normally healing, acute vs. chronic, skin wounds. In situ hybridization was performed to localize collagenase, stromelysin-1, stromelysin-2, matrilysin, urokinase plasminogen activator (uPA) and TIMP-1 mRNAs in 14 chronic venous ulcers and 10 normally healing wounds, representing different time points after wounding. Surgical wounds, made in piglets harvested at several time points, were studied as controls. Collagenase, stromelysin-1 and -2, as well as uPa, were expressed in keratinocytes in both acute and chronic wounds, while epithelial TIMP-1 mRNA was not detected in any chronic wound biopsies studied. However, TIMP-1 was expressed at the epithelial edges of both acute human and pig wounds. Our results suggest that the balance between metalloenzymes and their inhibitor TIMP-1, is disturbed, in poorly healing wounds.

Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers.

Although extracellular proteolysis is a prerequisite for normal wound healing, uncontrolled proteolytic tissue destruction appears to be a pathogenic factor in non-healing wounds. The aim of our study was to compare the activities of the serine proteinases of polymorphonuclear origin, elastase and cathepsin G, and the metalloproteinases, gelatinase and collagenase, in chronic leg ulcer exudate (10 patients) and acute wound fluid (6 patients). Serine proteinase activities were low in leg ulcer exudates but very high in some but not all acute wound fluids. Total collagenase activity, measured as activity against type I collagen monitored by SDS-PAGE and densitometry, was higher in chronic leg ulcer exudate than in acute wound fluid and its degree of autoactivation was relatively high. Doxycycline inhibition studies suggested that the collagenase activity in chronic leg ulcer exudate was MMP-1 ("fibroblast-type") and not MMP-8 ("neutrophil-type"). Zymographic analysis of the gelatinolytic enzymes in acute wound fluid showed a progressive increase from the day of operation to postoperative day 5, but the degree of activity was lower than in chronic leg ulcer exudate and the low molecular mass activation products were faint. The leg ulcer gelatinase profiles were characterized by high expression of 92/82- and 72/62-kDa duplex bands and by the presence of low molecular mass activation products. Leg ulcer collagenase seems to be derived from mononuclear rather than polymorphonuclear cells, which are known to be involved in acute wound healing. In conclusion, the present study shows that gelatinase and collagenase, but not elastase and cathepsin G are found in chronic leg ulcer exudate.