Alterations in Extracellular Remodeling and the Wound Fluid Environment

The extracellular matrix (ECM) is an important structural and functional scaffolding made up of proteins that are necessary for cell function, wound repair, epithelialization, blood vessel support, cell differentiation and signaling, and cellular migration. The ECM is particularly important in providing a substrate for keratinocytes to migrate and establish coverage in both acute and chronic wounds.39 Alterations in protease activity and the relation to abnormalities in ECM metabolism in wounds have been areas of active investigation in the past decade. In an early report evaluating wound fluid collected from patients with venous ulcers, the investigators determined that compared to acute wound fluid, the chronic wound fluid contained up to ten-fold increased levels of MMP-2 and MMP-9 as well as increased activity of the enzymes, suggesting high tissue turnover.45 Increased MMP-1 and gelatinase activity from the exudates of chronic venous leg ulcers also has been confirmed by other investigators, and the doxycycline inhibition studies suggested that the protease activity was that of fibroblasts, mononuclear cells, keratinocytes, or endothelial cells and not neutrophils.46 It is important to note that MMP's levels and activity, although abnormal in venous ulcers, is not specific to just venous disease, and alterations are found in other inflammatory wounds including burn and pressure ulcers.47 The excess proteolytic activity in venous leg ulcers has been found to degrade essential plasminogen, which is important in activating pro-MMP to MMP necessary for fibrinolysis and cell migration, and MMP's inhibit plasmin production by keratinocytes, which may lead to reduced cell migration.48 Of interest, a study evaluating fibroblasts cultured from venous ulcers determined that there was a marked reduction in MMP-1 and MMP-2 level and activity, and a significant increase in TIMP-1 and TIMP-2 production. The authors concluded that the inhibition of proteinase activity by TIMP in fibroblasts causes impaired function to reorganize the ECM in chronic wounds leading to delayed healing.49

The abnormalities in structure and healing process seen in lipodermatosclerotic skin have also been attributed to MMP pathophysiology. A study where dermal biopsies were obtained from liposclerotic skin and compared to healthy skin and analyzed by immunohistochemistry, reverse tran-scriptase polymerase chain reaction, immunoblot, and zymography found that lipodermatosclerotic skin had increased expression of mRNA and protein for MMP-1, MMP-2, and TIMP-1, and increased levels of active MMP-

2. The MMP-2 was localized predominantly in the basal and suprabasal layers of the epidermis, perivascular region, and reticular dermis, and had less TIMP-2 in the basement membrane of the diseased skin. The relevance of this study was that lipodermatosclerotic skin is characterized by elevated ECM turnover.50 The regulation of MMP production is complex. Posttranslational modifications of MMPs appear to be essential, and dermal fibroblasts and leukocytes are sources for MMPs, especially MMP-2, and likely regulated by TGF-P1.51 The interplay of MAPK with MMP activation has also been investigated in fibroblasts. The cytokine tumor necrosis factor alpha has been demonstrated to induce MMP-19 expression, which is inhibited by blocking MAPK pathways ERK1 and ERK2 with PD98059 and p38 with SB203580. In addition, adenovirus mediated induction of ERK 1 and ERK 2 in combination with p38 resulted in potent MMP-19 expression in fibroblasts and the activation of c-JNK also produced abundant pro-MMP-19. These data indicated the important regulatory functions of MAPK and proteolytic activity in dermal fibroblasts.52

The venous ulcer microenvironment consists of dermal fibroblasts, keratinocytes, inflammatory cells, ECM, bacteria, and microcirculation. An interesting aspect of the venous ulcer milieu is the presence of the chronic wound fluid. The wound fluid is known to have properties of excess protease activity as stated earlier. In addition, the venous ulcer wound fluid has been demonstrated to cause inhibition of fibroblast proliferation and induce changes of cellular senescence.34,53 The venous ulcer wound fluid has been demonstrated to inhibit the growth of fibroblasts with the majority of cells with a G1 and G2 DNA content in the cell cycle (quiescent state, unable to enter S phase). Proliferation of fibroblasts treated with chronic venous ulcer wound fluid could be reestablished by heat inactivation or reversed by removal and placement of cells in 10% serum.54 In addition to fibro-blasts, wound fluid from venous ulcers also inhibits the proliferation of endothelial cells and keratinocytes. Although the inhibitory component(s) and the responsible source(s) for production of wound fluid are not known, the active inhibitory substance can be fractionated and consist of a molecular weight of less than 30 kd.55 In neonatal fibroblasts, venous ulcer wound fluid was demonstrated to inhibit the expression of MAPK, specifically ERK 1 and ERK 2, with simultaneous decrease in proliferation.36 In addition, the mechanism of cell inhibition by wound fluid, in part, involves downregulation of phosphorylated retinoblastoma tumor suppression gene and cyclin D1, via inhibition of Ras-dependent MAPK pathway.56

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