Acute Wound Fluid Research Articles

Induction of MMP-1, MMP-3 and TIMP-1 in normal dermal fibroblasts by chronic venous leg ulcer wound fluid*

In the wound bed of chronic venous leg ulcers, an imbalance of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) may cause excessive proteolysis and impair wound granulation. Soluble mediators in the wound environment may be responsible for this imbalance. The in vitro effect of wound fluid from venous leg ulcers on dermal fibroblast production of MMP-1, MMP-3 and TIMP-1 was compared with the effect of acute wound fluid from two different sources: fluid from post-mastectomy axillary drains and fluid from skin graft donor sites. Significantly higher MMP-1 and MMP-3 levels were induced by chronic venous leg ulcer wound fluid compared with both types of acute wound fluid (P < 0.005). Chronic venous ulcer wound fluid reduced TIMP-1 protein levels significantly more than acute graft fluid (P < 0.05). Venous ulcer wound fluid significantly increased MMP-1 and MMP-3 production in dermal fibroblasts and reduced TIMP-1 production, confirming that mediators in the leg ulcer microenvironment can potentially induce excessive proteolysis in the ulcer dermis by altering the balance between MMPs and TIMPs. Inflammatory mediators including interleukin-1beta and tumour necrosis factor-alpha can induce these MMPs. Further work is required to confirm the factors responsible for the induction of a high MMP and low TIMP profile in fibroblasts by venous ulcer wound fluid.

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

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. To assess individual MMPs in chronic wound fluid (CWF) in order to develop improved treatments for chronic ulcers. 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. 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. 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.

Temporal and quantitative profiles of growth factors and metalloproteinases in acute wound fluid after mastectomy

Seventy-three samples of acute wound fluid were collected from 47 patients during the first 3 postoperative days (POD) following mastectomy for cancer (n=47 on POD-1, n=19 on POD-2, and n=7 POD-3). Samples were analyzed by enzyme-linked immunosorbent assay for growth factor levels (epidermal [EGF], platelet-derived [PDGF], basic fibroblast [bFGF], transforming growth factor-beta1 [TGF-beta1], vascular endothelial [VEGF]), interleukin-6 (IL-6), matrix metalloproteinases (MMPs-2, -3, -9), and the tissue inhibitor of metalloproteinase 1 (TIMP-1). The levels of EGF, bFGF, PDGF, and interleukin-6 peaked on POD-1, with a significant decrease by POD-3, while total and active MMP-2, MMP-3, and tissue inhibitor of metalloproteinase 1 showed a progressive and significant increase from days 1 to 3. The wounds that later developed an infection (11%) were found to have a significantly lower PDGF and EGF on day 1 (PDGF, median 169 pg/mL [range, 86-2,595]) than the noninfected wounds (2,098 [17-66,506] p<0.05, Mann-Whitney U-test). Sixty-two percent patients developed a seroma and the levels of bFGF were significantly less in these patients (441 pg/mL [45-4,108]) than in those patients where there was no seroma (807 [245-3,133] p<0.05). The levels of certain growth factors in acute wound fluid may be important markers for wound outcomes.

Upregulation of the Gamma‐2 Chain of Laminin‐332 (Laminin‐5) in Wounded Mouse Ears

Laminins, extracellular matrix components found in the basement membranes of tissues, are formed from combinations of α, β, and γ polypeptide chains that are separate gene products. Laminin-332 (formerly LN-5) attaches epithelial cells to basement membrane but also affects cell motility during wound healing. In vitro evidence suggests that human acute wound fluid upregulates all three gene products used to assemble laminin-332, but this correlation has not been studied in vivo. Mouse ear skin was wounded by exposure to the chemical warfare agent, bis(2-chloroethyl) sulfide (sulfur mustard, SM) and a time-course study followed to examine the chains of laminin-332 as potential biomarkers of injury. Skin biopsies 24, 72, and 168 hours after SM exposure were analyzed by histology, immunohistochemistry, real-time PCR, and Western blot analysis. SM exposure increased mRNA levels for laminin-γ 2 by 2-fold, 6-fold, and 3-fold for 24, 72, and 168 hours, respectively, but had no observable effect on laminin-α3 or laminin-β3 mRNA. Immunohistochemistry of tissue sections with chain-specific antibodies showed overlap of the three chains extracellularly, around the epithelial cells of the epidermis and those lining the sebaceous glands. Laminin-γ 2 also preferentially accumulated intracellularly. We speculate that the expression of laminin-γ 2 monomers expressed in the SM-treated ears function to enhance migration of the epithelial cells in a manner similar to that reported for invading carcinoma cells. *This work is supported by the USAMRAA under Contract No. DAMD17-02-C-0091 and NIH grant # ES07148.

Defective acute inflammation in Crohn's disease: a clinical investigation

The cause of Crohn's disease has not been mechanistically proven. We tested the hypothesis that the disease is a form of immunodeficiency caused by impaired innate immunity. We investigated inflammatory responses in patients and controls by quantifying neutrophil recruitment and cytokine production after acute trauma, interleukin 8 secretion by cultured monocyte-derived macrophages after exposure to inflammatory mediators, and local inflammatory and vascular changes in response to subcutaneous injection of heat-killed Escherichia coli. In patients with Crohn's disease, trauma to rectum, ileum, or skin led to abnormally low neutrophil accumulation (differences from healthy individuals of 79%, n=8, p=0.0003; 57%, n=3, p=0.05; 50%, n=13, p<0.0001, respectively) and lower production of proinflammatory interleukin 8 (63%, n=7, p=0.003; 63%, n=3, p=0.05; 45%, n=8, p<0.0001) and interleukin 1beta (50%, n=8, p=0.0005). Interleukin 8 secretion by cultured macrophages was reduced after exposure to acute wound fluid (38%, n=50, p<0.0001), C5a (48%, n=41, p=0.0005), or tumour necrosis factor alpha (52%, n=27, p<0.0001). Local inflammatory reaction to inoculation with E coli was attenuated, as quantified by changes in bloodflow (ileal disease 50%, n=6, p=0.01; colonic disease 77%, n=6, p=0.0003). This response was mediated by nitric oxide in controls, was increased by sildenafil in patients, and was not related to CARD15 genotype. In Crohn's disease, a constitutionally weak immune response predisposes to accumulation of intestinal contents that breach the mucosal barrier of the bowel wall, resulting in granuloma formation and chronic inflammation. Polymorphisms in CARD15 do not underlie this phenotype, but incapacitate the NOD2 pathway that can compensate for impairment of innate inflammation. Current treatment of secondary chronic inflammation might exaggerate the underlying lesion and promote chronic disease.

Effect of Chronic Wound Exudates and MMP-2/-9 Inhibitor on Angiogenesis In Vitro

New evidence suggests that matrix metalloproteinases (MMPs) may facilitate angiogenesis as well as function to generate angiogenesis inhibitors. In this study, the angiogenic effect of wound exudates from patients with venous insufficiency ulcers was examined in an in vitro angiogenesis model with and without synthetic MMP-2/-9 inhibitor. Wound exudates were obtained from 20 patients with venous insufficiency ulcers and 20 control patients with donor-site wounds after skin grafting for burns. In the angiogenesis model, suramin (20 microg/ml) was used in five wells without wound fluid as negative control, and vascular endothelial growth factor (1 microg/ml) was used in five other wells as positive control. Chronic wound fluids were analyzed without and with a synthetic MMP-2/-9 inhibitor with a concentration of 2 microM and 20 microM in the medium. The total length of tubules was calculated by map reader. Statistical analysis was performed using the Mann-Whitney test. The level of significance was considered to be p < 0.05. Chronic ulcer exudates inhibited angiogenesis significantly (490 +/- 130 microm) compared with acute wound fluids (1740 +/- 320 microm; p < 0.05). In wells with chronic wound exudates and high concentrations of MMP-2/-9 inhibitor, angiogenesis was stimulated significantly (870 +/- 220 microm, p < 0.05). In this model, reduced angiogenesis might be due to an antiangiogenic effect of MMP-2 and MMP-9. MMP-2/-9 inhibition results in a stimulation of angiogenesis and might be an approach for the treatment of patients with chronic wounds and reduced angiogenesis.

Increase of laminin 5 synthesis in human keratinocytes by acute wound fluid, inflammatory cytokines and growth factors, and lysophospholipids

Laminin 5 is known to induce the adhesion, spreading and migration of human keratinocytes. In skin wound healing, laminin 5 deposition beneath migrating keratinocytes occurs early and is followed by the formation of hemidesmosomes and then basement membrane. To identify factors that regulate the synthesis and secretion of laminin 5 by human keratinocytes during acute wound healing. Laminin 5 synthesis by human keratinocytes was determined by a specific sandwich enzyme-linked immunosorbent assay. To determine the total amount of laminin 5 synthesized, laminin 5 deposited on culture dishes and inside cells was solubilized by detergent solution and determined separately from conditioned medium, and the total laminin 5 synthesis was calculated. A quantitative polymerase chain reaction method was used to measure the expression levels of laminin 5 genes, LAMA3, LAMB3 and LAMC2, which correspond to the alpha3, beta3 and gamma2 chains of laminin 5. We also examined the effects of lysophospholipids, proinflammatory cytokines and growth factors, which are components in acute wound fluids, on laminin 5 synthesis in keratinocytes. Human acute wound fluid at days 1, 2 and 3 stimulated laminin 5 synthesis in cultured human keratinocytes in a concentration-dependent manner, although findings are restricted to one case. Human serum also increased laminin 5 production by human keratinocytes as strongly as the wound fluid did, suggesting that the major active components in acute wound fluid may be derived from those in human serum. Lysophospholipids such as lysophosphatidic acid (LPA), lysophosphatidylcholines (LPCs) and sphingosine-1-phosphate (S1P) increased laminin 5 synthesis in a concentration-dependent manner. Among growth factors, epidermal growth factor, insulin-like growth factor-1, interferon-gamma and keratinocyte growth factor increased laminin 5 production in keratinocytes, while platelet-derived growth factor, hepatocyte growth factor and basic fibroblast growth factor were ineffective. Although interleukin-1alpha had no effect, transforming growth factor (TGF)-alpha, tumour necrosis factor (TNF)-alpha and TGF-beta1 also stimulated laminin 5 synthesis, and TGF-alpha and TGF-beta1 showed a synergistic effect. Neutralizing antibodies to TGF-alpha and TGF-beta1 markedly inhibited the enhanced laminin 5 synthesis by human serum, suggesting that TGF-alpha and TGF-beta1 are important components to increase laminin 5 in human serum. In line with the increase of laminin 5 synthesis, the expression levels of all three laminin 5 genes were also augmented by TGF-alpha and TGF-beta1. Laminin 5 synthesis in human keratinocytes was augmented by inflammatory cytokines and growth factors such as TGF-alpha, TGF-beta1 and TNF-alpha, and lysophospholipids such as S1P, LPA and LPCs, which are supposed to be present in acute wound fluid. The increased laminin 5 protein in the wound area presumably enhances wound repair by stimulating adhesion and migration of keratinocytes on the wound bed and by facilitating basement membrane formation at the dermal-epidermal junction.

Neutrophil elastase in pressure ulcer fluid degrades fibronectin in the exudates

Background: Pressure ulcers are classified as chronic wounds, which do not heal in a timely fashion. Fibronectin is condensed in granulation tissue, and essential glycoprotein of wound healing. It has been proposed that fibronectin degradation may be involved in delaying wound healing. We have investigated whether pressure ulcer fluid (PUF) contains degraded fibronectin. In addition, we tried to identify the proteinase which contributes to fibronectin degradation in PUF.Methods: Fibronectin degradation and the presence of neutrophil elastase (NE) in PUF were determined by immunoblot analysis. Fibronectin degradation activity in PUF was determined in the presence of various proteinase inhibitors. NE activity was assessed using NE specific substrate.Results: Immunoblot analysis revealed that degraded fibronectin was observed in PUF samples but not in acute wound fluid (AWF). The PUF contained a proteinase capable of degrading freshly added fibronectin and its activity in PUF was blocked by a broad‐spectrum serine proteinase inhibitor or sivelestat, a specific neutrophil elastase inhibitor, but not by metalloproteinase and cysteine proteinase inhibitors. Immunoblot analysis of PUF using an antineutrophil elastase antibody revealed that neutrophil elastase was detected as three bands at molecular weights of ∼30 kDa, ∼38 kDa, and ∼54 kDa, indicating that neutrophil elastase in the exudates existed not only as free monomers, but also in polymers or complexes with other molecules.Conclusion: These results suggest that PUF contains a high level of neutrophil elastase which may be involved in the delay of the healing of pressure ulcer through the fibronectin degradation.

Iron and 8-Isoprostane Levels in Acute and Chronic Wounds

The purpose of this study was to determine differences in iron and iron protein (ferritin and transferrin) levels in chronic venous ulcers and acute wounds. The deleterious effect of iron in free-radical-induced tissue damage was indirectly examined by assessing 8-isoprostane levels and antioxidant status in wound fluid samples. Wound fluid samples from chronic leg ulcers in nonhealing and healing phases and wound fluid from mastectomy wounds were assayed for ferritin, transferrin, total iron, 8-isoprostane, and total antioxidant status. Immunohistochemistry and Perls' staining were performed on paired biopsies from chronic leg ulcers and on normal skin biopsies. Chronic wound fluid had significantly greater levels of ferritin (p < 0.05) and lower levels of transferrin (p < 0.001) than acute wound fluid and there was a significant reduction in the level of ferritin in healing compared to nonhealing chronic leg ulcers (p < 0.05). No significant differences were observed in the levels of total iron present in the wound fluids. Histologic staining showed consistently more ferritin and ferric iron in chronic wound tissue than in normal skin. Elevated levels of 8-isoprostane and antioxidants were observed for chronic wound fluid compared to acute wound fluid (p < 0.001). These results suggest the existence of an environment of oxidative stress in chronic wounds and the likely contribution of iron to exacerbating tissue damage and delaying healing in these wounds.

Growth factor profiles in intraperitoneal drainage fluid following colorectal surgery: relationship to wound healing and surgery.

Cytokines and growth factors are important at each stage of wound healing. This study aims to determine the changing profiles of these factors in intraperitoneal drainage, acute wound fluid, following colorectal surgery, and to correlate levels to wound healing and surgical outcomes. Acute wound fluid samples (n = 52 patients) were collected daily from postoperative day 1 until drain removal. Levels of cytokines (interleukins-6 and -1beta and tumor necrosis factor-alpha) and epidermal growth factor, platelet-derived growth factor, vascular endothelial derived growth factor, basic fibroblast growth factor, and transforming growth factor-beta1 were determined by enzyme-linked immunosorbent assay. A significant negative correlation emerged between the levels of interleukin-6, epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor and the postoperative day, e.g., basic fibroblast growth factor : day 1, 695, median (29-2,806, range) pg/ml; day 2, 249 (1-1,784); day 3, 94 (0-722); day 7, 22 (0-326) (p < 0.05, Spearman's correlation). Levels appeared to relate to the stage of wound healing. Several factors, in particular interleukin-1beta and tumor necrosis factor-alpha levels, correlated with surgical outcomes such as the need for a defunctioning stoma and/or postoperative complications. Cytokines and growth factors are involved in normal wound healing, and their levels in acute wound fluid may act as markers of wound healing and surgical outcome.

Profiles of matrix metalloproteinases and their tissue inhibitors in intraperitoneal drainage fluid: relationship to wound healing.

Matrix degradation and remodeling occurs during wound healing, thereby aiding tissue repair, angiogenesis, and cell migration. It is dependent on the balance between proteinases and their inhibitors, namely the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Acute wound fluid samples (n = 58 patients) were collected daily from the intraperitoneal drain placed after colorectal surgery from the first postoperative day until drain removal. Three laboratory techniques were performed: enzyme linked immunosorbent assays (MMP-1, MMP-3, TIMP-1, TIMP-2), gelatinase activity assays (MMP-2, MMP-9), and quenched fluorescent substrate hydrolysis (total MMP activity). Levels were correlated with each postoperative day, wound healing, and surgical outcome (p < 0.05, Spearman's correlation). Significant negative (MMP-9, MMP-3, MMP-8, TIMP-2, total MMP activity) and positive (MMP-2, TIMP-1) correlations were observed with the postoperative day, e.g., total MMP-9: day 1, median, 121 (range, 12-189) ng/ml; day 3, 46 (8-179); day 5, 31 (0-155), day 7, 20 (6-58). Differences were also observed with the type of operation, estimated blood loss, and length of operation and with postoperative complications. MMPs and TIMPs are involved in wound healing after elective colorectal cancer surgery and their levels in drain fluid may act as markers of wound healing and surgical outcome.

Evidence of oxidative stress in chronic venous ulcers.

Reactive oxygen species have been implicated in the impaired healing of chronic leg ulcers but little direct evidence is available. We have observed a significant (p < 0.01) elevation of the allantoin : uric acid percentage ratio, a marker of oxidative stress, in wound fluid from chronic leg ulcers (median 17, range 8-860) compared to both paired plasma (median 2, range 1-8) and acute surgical wound fluid (median 4, range 3-7). However, the allantoin : uric acid percentage ratio did not differ significantly between chronic wounds that healed and those that failed to heal. Neutrophil elastase was elevated 30- to 1300-fold in chronic wound fluid compared to plasma and there was a correlation (r(2) = 0.742) between wound fluid elastase and the allantoin : uric acid percentage ratio. Total antioxidant capacity of wound fluid, as measured with a chemiluminescence assay, did not show a correlation (r(2) = 0.03) with the observed oxidative stress. These observations suggest that conditions of localized oxidative stress, possibly related to neutrophil-associated production of reactive oxygen species, are present in chronic leg ulcers. It is possible that future therapeutic strategies aimed at reducing oxidative stress, in addition to good standard care, could improve healing rates of chronic wounds.

Effect of venous ulcer exudates on angiogenesis in vitro.

Angiogenesis, the formation of new from existing capillaries, is an important mechanism in venous ulcer healing. The aim of this study was to determine whether venous leg ulcer wound exudates stimulate or inhibit angiogenesis. Fluid exudate was obtained from 16 venous ulcers over a 4-h interval. Five of the ulcers had not healed after more than 1 year of compression bandaging, and five were rapidly healing ulcers. As a control, acute wound fluids were collected from subcutaneous drains in seven patients. Vascular endothelial growth factor (VEGF) at 2 ng/ml acted as a positive control. Tubules stained with an antiendothelial antibody were quantified using an image analysis system. The extent of angiogenesis was expressed as the ratio of the mean tubule length in the test wells over that in blank control wells. Venous ulcer exudates significantly inhibited angiogenesis (mean (95 per cent confidence interval) 0.72 (0.48 to 0.96)) compared with acute wound fluids (2.48 (0.86 to 4.10)) (P < 0.002) and VEGF (1.47 (1.32 to 1.61)) (P = 0.01). Exudates from the five non-healing venous ulcers inhibited angiogenesis (0.31 (0.15 to 0.46)) significantly more than exudates from the five rapidly healing venous ulcers (0.93 (0.21 to 1.65)) (P = 0.03). Fluid exudate from venous ulcers, in particular those that healed slowly, inhibited experimental angiogenesis in this study.

Differential Expression of Matrix Metalloproteinases During Impaired Wound Healing of the Diabetes Mouse

A number of studies have shown elevated matrix metalloproteinase expression in chronic wound fluid compared to an acute wound; however, little has been done to characterize animal models in a similar manner and thus determine their usefulness. The diabetes mouse is an animal model of type II diabetes that shows impaired dermal wound healing and has been proposed as a model of human impaired wound healing. In this study we have determined the mRNA and protein expression profiles of matrix metalloproteinases 2, 3, and 9 during the first 10 d of dermal healing for the diabetes mouse and its normally healing littermate. Additionally, human wound fluid from diabetic chronic wounds and acute surgical wounds were studied to enable a comparison of the model to the human condition. We show that during the early stages of wound healing the diabetes mouse possesses significantly reduced protein levels of pro-matrix metalloproteinases 2 and 9 within the wound tissue and active matrix metalloproteinase 3 within the fluid. Pro-matrix metalloproteinase 3 levels are also significantly reduced in the diabetes mouse during the later stages of healing. These differences may be contributing to the impaired healing of the diabetes mouse; however, they differ from the human data presented here, which show elevated matrix metalloproteinase 2 and reduced matrix metalloproteinase 9 in human diabetic chronic wound fluid compared to acute wound fluid. Therefore, although clearly showing the importance of appropriate matrix metalloproteinase regulation for normal acute wound healing to occur, the diabetes mouse may not be an ideal model for study of matrix metalloproteinase involvement in human chronic wound healing.

Proteinases, their inhibitors, and cytokine profiles in acute wound fluid

Proteinases, their inhibitors, and cytokine profiles in acute wound fluid

Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase.

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors.

To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 microg MMP Eq/ml) compared to acute wounds (median 0.76 microg MMP Eq/ml) (p < 0.001). The addition of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase-9 with smaller elevations of matrix metalloproteinase-2. In addition tissue inhibitor of metalloproteinase-1 levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase-1 levels were compared to protease levels there was an inverse correlation (p = 0.02, r = - 0.78). In vitro degradation of epidermal growth factor was measured by addition of 125I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.1%) compared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0. 001, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collected from 15 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median 12%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers (p < 0.01). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.

Interleukin-1α and Collagenase Activity Are Elevated in Chronic Wounds

Interleukin-1-alpha (IL-1alpha) is a member of a family of proinflammatory polypeptide mediators that has been shown in vitro to stimulate collagenase production. Collagenase is a proteolytic enzyme classified as one of the matrix metalloproteinases (MMP-1) that specifically recognizes and cleaves collagen. Therefore, the objective of this study was to compare the levels of these two proteins in chronic wounds as possible factors in the pathogenesis of chronic wounds. Fluids from 10 chronic wounds were collected before and after a 1-week treatment with a hydroactive dressing (Cutinova cavity). In addition, fluids were collected from 20 acute wounds for comparison. IL-1alpha and MMP-1 levels were quantified using sandwich ELISA. Collagenase activity was measured using a radiolabeled collagen as substrate. Clinically, the chronic wounds showed decreased area (-21.0 cm2) and reduced volume (-134.5 cm3) by 4 weeks after treatment with the hydroactive dressing. There were no significant differences in the protein concentrations between acute wound fluids (21.0 +/- 3.0 mg/ml) and chronic wound fluids before and after treatment with the hydroactive dressing (18.3 +/- 5.5 and 25.2 +/- 7.6 mg/ml, respectively). Levels of IL-1alpha in the acute wound fluids were low (0.019 pg/mg), whereas in the chronic wound fluid before treatment they had been significantly elevated (44.9 + 21.8 pg/mg). Following treatment with the hydroactive dressing, the IL-1alpha levels dropped to 10.3 + 3.3 pg/mg (p < 0.05). Collagenase activity was not detectable in acute wound fluid, elevated in pretreatment chronic wounds (12.9 + 3.4 units), and decreased in chronic wounds after treatment (11.4 + 3.3 units). This study correlated clinical healing of chronic wounds with biochemical changes in the ulcer microenvironment. As the chronic wounds began to heal, there was a significant decrease in the IL-1alpha levels and collagenase activity, thus suggesting that these two proteins may contribute to the lack of healing characteristic of chronic wounds.

Interleukin-1α and Collagenase Activity Are Elevated in Chronic Wounds

Interleukin-1-alpha (IL-1alpha) is a member of a family of proinflammatory polypeptide mediators that has been shown in vitro to stimulate collagenase production. Collagenase is a proteolytic enzyme classified as one of the matrix metalloproteinases (MMP-1) that specifically recognizes and cleaves collagen. Therefore, the objective of this study was to compare the levels of these two proteins in chronic wounds as possible factors in the pathogenesis of chronic wounds. Fluids from 10 chronic wounds were collected before and after a 1-week treatment with a hydroactive dressing (Cutinova cavity). In addition, fluids were collected from 20 acute wounds for comparison. IL-1alpha and MMP-1 levels were quantified using sandwich ELISA. Collagenase activity was measured using a radiolabeled collagen as substrate. Clinically, the chronic wounds showed decreased area (-21.0 cm2) and reduced volume (-134.5 cm3) by 4 weeks after treatment with the hydroactive dressing. There were no significant differences in the protein concentrations between acute wound fluids (21.0 +/- 3.0 mg/ml) and chronic wound fluids before and after treatment with the hydroactive dressing (18.3 +/- 5.5 and 25.2 +/- 7.6 mg/ml, respectively). Levels of IL-1alpha in the acute wound fluids were low (0.019 pg/mg), whereas in the chronic wound fluid before treatment they had been significantly elevated (44.9 + 21.8 pg/mg). Following treatment with the hydroactive dressing, the IL-1alpha levels dropped to 10.3 + 3.3 pg/mg (p < 0.05). Collagenase activity was not detectable in acute wound fluid, elevated in pretreatment chronic wounds (12.9 + 3.4 units), and decreased in chronic wounds after treatment (11.4 + 3.3 units). This study correlated clinical healing of chronic wounds with biochemical changes in the ulcer microenvironment. As the chronic wounds began to heal, there was a significant decrease in the IL-1alpha levels and collagenase activity, thus suggesting that these two proteins may contribute to the lack of healing characteristic of chronic wounds.

Syndecans, Heparan Sulfate Proteoglycans, Maintain the Proteolytic Balance of Acute Wound Fluids

An imbalance between proteases and antiproteases is thought to play a role in the inflammatory injury that regulates wound healing. The activities of some proteases and antiproteases found in inflammatory fluids can be modified in vitro by heparin, a mast cell-derived glycosaminoglycan. Because syndecans, a family of cell surface heparan sulfate proteoglycans, are the major cellular source of heparin-like glycosaminoglycan, we asked whether syndecans modify protease activities in vivo. Syndecan-1 and syndecan-4 ectodomains are shed into acute human dermal wound fluids (Subramanian, S. V., Fitzgerald, M. L., and Bernfield, M. (1997) J. Biol. Chem. 272, 14713-14720). Moreover, purified syndecan-1 ectodomain binds cathepsin G (Kd = 56 nM) and elastase (Kd = 35 nM) tightly and reduces the affinity of these proteases for their physiological inhibitors. Purified syndecan-1 ectodomain protects cathepsin G from inhibition by alpha1-antichymotrypsin and squamous cell carcinoma antigen 2 and elastase from inhibition by alpha1-proteinase inhibitor by decreasing second order rate constants for protease-antiprotease associations (kass) by 3700-, 32-, and 60-fold, respectively. Both enzymatic degradation of heparan sulfate and immunodepletion of the syndecan-1 and -4 in wound fluid reduce these proteolytic activities in the fluid, indicating that the proteases in the wound environment are regulated by interactions with syndecan ectodomains. Thus, syndecans are shed into acute wound fluids, where they can modify the proteolytic balance of the fluid. This suggests a novel physiological role for these soluble heparan sulfate proteoglycans.